HUMAN PAPILLOMAVIRUS TYPE 31 CHIMERIC PROTEIN AND USE THEREOF
20240082382 ยท 2024-03-14
Inventors
Cpc classification
C07K7/00
CHEMISTRY; METALLURGY
C07K19/00
CHEMISTRY; METALLURGY
C12N2710/20022
CHEMISTRY; METALLURGY
C12N2710/20034
CHEMISTRY; METALLURGY
C12N15/70
CHEMISTRY; METALLURGY
C12N2710/14043
CHEMISTRY; METALLURGY
A61K2039/545
HUMAN NECESSITIES
International classification
C07K7/00
CHEMISTRY; METALLURGY
Abstract
The present invention relates to a human papillomavirus type 31 chimeric protein and a use thereof. Specifically, the present invention relates to a human papillomavirus chimeric protein, containing or being composed of an HPV31L1 protein or HPV31L1 protein mutant, and a polypeptide derived from an HPV73L2 protein and inserted into the HPV31L1 protein or HPV31L1 protein mutant, wherein the HPV31L1 protein is as shown in SEQ ID No. 1, and the HPV73L2 protein is as shown in SEQ ID No. 2.
Claims
1. A human papillomavirus chimeric protein comprising or consisting of a HPV type 31L1 protein or a mutant of the HPV type 31L1 protein, and a polypeptide from a HPV type 73L2 protein inserted into the HPV type 31L1 protein or the mutant of the HPV type 31L1 protein, wherein the HPV type 31L1 protein is as shown in SEQ ID No. 1, and the HPV type 73L2 protein is as shown in SEQ ID No. 2.
2. The human papillomavirus chimeric protein according to claim 1, wherein the amino acid sequence of the human papillomavirus chimeric protein is as shown in any one of SEQ ID Nos. 18-33.
3. A polynucleotide encoding the human papillomavirus chimeric protein according to claim 1.
4. A vector comprising the polynucleotide according to claim 3.
5. A cell comprising the vector according to claim 4.
6. A polymer which is a chimeric pentamer or chimeric virus-like particle comprising the human papillomavirus chimeric protein according to claim 1, or formed by the human papillomavirus chimeric protein according to claim 1.
7. (canceled)
8. A vaccine for the prevention of papillomavirus infection and/or a papillomavirus infection-induced disease, comprising the human papillomavirus chimeric protein according to claim 1 or the polymer according to claim 6, an adjuvant, as well as an excipient or carrier for vaccines.
9. The vaccine for the prevention of papillomavirus infection and/or a papillomavirus infection-induced disease according to claim 8, further comprising at least one virus-like particle or chimeric virus-like particle of HPV of the mucosa-tropic group and/or skin-tropic group.
10. (canceled)
11. The human papillomavirus chimeric protein according to claim 1, wherein the polypeptide from the HPV type 73L2 protein is as shown in SEQ ID No. 15, SEQ ID No. 16 or SEQ ID No.17.
12. The human papillomavirus chimeric protein according to claim 1, wherein the polypeptide from the HPV type 73L2 protein is inserted into the DE loop or h4 region of the HPV type 31L1 protein or the mutant of the HPV type 31L1 protein.
13. The human papillomavirus chimeric protein according to claim 1, wherein the polypeptide from the HPV type 73L2 protein is inserted between amino acids 132 and 133, or between amino acids 134 and 135, or between amino acids 136 and 137, or between amino acids 137 and 138, or between amino acids 432 and 433, or between amino acids 434 and 435, or between amino acids 435 and 436 of the HPV type 31L1 protein or the mutant of the HPV type 31L1 protein by direct insertion; or wherein the polypeptide from the HPV type 73L2 protein is inserted into the region of amino acids 132 to 136, or the region of amino acids 135 to 139, or the region of amino acids 428 to 431, or the region of amino acids 431 to 434 of the HPV type 31L1 protein or the mutant of the HPV type 31L1 protein by non-isometric substitution.
14. The human papillomavirus chimeric protein according to claim 1, wherein the polypeptide from the HPV type 73L2 protein comprises a linker of 1 to 3 amino acid residues in length at its N-terminus and/or C-terminus.
15. The human papillomavirus chimeric protein according to claim 1, wherein the polypeptide from the HPV type 73L2 protein comprises a linker having 1 to 3 amino acids selected from the group consisting of glycine, serine, alanine and proline.
16. The human papillomavirus chimeric protein according to claim 1, wherein the mutant of the HPV type 31L1 protein comprises any one or more mutations selected from i) to iii), compared with the HPV type 31L1 protein as shown in SEQ ID No. 1: i) any one or more substitution mutation(s) selected from the group consisting of T.sub.274N, R475G, R483G, R496G, K477S, K497S, K501S, K479A, K482A, K498A, K495G, K500G and R473G; ii) truncation mutation of 2, 4, 5, 8 or 10 amino acids truncated at the N-terminus; and iii) truncation mutation of 29 amino acids truncated at the C-terminus.
17. The human papillomavirus chimeric protein according to claim 1, wherein the mutant of the HPV type 31L1 protein is any one selected from the variants as shown in SEQ ID Nos. 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 and 14.
18. The polynucleotide according to claim 3, wherein the sequence of the polynucleotide is whole-gene optimized using E. coli codons or whole-gene optimized using insect cell codons.
19. The polynucleotide according to claim 3, wherein the sequence of the polynucleotide is as shown in any one of SEQ ID No. 37 to SEQ ID No. 52.
20. A method for prevention of papillomavirus infection and/or papillomavirus infection-induced diseases, including administering to a subject in need thereof a preventively effective amount of the human papillomavirus chimeric protein according to claim 1.
21. The method of claim 20, wherein the papillomavirus infection-induced diseases are selected from the group consisting of cervical cancer, vaginal cancer, vulval cancer, penile cancer, perianal cancer, oropharyngeal cancer, tonsil cancer and oral cancer.
22. The method of claim 20, wherein the papillomavirus infection is an infection selected from one or more of the following papillomavirus types: HPV16, HPV18, HPV26, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV53, HPV56, HPV58, HPV59, HPV66, HPV68, HPV70, HPV73, HPV6, HPV11, HPV2, HPV5, HPV27 and HPV57.
Description
DESCRIPTION OF THE DRAWINGS
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DETAILED DESCRIPTION OF THE INVENTION
[0077] The present invention will be further illustrated by the non-limiting examples below. It is well known to those skilled in the art that many modifications can be made to the present invention without departing from the spirit of the present invention, and such modifications also fall within the scope of the present invention. The following examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention, as the embodiments are necessarily diverse. The terms used in the present specification are intended only to describe particular embodiments but not as limitations. The scope of the present invention has been defined in the appended claims.
[0078] Unless otherwise specified, all the technical and scientific terms used in the present specification have the same meaning as those generally understood by those skilled in the technical field to which the present application relates. Preferred methods and materials of the present invention are described below, but any method and material similar or equivalent to the methods and materials described in the present specification can be used to implement or test the present invention. Unless otherwise specified, the following experimental methods are conventional methods or methods described in product specifications. Unless otherwise specified, the experimental materials used are easily available from commercial companies. All published literatures referred to in the present specification are incorporated here by reference to reveal and illustrate the methods and/or materials in the published literatures.
Example 1: Sequence Analysis of L1 of HPV31 Variant Strains
[0079] The keyword major capsid protein L1 [Human papillomavirus type 31] or late protein L1 [Human papillomavirus type 31] was entered into NCBI Genbank to obtain 19 variant strains of HPV31L1 existing in nature, and the amino acid sequences were aligned using DNAMAN software (Table 1). It was found that the amino acids at positions 15, 179, 181, 194, 267, 274, 432, 439 of L1 were mutated, in which the positions 267 (mutation frequency 53%) and 274 (mutation frequency 89%) were high-frequency mutation sites, and the amino acid mutation frequencies of other sites were between 5%15%. For the amino acids at positions 267 and 274, mutations from threonine (T) to alanine (A) at position 267 accounted for 53%, and mutations from threonine (T) to asparagine (N) at position 274 accounted for 89%. Therefore, A and T were the dominant amino acids at positions 267 and 274, respectively.
TABLE-US-00001 TABLE 1 Alignment of amino acid sequences of different HPV31 L1 variant strains 31L1 Sequence Amino acid No. in L1 No. 15 179 181 194 267 274 432 439 P17388.1 P N I T T T T E (original) AIG59271.1 N A N AIG59269.1 L A N AIG59267.1 A N D AIG59263.1 A N AIG59261.1 S AIG59259.1 N N AIG59257.1 N A N AIG59255.1 A N AIG59253.1 N AIG59251.1 S AIG59249.1 A N AIG59247.1 N AIG59245.1 N A AIG59243.1 N AIG59241.1 A N AIG59239.1 N AIG59237.1 T A N AIG59235.1 N N AIG59233.1 A N *The amino acids represented by hyphens () were the same as the amino acids in the corresponding positions of the original HPV31L1.
Example 2: Immunoactivity Detection of Different Types of RG-1 Epitope Peptides
[0080] HPV35, -39, -51, -53, -56, -68, -73, -82 RG-1 epitope peptides were synthesized using chemical synthesis, and the sequences of the epitope peptides were as shown in Table 1. The polypeptides were synthesized by GL Biochem (Shanghai) Co., Ltd. In order to improve the immunogenicity of the synthetic peptides, each synthetic peptide was coupled with keyhole limpet hemocyanin (KLH) after activation of carboxyl group by 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC, CAS No. 25952-53-8).
[0081] New Zealand white rabbits weighing 2.0-2.5 kg were randomly divided into groups, 2-4 rabbits per group. Four days before immunization, 15 mg of inactivated DH5a (PBS containing 0.5% v/v formaldehyde, treated at 37 C. for 24-48 h) thoroughly mixed with an equal volume of Freund's complete adjuvant was injected subcutaneously at multiple sites on the back for immunostimulation. The first immunization was performed by subcutaneous injection of 1 mg of KLH-polypeptide thoroughly mixed with an equal volume of Freund's complete adjuvant at multiple sites on the back and inner thigh. Booster immunization was performed for 4 times at an interval of 2 weeks, and the antigen of the booster immunization was 0.5 mg of KLH-polypeptide thoroughly mixed with an equal volume of Freund's incomplete adjuvant. Blood was collected 2 weeks after the last immunization and serum was isolated.
[0082] 17 types of HPV pseudoviruses were used to detect the titers of neutralizing antibodies in the immune serum, and the results were as shown in Table 3. The 73RG-1 epitope peptide had the best immune activity, and its antiserum could neutralize all 17 types used for detection, in which the titers of neutralizing antibodies of HPV45, -18, -16 were all above 10.sup.3, and the titers of neutralizing antibodies of HPV68, -57, -59, -39, -5 were between 500 and 1000.
[0083] Methods of polypeptide synthesis, pseudovirus preparation and pseudoviral neutralization experiments were all publicly available, for example, the patents CN 104418942A and 108676057A.
TABLE-US-00002 TABLE2 Sequencesofdifferenttypesof RG-1epitopepeptidessynthesized Sequenceof Type syntheticpeptide SEQIDNO. HPV35 TQLYRTCKAAGTCPPDVIPKVEG 53 HPV39 STLYRTCKQSGTCPPDVVDKVEG 54 HPV51 TQLYSTCKAAGTCPPDVVNKVEG 55 HPV53 TQLYQTCKQSGTCPEDVINKIEH 56 HPV56 TQLYKTCKLSGTCPEDVVNKIEQ 57 HPV68 STLYKTCKQSGTCPPDVINKVEG 58 HPV73 TQLYKTCKQAGTCPPDVIPKVEG 59 HPV82 TQLYSTCKAAGTCPPDVIPKVKG 60
TABLE-US-00003 TABLE 3 Titers of serum neutralizing antibodies induced by different RG1-KLH conjugated peptides in rabbits 35RG- 39RG- 51RG- 53RG- 56RG- 68RG- 73RG- 82RG- 1 1 1 1 1 1 1 1 of 7 HPV 18 ND* ND ND ND 50 25 1200 100
subgenus HPV 39 ND 25 ND ND 100 100 500 400 HPV 45 25 25 25 ND 1200 1600 3600 400 HPV 59 ND ND ND 100 25 ND 600 ND HPV 68 ND ND ND ND 75 425 800 100 9 HPV 16 ND ND ND ND ND ND 1200 50 subgenus HPV 31 ND ND ND ND ND ND 200 25 HPV 33 ND ND ND ND ND ND 25 25 HPV 35 25 ND ND ND ND ND 300 100 HPV 52 ND ND ND ND ND ND 200 50 HPV 58 ND ND ND ND 25 ND 425 100 10 HPV 6 ND ND ND ND 25 ND 75 50 subgenus HPV 11 ND 25 ND 25 ND 25 125 50 4 HPV 2 25 125 ND 50 ND ND 400 50 subgenus HPV 27 50 50 25 50 50 50 200 25 HPV 57 75 50 50 50 75 50 800 125 1 HPV 5 50 50 25 ND 50 200 500 225 subgenus
indicates data missing or illegible when filed
Example 3: Synthesis of Genes of the HPV31L1 Protein and Mutants thereof and Construction of Expression Vectors
[0084] There were a total of 11 types of HPV31L1 protein and mutants, namely: [0085] 1) Original 31L1: its amino acid sequence was as shown in SEQ ID No. 1, and the nucleotide sequence encoding the 31L1 original protein was optimized with insect cell codons and constructed by whole-gene synthesis; [0086] 2) T.sub.274N mutant: the threonine at position 274 of the sequence SEQ ID No. 1 was mutated to asparagine, its amino acid sequence was as shown in SEQ ID No. 3, and the nucleotide sequence encoding the T.sub.274N mutant was optimized with insect cell codons and constructed by whole-gene synthesis; [0087] 3) 31L1MC mutant: 29 amino acids at the C-terminus of HPV31L1 were truncated, its amino acid sequence was as shown in SEQ ID No. 5, and the nucleotide sequence encoding 31L1MC was optimized with insect cell codons and constructed by whole-gene synthesis, the nucleotide sequence was as shown in SEQ ID No. 34; [0088] 4) T.sub.274NC mutant: the threonine at position 274 of the sequence SEQ ID No. 5 was mutated to asparagine, its amino acid sequence was as shown in SEQ ID No. 6, and the nucleotide sequence encoding T.sub.274NC was optimized with insect cell codons and constructed by whole-gene synthesis, the nucleotide sequence was as shown in SEQ ID No. 35; [0089] 5) T.sub.267AC mutant: the threonine at position 267 of the sequence SEQ ID No. 5 was mutated to alanine, and the nucleotide sequence encoding T.sub.267AC was optimized with insect cell codons and constructed by whole-gene synthesis; [0090] 6) T.sub.267AT.sub.274NC mutant: the threonine at position 267 of the sequence SEQ ID No. 6 was mutated to alanine, and the nucleotide sequence encoding T.sub.267AT.sub.274NC was optimized with insect cell codons and constructed by whole-gene synthesis; [0091] 7) T.sub.274NN2C mutant: 2 amino acids at the N-terminus of the sequence as shown in SEQ ID No. 6 were truncated, its sequence was as shown in SEQ ID No. 7, and the nucleotide sequence encoding T.sub.274NN2C was optimized with insect cell codons and constructed by whole-gene synthesis; [0092] 8) T.sub.274NN4C mutant: 4 amino acids at the N-terminus of the sequence as shown in SEQ ID No. 6 were truncated, its amino acid sequence was as shown in SEQ ID No. 8, and the nucleotide sequence encoding T.sub.274NN4C was optimized with insect cell codons and constructed by whole-gene synthesis, the nucleotide sequence was as shown in SEQ ID No. 36; [0093] 9) T.sub.274NN5C mutant: 5 amino acids at the N-terminus of the sequence as shown in SEQ ID No. 6 were truncated, its sequence was as shown in SEQ ID No. 9, and the nucleotide sequence encoding T.sub.274NN5C was optimized with insect cell codons and constructed by whole-gene synthesis; [0094] 10) T.sub.274NN8C mutant: 8 amino acids at the N-terminus of the sequence as shown in SEQ ID No. 6 were truncated, its sequence was as shown in SEQ ID No. 10, and the nucleotide sequence encoding T.sub.274NN8C was optimized with insect cell codons and constructed by whole-gene synthesis; [0095] 11) T.sub.274NN10C mutant: 10 amino acids at the N-terminus of the sequence as shown in SEQ ID No. 6 were truncated, its sequence was as shown in SEQ ID No. 11, and the nucleotide sequence encoding T.sub.274NN10C was optimized with insect cell codons and constructed by whole-gene synthesis.
[0096] The genes of HPV31L1 protein and mutants optimized with insect cell codons were digested by BamHI/Xbal and inserted into the commercial expression vector pFastBac1 (produced by Invitrogen), respectively. Expression vectors comprising the chimeric protein genes were obtained, namely pFastBac1-31L1, pFastBac1-T.sub.274N, pFastBac1-31L1MC, pFastBac1-T.sub.274NC, pFastBac1-T.sub.267AC, pFastBac1-T.sub.267AT.sub.274NC, pFastBac1-T.sub.274NN2C, pFastBac1-T.sub.274NN4C, pFastBac1-T.sub.274NN5C, pFastBac1-T.sub.274NN8C, and pFastBac1-T.sub.274NN10C. The above methods of enzymatic digestion, ligation and construction of clones were all well known, for example, the patent CN 101293918 B.
Example 4: Synthesis of Genes of the HPV31L1 Chimeric Protein and Mutants thereof and Construction of Expression Vectors
[0097] There were a total of 16 types of chimeric proteins and mutants, namely: [0098] 1) Chimeric L1 protein 31L1DE.sub.132-136/dE: the backbone was T.sub.274NN4C (i.e., 4 amino acids at the N-terminus were truncated and 29 amino acids at the C-terminus were truncated on the basis of mutation of threonine at position 274 to asparagine, its sequence was as shown in SEQ ID No. 8), where the region of aa. 133-135 was deleted, and the polypeptide of aa. 18-38 of HPV type 73L2 protein comprising a GP linker at the N-terminus was fused between aa. 132/136 (inserted at the region of aa. 132-136 of SEQ ID No. 8 by non-isometric substitution). The amino acid sequence of the inserted fragment was the sequence as shown in SEQ ID No. 15 with glycine-proline added at the N-terminus, and the amino acid sequence of 31L1DE.sub.132-136/dE chimeric protein was as shown in SEQ ID No. 18. The polynucleotide sequence encoding 31L1DE.sub.132-136/dE was optimized with insect cell codons and constructed by whole-gene synthesis, and its sequence was as shown in SEQ ID No. 37; [0099] 2) Chimeric L1 protein 31L1DE.sub.132-136/dES: the backbone was T.sub.274NN4C (its sequence was as shown in SEQ ID No. 8), where the region of aa. 133-135 was deleted, and the polypeptide of aa. 19-35 of HPV type 73L2 protein comprising a GP linker at the N-terminus was fused between aa. 132/136 (inserted at the region of aa. 132-136 of SEQ ID No. 8 by non-isometric substitution). The amino acid sequence of the inserted fragment was the sequence as shown in SEQ ID No. 17 with glycine-proline added at the N-terminus, and the amino acid sequence of 31L1DE.sub.132-136/dES chimeric protein was as shown in SEQ ID No. 19. The polynucleotide sequence encoding 31L1DE.sub.132-136/dES was optimized with insect cell codons and constructed by whole-gene synthesis, and its sequence was as shown in SEQ ID No. 38; [0100] 3) Chimeric L1 protein 31L1DE.sub.132-136/dE-CS1: the backbone was T.sub.274NN4C-CS1 (i.e., 4 amino acids at the N-terminus were truncated and the basic acids within 29 amino acids at the C-terminus were substituted on the basis of mutation of threonine at position 274 to asparagine, its sequence was as shown in SEQ ID No. 12), where the region of aa. 133-135 was deleted, and the polypeptide of aa. 18-38 of HPV type 73L2 protein comprising a GP linker at the N-terminus was fused between aa. 132/136 (inserted at the region of aa. 132-136 of SEQ ID No. 12 by non-isometric substitution). The amino acid sequence of the inserted fragment was the sequence as shown in SEQ ID No. 15 with glycine-proline added at the N-terminus, and the amino acid sequence of 31L1DE.sub.132-136/dE-CS1 chimeric protein was as shown in SEQ ID No. 20. The polynucleotide sequence encoding 31L1DE.sub.132-136/dE-CS1 was optimized with insect cell codons and constructed by whole-gene synthesis, and its sequence was as shown in SEQ ID No. 39; [0101] 4) Chimeric L1 protein 31L1DE.sub.132-136/dES-CS1: the backbone was T.sub.274NN4C-CS1 (its sequence was as shown in SEQ ID No. 12), where the region of aa. 133-135 was deleted, and the polypeptide of aa. 19-35 of HPV type 73L2 protein comprising a GP linker at the N-terminus was fused between aa. 132/136 (inserted at the region of aa. 132-136 of SEQ ID No. 12 by non-isometric substitution). The amino acid sequence of the inserted fragment was the sequence as shown in SEQ ID No. 17 with glycine-proline added at the N-terminus, and the amino acid sequence of 31L1DE.sub.132-136/dES-CS1 chimeric protein was as shown in SEQ ID No. 21. The polynucleotide sequence encoding 31L1DE.sub.132-136/dES-CS1 was optimized with insect cell codons and constructed by whole-gene synthesis, and its sequence was as shown in SEQ ID No. 40; [0102] 5) Chimeric L1 protein 31L1DE.sub.132-136/dE-CS2: the backbone was T.sub.274NN4C-CS2 (i.e., 4 amino acids at the N-terminus were truncated and the basic acids within 29 amino acids at the C-terminus were substituted on the basis of mutation of threonine at position 274 to asparagine, its sequence was as shown in SEQ ID No. 13), where the region of aa. 133-135 was deleted, and the polypeptide of aa. 18-38 of HPV type 73L2 protein comprising a GP linker at the N-terminus was fused between aa. 132/136 (inserted at the region of aa. 132-136 of SEQ ID No. 13 by non-isometric substitution). The amino acid sequence of the inserted fragment was the sequence as shown in SEQ ID No. 15 with glycine-proline added at the N-terminus, and the amino acid sequence of 31L1DE.sub.132-136/dE-CS2 chimeric protein was as shown in SEQ ID No. 22. The polynucleotide sequence encoding 31L1DE.sub.132-136/dE-CS2 was optimized with insect cell codons and constructed by whole-gene synthesis, and its sequence was as shown in SEQ ID No. 41; [0103] 6) Chimeric L1 protein 31L1DE.sub.132-136/dES-CS2: the backbone was T.sub.274NN4C-CS2 (its sequence was as shown in SEQ ID No. 13), where the region of aa. 133-135 was deleted, and the polypeptide of aa. 19-35 of HPV type 73L2 protein comprising a GP linker at the N-terminus was fused between aa. 132/136 (inserted at the region of aa. 132-136 of SEQ ID No. 13 by non-isometric substitution). The amino acid sequence of the inserted fragment was the sequence as shown in SEQ ID No. 17 with glycine-proline added at the N-terminus, and the amino acid sequence of 31L1DE.sub.132-136/dES-CS2 chimeric protein was as shown in SEQ ID No. 23. The polynucleotide sequence encoding 31L1DE.sub.132-136/dES-CS2 was optimized with insect cell codons and constructed by whole-gene synthesis, and its sequence was as shown in SEQ ID No. 42; [0104] 7) Chimeric L1 protein 31L1DE.sub.132-136/dE-CS3: the backbone was T.sub.274NN4C-CS3 (i.e., 4 amino acids at the N-terminus were truncated and the basic acids within 29 amino acids at the C-terminus were substituted on the basis of mutation of threonine at position 274 to asparagine, its sequence was as shown in SEQ ID No. 14), where the region of aa. 133-135 was deleted, and the polypeptide of aa. 18-38 of HPV type 73L2 protein comprising a GP linker at the N-terminus was fused between aa. 132/136 (inserted at the region of aa. 132-136 of SEQ ID No. 14 by non-isometric substitution). The amino acid sequence of the inserted fragment was the sequence as shown in SEQ ID No. 15 with glycine-proline added at the N-terminus, and the amino acid sequence of 31L1DE.sub.132-136/dE-CS3 chimeric protein was as shown in SEQ ID No. 24. The polynucleotide sequence encoding 31L1DE.sub.132-136/dE-CS3 was optimized with insect cell codons and constructed by whole-gene synthesis, and its sequence was as shown in SEQ ID No. 43; [0105] 8) Chimeric L1 protein 31L1DE.sub.132-136/dES-CS3: the backbone was T.sub.274NN4C-CS3 (its sequence was as shown in SEQ ID No. 14), where the region of aa. 133-135 was deleted, and the polypeptide of aa. 19-35 of HPV type 73L2 protein comprising a GP linker at the N-terminus was fused between aa. 132/136 (inserted at the region of aa. 132-136 of SEQ ID No. 14 by non-isometric substitution). The amino acid sequence of the inserted fragment was the sequence as shown in SEQ ID No. 17 with glycine-proline added at the N-terminus, and the amino acid sequence of 31L1DE.sub.132-136/dES-CS3 chimeric protein was as shown in SEQ ID No. 25. The polynucleotide sequence encoding 31L1DE.sub.132-136/dES-CS3 was optimized with insect cell codons and constructed by whole-gene synthesis, and its sequence was as shown in SEQ ID No. 44; [0106] 9) Chimeric L1 protein 31L1h4.sub.428-431/dE: the backbone was T.sub.274NN4C (i.e., 4 amino acids at the N-terminus were truncated and 29 amino acids at the C-terminus were truncated on the basis of mutation of threonine at position 274 to asparagine, its sequence was as shown in SEQ ID No. 8), where the region of aa. 429-430 was deleted, and the polypeptide of aa. 19-39 of HPV type 73L2 protein was fused between aa. 428/431 (inserted at the region of aa. 428-431 of SEQ ID No. 8 by non-isometric substitution). The amino acid sequence of the inserted fragment was the sequence as shown in SEQ ID No. 16, and the amino acid sequence of 31L1h4.sub.428-431/dE chimeric protein was as shown in SEQ ID No. 26. The polynucleotide sequence encoding 31L1h4.sub.428-431/dE was optimized with insect cell codons and constructed by whole-gene synthesis, and its sequence was as shown in SEQ ID No. 45; [0107] 10) Chimeric L1 protein 31L1h4.sub.428-431/dES: the backbone was T.sub.274NN4C (its sequence was as shown in SEQ ID No. 8), where the region of aa. 429-430 was deleted, and the polypeptide of aa. 19-35 of HPV type 73L2 protein was fused between aa. 428/431 (inserted at the region of aa. 428-431 of SEQ ID No. 8 by non-isometric substitution). The amino acid sequence of the inserted fragment was the sequence as shown in SEQ ID No. 17, and the amino acid sequence of 31L1h4.sub.428-431/dES chimeric protein was as shown in SEQ ID No. 27. The polynucleotide sequence encoding 31L1h4.sub.428-431/dES was optimized with insect cell codons and constructed by whole-gene synthesis, and its sequence was as shown in SEQ ID No. 46; [0108] 11) Chimeric L1 protein 31L1h4.sub.428-431/dE-CS1: the backbone was T.sub.274NN4C-CS1 (i.e., 4 amino acids at the N-terminus were truncated and the basic acids within 29 amino acids at the C-terminus were substituted on the basis of mutation of threonine at position 274 to asparagine, its sequence was as shown in SEQ ID No. 12), where the region of aa. 429-430 was deleted, and the polypeptide of aa. 19-39 of HPV type 73L2 protein was fused between aa. 428/431 (inserted at the region of aa. 428-431 of SEQ ID No. 12 by non-isometric substitution). The amino acid sequence of the inserted fragment was the sequence as shown in SEQ ID No. 16, and the amino acid sequence of 31L1h4.sub.428-431/dE-CS1 chimeric protein was as shown in SEQ ID No. 28. The polynucleotide sequence encoding 31L1h4.sub.428-431/dE-CS1 was optimized with insect cell codons and constructed by whole-gene synthesis, and its sequence was as shown in SEQ ID No. 47; [0109] 12) Chimeric L1 protein 31L1h4.sub.428-431/dES-CS1: the backbone was T.sub.274NN4C-CS1 (its sequence was as shown in SEQ ID No. 12), where the region of aa. 429-430 was deleted, and the polypeptide of aa. 19-35 of HPV type 73L2 protein was fused between aa. 428/431 (inserted at the region of aa. 428-431 of SEQ ID No. 12 by non-isometric substitution). The amino acid sequence of the inserted fragment was the sequence as shown in SEQ ID No. 17, and the amino acid sequence of 31L1h4.sub.428-431/dES-CS1 chimeric protein was as shown in SEQ ID No. 29. The polynucleotide sequence encoding 31L1h4.sub.428-431/dES-CS1 was optimized with insect cell codons and constructed by whole-gene synthesis, and its sequence was as shown in SEQ ID No. 48; [0110] 13) Chimeric L1 protein 31L1h4.sub.428-431/dE-CS2: the backbone was T.sub.274NN4C-CS2 (i.e., 4 amino acids at the N-terminus were truncated and the basic acids within 29 amino acids at the C-terminus were substituted on the basis of mutation of threonine at position 274 to asparagine, its sequence was as shown in SEQ ID No. 13), where the region of aa. 429-430 was deleted, and the polypeptide of aa. 19-39 of HPV type 73L2 protein was fused between aa. 428/431 (inserted at the region of aa. 428-431 of SEQ ID No. 13 by non-isometric substitution). The amino acid sequence of the inserted fragment was the sequence as shown in SEQ ID No. 16, and the amino acid sequence of 31L1h4.sub.428-431/dE-CS2 chimeric protein was as shown in SEQ ID No. 30. The polynucleotide sequence encoding 31L1h4.sub.428-431/dE-CS2 was optimized with insect cell codons and constructed by whole-gene synthesis, and its sequence was as shown in SEQ ID No. 49; [0111] 14) Chimeric L1 protein 31L1h4.sub.428-431/dES-CS2: the backbone was T.sub.274NN4C-CS2 (its sequence was as shown in SEQ ID No. 13), where the region of aa. 429-430 was deleted, and the polypeptide of aa. 19-35 of HPV type 73L2 protein was fused between aa. 428/431 (inserted at the region of aa. 428-431 of SEQ ID No. 13 by non-isometric substitution). The amino acid sequence of the inserted fragment was the sequence as shown in SEQ ID No. 17, and the amino acid sequence of 31L1h4.sub.428-431/dES-CS2 chimeric protein was as shown in SEQ ID No. 31. The polynucleotide sequence encoding 31L1h4.sub.428-431/dES-CS2 was optimized with insect cell codons and constructed by whole-gene synthesis, and its sequence was as shown in SEQ ID No. 50; [0112] 15) Chimeric L1 protein 31L1h4.sub.428-431/dE-CS3: the backbone was T.sub.274NN4C-CS3 (i.e., 4 amino acids at the N-terminus were truncated and the basic acids within 29 amino acids at the C-terminus were substituted on the basis of mutation of threonine at position 274 to asparagine, the sequence was as shown in SEQ ID No. 14), where the region of aa. 429-430 was deleted, and the polypeptide of aa. 19-39 of HPV type 73L2 protein was fused between aa. 428/431 (inserted at the region of aa. 428-431 of SEQ ID No. 13 by non-isometric substitution). The amino acid sequence of the inserted fragment was the sequence as shown in SEQ ID No. 16, and the amino acid sequence of 31L1h4.sub.428-431/dE-CS3 chimeric protein was as shown in SEQ ID No. 32. The polynucleotide sequence encoding 31L1h4.sub.428-431/dE-CS3 was optimized with insect cell codons and constructed by whole-gene synthesis, and its sequence was as shown in SEQ ID No. 51; [0113] 16) Chimeric L1 protein 31L1h4.sub.428-431/dES-CS3: the backbone was T.sub.274NN4C-CS3 (its sequence was as shown in SEQ ID No. 14), where the region of aa. 429-430 was deleted, and the polypeptide of aa. 19-35 of HPV type 73L2 protein was fused between aa. 428/431 (inserted at the region of aa. 428-431 of SEQ ID No. 14 by non-isometric substitution). The amino acid sequence of the inserted fragment was the sequence as shown in SEQ ID No. 17, and the amino acid sequence of 31L1h4.sub.428-431/dES-CS3 chimeric protein was as shown in SEQ ID No. 33. The polynucleotide sequence encoding 31L1h4.sub.428-431/dES-CS3 was optimized with insect cell codons and constructed by whole-gene synthesis, and its sequence was as shown in SEQ ID No. 52.
[0114] The genes of HPV31L1 protein and mutants optimized with insect cell codons were digested by BamHI/Xbal and inserted into the commercial expression vector pFastBac1 (produced by Invitrogen), respectively. Expression vectors comprising the chimeric protein genes were obtained, namely pFastBac1-31L1DE.sub.132-136/dE, pFastBac1-31L1DE.sub.132-136/dES, pFastBac1-31L1DE.sub.132-136/dE-CS1, pFastBac1-31L1DE.sub.132-136/dES-CS1, pFastBac1-31L1DE.sub.132-136/dE-CS2, pFastBac1-31L1DE.sub.132-136/dES-CS2, pFastBac1-31L1DE.sub.132-136/dE-CS3, pFastBac1-31L1DE.sub.132-136/dES-CS3, pFastBac1-31L1h4.sub.428-431/dE, pFastBac1-31L1h4.sub.428-431/dES, pFastBac1-31L1h4.sub.428-431/dE-CS1, pFastBac1-31L1h4.sub.428-431/dES-CS1, pFastBac1-31L1h4.sub.428-431/dE-CS2, pFastBac1-31L1h4.sub.428-431/dES-CS2, pFastBac1-31L1h4.sub.428-431/dE-CS3, and pFastBac1-31L1h4.sub.428-431/dES-CS3. The above methods of enzyme digestion, ligation and construction of clones were all well known, for example, the patent CN 101293918 B.
[0115] The amino acid sequences involved in the present invention were as described below:
TABLE-US-00004 HPV31L1 SEQIDNo.1 MSLWRPSEATVYLPPVPVSKVVSTDEYVTRTNIYYHAGSARLLTVGHPYYSIPKSDNPKK IVVPKVSGLQYRVFRVRLPDPNKFGFPDTSFYNPETQRLVWACVGLEVGRGQPLGVGISG HPLLNKFDDTENSNRYAGGPGTDNRECISMDYKQTQLCLLGCKPPIGEHWGKGSPCSNNA ITPGDCPPLELKNSVIQDGDMVDTGFGAMDFTALQDTKSNVPLDICNSICKYPDYLKMVA EPYGDTLFFYLRREQMFVRHFFNRSGTVGESVPTDLYIKGSGSTATLANSTYFPTPSGSM VTSDAQIFNKPYWMQRAQGHNNGICWGNQLFVTVVDTTRSTNMSVCAAIANSDTTFKSSN FKEYLRHGEEFDLQFIFQLCKITLSADIMTYIHSMNPAILEDWNFGLTTPPSGSLEDTYR FVTSQAITCQKTAPQKPKEDPFKDYVFWEVNLKEKFSADLDQFPLGRKFLLQAGYRARPK FKAGKRSAPSASTTTPAKRKKTKK HPV73L2 SEQIDNo.2 MRRKRDTHIRKKRASATQLYKTCKQAGTCPPDVIPKVEGSTIADNILKYGSIGVFFGGLG IGSGSGSGGRTGYVPLSTGTPSKPVEMPLQPIRPSVVTSVGPSDSSIVSLVEESSFIESG IPGPTSIVPSTSGFDITTSVNSTPAIIDVSAISDTTQISVTTFKNPTFTDPSVLQPPPPL EASGRLLFSNDTVTTHSYENIPLDTFVVTTDHNSIVSSTPIPGRQPAARLGLYGRAIQQV KVVDPAFLTTPTRLVTYDNPAFEGLQDTTLEFQHSDLHNAPDSDELDIVKLHRPALTSRK TGIRVSRLGQRATLSTRSGKRIGAKVHFYHDISPIPINDIEMQPLVTPQTPSIVTGSSIN DGLYDVFLENDVEDTVVQQTYTPTSIHSNSLVSSDVSTATANTTIPFSTGLDTHPGPDIA LPLPSTETIFTPIVPLQPAGPIYIYGSGFILHPSYYLLKRKRKRLSYSFTDVATY T.sub.274N SEQIDNo.3 MSLWRPSEATVYLPPVPVSKVVSTDEYVTRTNIYYHAGSARLLTVGHPYYSIPKSDNPKK IVVPKVSGLQYRVFRVRLPDPNKFGFPDTSFYNPETQRLVWACVGLEVGRGQPLGVGISG HPLINKEDDTENSNRYAGGPGTDNRECISMDYKQTQLCLLGCKPPIGEHWGKGSPCSNNA ITPGDCPPLELKNSVIQDGDMVDTGFGAMDFTALQDTKSNVPLDICNSICKYPDYLKMVA EPYGDTLFFYLRREQMFVRHFFNRSGTVGESVPNDLYIKGSGSTATLANSTYFPTPSGSM VTSDAQIFNKPYWMQRAQGHNNGICWGNQLFVTVVDTTRSTNMSVCAAIANSDTTFKSSN FKEYLRHGEEFDLQFIFQLCKITLSADIMTYIHSMNPAILEDWNFGLTTPPSGSLEDTYR FVTSQAITCQKTAPQKPKEDPFKDYVFWEVNLKEKFSADLDQFPLGRKFLLQAGYRARPK FKAGKRSAPSASTTTPAKRKKTKK T.sub.274NN4 SEQIDNo.4 MRPSEATVYLPPVPVSKVVSTDEYVTRTNIYYHAGSARLLTVGHPYYSIPKSDNPKK IVVPKVSGLQYRVFRVRLPDPNKFGFPDTSFYNPETQRLVWACVGLEVGRGQPLGVGISG HPLLNKFDDTENSNRYAGGPGTDNRECISMDYKQTQLCLLGCKPPIGEHWGKGSPCSNNA ITPGDCPPLELKNSVIQDGDMVDTGFGAMDFTALQDTKSNVPLDICNSICKYPDYLKMVA EPYGDTLFFYLRREQMFVRHFFNRSGTVGESVPNDLYIKGSGSTATLANSTYFPTPSGSM VTSDAQIFNKPYWMQRAQGHNNGICWGNQLFVTVVDTTRSTNMSVCAAIANSDTTFKSSN FKEYLRHGEEFDLQFIFQLCKITLSADIMTYIHSMNPAILEDWNFGLTTPPSGSLEDTYR FVTSQAITCQKTAPQKPKEDPFKDYVFWEVNLKEKFSADLDQFPLGRKFLLQAGYRARPK FKAGKRSAPSASTTTPAKRKKTKK 31L1C29 SEQIDNo.5 MSLWRPSEATVYLPPVPVSKVVSTDEYVTRTNIYYHAGSARLLTVGHPYYSIPKSDNPKK IVVPKVSGLQYRVFRVRLPDPNKFGFPDTSFYNPETQRLVWACVGLEVGRGQPLGVGISG HPLLNKFDDTENSNRYAGGPGTDNRECISMDYKQTQLCLLGCKPPIGEHWGKGSPCSNNA ITPGDCPPLELKNSVIQDGDMVDTGFGAMDFTALQDTKSNVPLDICNSICKYPDYLKMVA EPYGDTLFFYLRREQMFVRHFFNRSGTVGESVPTDLYIKGSGSTATLANSTYFPTPSGSM VTSDAQIFNKPYWMQRAQGHNNGICWGNQLFVTVVDTTRSTNMSVCAAIANSDTTFKSSN FKEYLRHGEEFDLQFIFQLCKITLSADIMTYIHSMNPAILEDWNFGLTTPPSGSLEDTYR FVTSQAITCQKTAPQKPKEDPFKDYVFWEVNLKEKFSADLDQFPLGRKFLLQAGY T.sub.274NC29 SEQIDNo.6 MSLWRPSEATVYLPPVPVSKVVSTDEYVTRTNIYYHAGSARLLTVGHPYYSIPKSDNPKK IVVPKVSGLQYRVFRVRLPDPNKFGFPDTSFYNPETQRLVWACVGLEVGRGQPLGVGISG HPLLNKFDDTENSNRYAGGPGTDNRECISMDYKQTQLCLLGCKPPIGEHWGKGSPCSNNA ITPGDCPPLELKNSVIQDGDMVDTGFGAMDFTALQDTKSNVPLDICNSICKYPDYLKMVA EPYGDTLFFYLRREQMFVRHFFNRSGTVGESVPNDLYIKGSGSTATLANSTYFPTPSGSM VTSDAQIFNKPYWMQRAQGHNNGICWGNQLFVTVVDTTRSTNMSVCAAIANSDTTFKSSN FKEYLRHGEEFDLQFIFQLCKITLSADIMTYIHSMNPAILEDWNFGLTTPPSGSLEDTYR FVTSQAITCQKTAPQKPKEDPFKDYVFWEVNLKEKFSADLDQFPLGRKFLLQAGY T.sub.274NN2C29 SEQIDNo.7 MLWRPSEATVYLPPVPVSKVVSTDEYVTRTNIYYHAGSARLLTVGHPYYSIPKSDNPKK IVVPKVSGLQYRVFRVRLPDPNKFGFPDTSFYNPETQRLVWACVGLEVGRGQPLGVGISG HPLLNKFDDTENSNRYAGGPGTDNRECISMDYKQTQLCLLGCKPPIGEHWGKGSPCSNNA ITPGDCPPLELKNSVIQDGDMVDTGFGAMDFTALQDTKSNVPLDICNSICKYPDYLKMVA EPYGDTLFFYLRREQMFVRHFFNRSGTVGESVPNDLYIKGSGSTATLANSTYFPTPSGSM VTSDAQIFNKPYWMQRAQGHNNGICWGNQLFVTVVDTTRSTNMSVCAAIANSDTTFKSSN FKEYLRHGEEFDLQFIFQLCKITLSADIMTYIHSMNPAILEDWNFGLITPPSGSLEDTYR FVTSQAITCQKTAPQKPKEDPFKDYVFWEVNLKEKFSADLDQFPLGRKFLLQAGY T.sub.274NN4C29 SEQIDNo.8 MRPSEATVYLPPVPVSKVVSTDEYVTRTNIYYHAGSARLLTVGHPYYSIPKSDNPKK IVVPKVSGLQYRVFRVRLPDPNKFGFPDTSFYNPETQRLVWACVGLEVGRGQPLGVGISG HPLLNKFDDTENSNRYAGGPGTDNRECISMDYKQTQLCLLGCKPPIGEHWGKGSPCSNNA ITPGDCPPLELKNSVIQDGDMVDTGFGAMDFTALQDTKSNVPLDICNSICKYPDYLKMVA EPYGDTLFFYLRREQMFVRHFFNRSGTVGESVPNDLYIKGSGSTATLANSTYFPTPSGSM VTSDAQIFNKPYWMQRAQGHNNGICWGNQLFVTVVDTTRSTNMSVCAAIANSDTTFKSSN FKEYLRHGEEFDLQFIFQLCKITLSADIMTYIHSMNPAILEDWNFGLTTPPSGSLEDTYR FVTSQAITCQKTAPQKPKEDPFKDYVFWEVNLKEKFSADLDQFPLGRKFLLQAGY T.sub.274NN5C29 SEQIDNo.9 MPSEATVYLPPVPVSKVVSTDEYVTRTNIYYHAGSARLLTVGHPYYSIPKSDNPKK IVVPKVSGLQYRVFRVRLPDPNKFGFPDTSFYNPETQRLVWACVGLEVGRGQPLGVGISG HPLLNKFDDTENSNRYAGGPGTDNRECISMDYKQTQLCLLGCKPPIGEHWGKGSPCSNNA ITPGDCPPLELKNSVIQDGDMVDTGFGAMDFTALQDTKSNVPLDICNSICKYPDYLKMVA EPYGDTLFFYLRREQMFVRHFFNRSGTVGESVPNDLYIKGSGSTATLANSTYFPTPSGSM VTSDAQIFNKPYWMQRAQGHNNGICWGNQLFVTVVDTTRSTNMSVCAAIANSDTTEKSSN FKEYLRHGEEFDLQFIFQLCKITLSADIMTYIHSMNPAILEDWNFGLTTPPSGSLEDTYR FVTSQAITCQKTAPQKPKEDPFKDYVFWEVNLKEKFSADLDQFPLGRKFLLQAGY T.sub.274NN8C29 SEQIDNo.10 MATVYLPPVPVSKVVSTDEYVTRTNIYYHAGSARLLTVGHPYYSIPKSDNPKK IVVPKVSGLQYRVFRVRLPDPNKFGFPDTSFYNPETQRLVWACVGLEVGRGQPLGVGISG HPLLNKFDDTENSNRYAGGPGTDNRECISMDYKQTQLCLLGCKPPIGEHWGKGSPCSNNA ITPGDCPPLELKNSVIQDGDMVDTGFGAMDFTALQDTKSNVPLDICNSICKYPDYLKMVA EPYGDTLFFYLRREQMFVRHFFNRSGTVGESVPNDLYIKGSGSTATLANSTYFPTPSGSM VTSDAQIFNKPYWMQRAQGHNNGICWGNQLFVTVVDTTRSTNMSVCAAIANSDTTEKSSN FKEYLRHGEEFDLQFIFQLCKITLSADIMTYIHSMNPAILEDWNFGLITPPSGSLEDTYR FVTSQAITCQKTAPQKPKEDPFKDYVFWEVNLKEKFSADLDQFPLGRKFLLQAGY T.sub.274NN10C29 SEQIDNo.11 MVYLPPVPVSKVVSTDEYVTRTNIYYHAGSARLLTVGHPYYSIPKSDNPKK IVVPKVSGLQYRVFRVRLPDPNKFGFPDTSFYNPETQRLVWACVGLEVGRGQPLGVGISG HPLLNKFDDTENSNRYAGGPGTDNRECISMDYKQTQLCLLGCKPPIGEHWGKGSPCSNNA ITPGDCPPLELKNSVIQDGDMVDTGFGAMDFTALQDTKSNVPLDICNSICKYPDYLKMVA EPYGDTLFFYLRREQMFVRHFFNRSGTVGESVPNDLYIKGSGSTATLANSTYFPTPSGSM VISDAQIFNKPYWMQRAQGHNNGICWGNQLFVTVVDTTRSTNMSVCAAIANSDTTFKSSN FKEYLRHGEEFDLQFIFQLCKITLSADIMTYIHSMNPAILEDWNFGLTTPPSGSLEDTYR FVTSQAITCQKTAPQKPKEDPFKDYVFWEVNLKEKFSADLDQFPLGRKFLLQAGY T.sub.274N-CS1 SEQIDNo.12 MRPSEATVYLPPVPVSKVVSTDEYVTRTNIYYHAGSARLLTVGHPYYSIPKSDNPKKIVV PKVSGLQYRVFRVRLPDPNKFGFPDTSFYNPETQRLVWACVGLEVGRGQPLGVGISGHPL LNKFDDTENSNRYAGGPGTDNRECISMDYKQTQLCLLGCKPPIGEHWGKGSPCSNNAITP GDCPPLELKNSVIQDGDMVDTGFGAMDFTALQDTKSNVPLDICNSICKYPDYLKMVAEPY GDTLFFYLRREQMFVRHFFNRSGTVGESVPNDLYIKGSGSTATLANSTYFPTPSGSMVTS DAQIFNKPYWMQRAQGHNNGICWGNQLFVTVVDTTRSTNMSVCAAIANSDTTFKSSNFKE YLRHGEEFDLQFIFQLCKITLSADIMTYIHSMNPAILEDWNFGLTTPPSGSLEDTYRFVT SQAITCQKTAPQKPKEDPFKDYVFWEVNLKEKFSADLDQFPLGRKFLLQAGYRAGPSFAA GAGSAPSASTTTPAGGSATGS T.sub.274N-CS2 SEQIDNo.13 MRPSEATVYLPPVPVSKVVSTDEYVTRTNIYYHAGSARLLTVGHPYYSIPKSDNPKKIVV PKVSGLQYRVFRVRLPDPNKFGFPDTSFYNPETQRLVWACVGLEVGRGQPLGVGISGHPL LNKFDDTENSNRYAGGPGTDNRECISMDYKQTQLCLLGCKPPIGEHWGKGSPCSNNAITP GDCPPLELKNSVIQDGDMVDTGFGAMDFTALQDTKSNVPLDICNSICKYPDYLKMVAEPY GDTLFFYLRREQMFVRHFFNRSGTVGESVPNDLYIKGSGSTATLANSTYFPTPSGSMVTS DAQIFNKPYWMQRAQGHNNGICWGNQLFVTVVDTTRSTNMSVCAAIANSDTTFKSSNFKE YLRHGEEFDLQFIFQLCKITLSADIMTYIHSMNPAILEDWNFGLTTPPSGSLEDTYRFVT SQAITCQKTAPQKPKEDPFKDYVFWEVNLKEKFSADLDQFPLGRKFLLQAGYGAGPSFAA GAGSAPSASTTTPAGGSATGS T.sub.274N-CS3 SEQIDNo.14 MRPSEATVYLPPVPVSKVVSTDEYVTRTNIYYHAGSARLLTVGHPYYSIPKSDNPKKIVV PKVSGLQYRVFRVRLPDPNKFGFPDTSFYNPETQRLVWACVGLEVGRGQPLGVGISGHPL LNKFDDTENSNRYAGGPGTDNRECISMDYKQTQLCLLGCKPPIGEHWGKGSPCSNNAITP GDCPPLELKNSVIQDGDMVDTGFGAMDFTALQDTKSNVPLDICNSICKYPDYLKMVAEPY GDTLFFYLRREQMFVRHFFNRSGTVGESVPNDLYIKGSGSTATLANSTYFPTPSGSMVTS DAQIFNKPYWMQRAQGHNNGICWGNQLFVTVVDTTRSTNMSVCAAIANSDTTFKSSNFKE YLRHGEEFDLQFIFQLCKITLSADIMTYIHSMNPAILEDWNFGLTTPPSGSLEDTYRFVT SQAITCQKTAPQKPKEDPFKDYVFWEVNLKEKFSADLDQFPLGRKFLLQAGYRAGPSFKA GAGSAPSASTTTPAGGSATGS 73L2aa.18-38 SEQIDNo.15 QLYKTCKQAGTCPPDVIPKVE 73L2aa.19-39 SEQIDNo.16 LYKTCKQAGTCPPDVIPKVEG 73L2aa.19-35 SEQIDNo.17 LYKTCKQAGTCPPDVIP 31L1DE.sub.132-136/dE SEQIDNo.18 MRPSEATVYLPPVPVSKVVSTDEYVTRTNIYYHAGSARLLTVGHPYYSIPKSDNPKKIVV PKVSGLQYRVFRVRLPDPNKFGFPDTSFYNPETQRLVWACVGLEVGRGQPLGVGISGHPL LNKFDDTENSNRGPQLYKTCKQAGTCPPDVIPKVEGPGTDNRECISMDYKQTQLCLLGCK PPIGEHWGKGSPCSNNAITPGDCPPLELKNSVIQDGDMVDTGFGAMDFTALQDTKSNVPL DICNSICKYPDYLKMVAEPYGDTLFFYLRREQMFVRHFFNRSGTVGESVPNDLYIKGSGS TATLANSTYFPTPSGSMVTSDAQIFNKPYWMQRAQGHNNGICWGNQLFVTVVDTTRSTNM SVCAAIANSDTTFKSSNFKEYLRHGEEFDLQFIFQLCKITLSADIMTYIHSMNPAILEDW NFGLTTPPSGSLEDTYRFVTSQAITCQKTAPQKPKEDPFKDYVFWEVNLKEKFSADLDQF PLGRKFLLQAGY 31L1DE.sub.132-136/dES SEQIDNo.19 MRPSEATVYLPPVPVSKVVSTDEYVTRTNIYYHAGSARLLTVGHPYYSIPKSDNPKKIVV PKVSGLQYRVFRVRLPDPNKFGFPDTSFYNPETQRLVWACVGLEVGRGQPLGVGISGHPL LNKFDDTENSNRGPLYKTCKQAGTCPPDVIPGPGTDNRECISMDYKQTQLCLLGCKPPIG EHWGKGSPCSNNAITPGDCPPLELKNSVIQDGDMVDTGFGAMDFTALQDTKSNVPLDICN SICKYPDYLKMVAEPYGDTLFFYLRREQMFVRHFFNRSGTVGESVPNDLYIKGSGSTATL ANSTYFPTPSGSMVTSDAQIFNKPYWMQRAQGHNNGICWGNQLFVTVVDTTRSTNMSVCA AIANSDTTFKSSNFKEYLRHGEEFDLQFIFQLCKITLSADIMTYIHSMNPAILEDWNFGL TTPPSGSLEDTYRFVTSQAITCQKTAPQKPKEDPFKDYVFWEVNLKEKFSADLDQFPLGR KFLLQAGY 31L1DE.sub.132-136/dE-CS1 SEQIDNo.20 MRPSEATVYLPPVPVSKVVSTDEYVTRTNIYYHAGSARLLTVGHPYYSIPKSDNPKKIVV PKVSGLQYRVFRVRLPDPNKFGFPDTSFYNPETQRLVWACVGLEVGRGQPLGVGISGHPL LNKFDDTENSNRGPQLYKTCKQAGTCPPDVIPKVEGPGTDNRECISMDYKQTQLCLLGCK PPIGEHWGKGSPCSNNAITPGDCPPLELKNSVIQDGDMVDTGFGAMDFTALQDTKSNVPL DICNSICKYPDYLKMVAEPYGDTLFFYLRREQMFVRHFENRSGTVGESVPNDLYIKGSGS TATLANSTYFPTPSGSMVTSDAQIFNKPYWMQRAQGHNNGICWGNQLFVTVVDTTRSTNM SVCAAIANSDTTFKSSNFKEYLRHGEEFDLQFIFQLCKITLSADIMTYIHSMNPAILEDW NFGLTTPPSGSLEDTYRFVTSQAITCQKTAPQKPKEDPFKDYVFWEVNLKEKFSADLDQF PLGRKFLLQAGYRAGPSFAAGAGSAPSASTTTPAGGSATGS 31L1DE.sub.132-136/dES-CS1 SEQIDNo.21 MRPSEATVYLPPVPVSKVVSTDEYVTRTNIYYHAGSARLLTVGHPYYSIPKSDNPKKIVV PKVSGLQYRVFRVRLPDPNKFGFPDTSFYNPETQRLVWACVGLEVGRGQPLGVGISGHPL LNKFDDTENSNRGPLYKTCKQAGTCPPDVIPGPGTDNRECISMDYKQTQLCLLGCKPPIG EHWGKGSPCSNNAITPGDCPPLELKNSVIQDGDMVDTGFGAMDFTALQDTKSNVPLDICN SICKYPDYLKMVAEPYGDTLFFYLRREQMFVRHFFNRSGTVGESVPNDLYIKGSGSTATL ANSTYFPTPSGSMVTSDAQIFNKPYWMQRAQGHNNGICWGNQLFVTVVDTTRSTNMSVCA AIANSDTTFKSSNFKEYLRHGEEFDLQFIFQLCKITLSADIMTYIHSMNPAILEDWNFGL TTPPSGSLEDTYRFVTSQAITCQKTAPQKPKEDPFKDYVFWEVNLKEKFSADLDQFPLGR KFLLQAGYRAGPSFAAGAGSAPSASTTTPAGGSATGS 31L1DE.sub.132-136/dE-CS2 SEQIDNo.22 MRPSEATVYLPPVPVSKVVSTDEYVTRTNIYYHAGSARLLTVGHPYYSIPKSDNPKKIVV PKVSGLQYRVFRVRLPDPNKFGFPDTSFYNPETQRLVWACVGLEVGRGQPLGVGISGHPL LNKFDDTENSNRGPQLYKTCKQAGTCPPDVIPKVEGPGTDNRECISMDYKQTQLCLLGCK PPIGEHWGKGSPCSNNAITPGDCPPLELKNSVIQDGDMVDTGFGAMDFTALQDTKSNVPL DICNSICKYPDYLKMVAEPYGDTLFFYLRREQMFVRHFFNRSGTVGESVPNDLYIKGSGS TATLANSTYFPTPSGSMVTSDAQIFNKPYWMQRAQGHNNGICWGNQLFVTVVDTTRSTNM SVCAAIANSDTTFKSSNFKEYLRHGEEFDLQFIFQLCKITLSADIMTYIHSMNPAILEDW NFGLTTPPSGSLEDTYRFVTSQAITCQKTAPQKPKEDPFKDYVFWEVNLKEKFSADLDQF PLGRKFLLQAGYGAGPSFAAGAGSAPSASTTTPAGGSATGS 31L1DE.sub.132-136/dES-CS2 SEQIDNo.23 MRPSEATVYLPPVPVSKVVSTDEYVTRTNIYYHAGSARLLTVGHPYYSIPKSDNPKKIVV PKVSGLQYRVFRVRLPDPNKFGFPDTSFYNPETQRLVWACVGLEVGRGQPLGVGISGHPL LNKFDDTENSNRGPLYKICKQAGTCPPDVIPGPGTDNRECISMDYKQTQLCLLGCKPPIG EHWGKGSPCSNNAITPGDCPPLELKNSVIQDGDMVDTGFGAMDFTALQDTKSNVPLDICN SICKYPDYLKMVAEPYGDTLFFYLRREQMFVRHFFNRSGTVGESVPNDLYIKGSGSTATL ANSTYFPTPSGSMVTSDAQIFNKPYWMQRAQGHNNGICWGNQLFVTVVDTTRSTNMSVCA AIANSDTTFKSSNFKEYLRHGEEFDLQFIFQLCKITLSADIMTYIHSMNPAILEDWNFGL TTPPSGSLEDTYRFVTSQAITCQKTAPQKPKEDPFKDYVFWEVNLKEKFSADLDQFPLGR KFLLQAGYGAGPSFAAGAGSAPSASTTTPAGGSATGS 31L1DE.sub.132-136/dE-CS3 SEQIDNo.24 MRPSEATVYLPPVPVSKVVSTDEYVTRTNIYYHAGSARLLTVGHPYYSIPKSDNPKKIVV PKVSGLQYRVFRVRLPDPNKFGFPDTSFYNPETQRLVWACVGLEVGRGQPLGVGISGHPL LNKFDDTENSNRGPQLYKTCKQAGTCPPDVIPKVEGPGTDNRECISMDYKQTQLCLLGCK PPIGEHWGKGSPCSNNAITPGDCPPLELKNSVIQDGDMVDTGFGAMDFTALQDTKSNVPL DICNSICKYPDYLKMVAEPYGDTLFFYLRREQMFVRHFFNRSGTVGESVPNDLYIKGSGS TATLANSTYFPTPSGSMVTSDAQIFNKPYWMQRAQGHNNGICWGNQLFVTVVDTTRSTNM SVCAAIANSDTTFKSSNFKEYLRHGEEFDLQFIFQLCKITLSADIMTYIHSMNPAILEDW NFGLTTPPSGSLEDTYRFVTSQAITCQKTAPQKPKEDPFKDYVFWEVNLKEKFSADLDQF PLGRKFLLQAGYRAGPSFKAGAGSAPSASTTTPAGGSATGS 31L1DE.sub.132-136/dES-CS3 SEQIDNo.25 MRPSEATVYLPPVPVSKVVSTDEYVTRINIYYHAGSARLLTVGHPYYSIPKSDNPKKIVV PKVSGLQYRVFRVRLPDPNKFGFPDTSFYNPETQRLVWACVGLEVGRGQPLGVGISGHPL LNKFDDTENSNRGPLYKTCKQAGTCPPDVIPGPGTDNRECISMDYKQTQLCLLGCKPPIG EHWGKGSPCSNNAITPGDCPPLELKNSVIQDGDMVDTGFGAMDFTALQDTKSNVPLDICN SICKYPDYLKMVAEPYGDTLFFYLRREQMFVRHFFNRSGTVGESVPNDLYIKGSGSTATL ANSTYFPTPSGSMVTSDAQIFNKPYWMQRAQGHNNGICWGNQLFVTVVDTTRSTNMSVCA AIANSDTTFKSSNFKEYLRHGEEFDLQFIFQLCKITLSADIMTYIHSMNPAILEDWNFGL TTPPSGSLEDTYRFVTSQAITCQKTAPQKPKEDPFKDYVFWEVNLKEKFSADLDQFPLGR KFLLQAGYRAGPSFKAGAGSAPSASTTTPAGGSATGS 31L1h4.sub.428-431/dE SEQIDNo.26 MRPSEATVYLPPVPVSKVVSTDEYVTRTNIYYHAGSARLLTVGHPYYSIPKSDNPKKIVV PKVSGLQYRVFRVRLPDPNKFGFPDTSFYNPETQRLVWACVGLEVGRGQPLGVGISGHPL LNKEDDTENSNRYAGGPGTDNRECISMDYKQTQLCLLGCKPPIGEHWGKGSPCSNNAITP GDCPPLELKNSVIQDGDMVDTGFGAMDFTALQDTKSNVPLDICNSICKYPDYLKMVAEPY GDTLFFYLRREQMFVRHFFNRSGTVGESVPNDLYIKGSGSTATLANSTYFPTPSGSMVTS DAQIFNKPYWMQRAQGHNNGICWGNQLFVTVVDTTRSTNMSVCAAIANSDTTFKSSNFKE YLRHGEEFDLQFIFQLCKITLSADIMTYIHSMNPAILEDWNFGLTTPPSGSLEDTYRFVT SQAITCQKLYKTCKQAGTCPPDVIPKVEGPQKPKEDPFKDYVFWEVNLKEKFSADLDQFP LGRKFLLQAGY 31L1h4.sub.428-431/dES SEQIDNo.27 MRPSEATVYLPPVPVSKVVSTDEYVTRTNIYYHAGSARLLTVGHPYYSIPKSDNPKKIVV PKVSGLQYRVFRVRLPDPNKFGFPDTSFYNPETQRLVWACVGLEVGRGQPLGVGISGHPL LNKFDDTENSNRYAGGPGTDNRECISMDYKQTQLCLLGCKPPIGEHWGKGSPCSNNAITP GDCPPLELKNSVIQDGDMVDTGFGAMDFTALQDTKSNVPLDICNSICKYPDYLKMVAEPY GDTLFFYLRREQMFVRHFFNRSGTVGESVPNDLYIKGSGSTATLANSTYFPTPSGSMVTS DAQIFNKPYWMQRAQGHNNGICWGNQLFVTVVDTTRSTNMSVCAAIANSDTTFKSSNFKE YLRHGEEFDLQFIFQLCKITLSADIMTYIHSMNPAILEDWNFGLITPPSGSLEDTYRFVT SQAITCQKLYKTCKQAGTCPPDVIPPQKPKEDPFKDYVFWEVNLKEKFSADLDQFPLGRK FLLQAGY 31L1h4.sub.428-431/dE-CS1 SEQIDNo.28 MRPSEATVYLPPVPVSKVVSTDEYVTRTNIYYHAGSARLLTVGHPYYSIPKSDNPKKIVV PKVSGLQYRVFRVRLPDPNKFGFPDTSFYNPETQRLVWACVGLEVGRGQPLGVGISGHPL LNKFDDTENSNRYAGGPGTDNRECISMDYKQTQLCLLGCKPPIGEHWGKGSPCSNNAITP GDCPPLELKNSVIQDGDMVDTGFGAMDFTALQDTKSNVPLDICNSICKYPDYLKMVAEPY GDTLFFYLRREQMFVRHFFNRSGTVGESVPNDLYIKGSGSTATLANSTYFPTPSGSMVTS DAQIFNKPYWMQRAQGHNNGICWGNQLFVTVVDTTRSTNMSVCAAIANSDTTFKSSNFKE YLRHGEEFDLQFIFQLCKITLSADIMTYIHSMNPAILEDWNFGLITPPSGSLEDTYRFVT SQAITCQKLYKTCKQAGTCPPDVIPKVEGPQKPKEDPFKDYVFWEVNLKEKFSADLDQFP LGRKFLLQAGYRAGPSFAAGAGSAPSASTTTPAGGSATGS 31L1h4.sub.428-431/dES-CS1 SEQIDNo.29 MRPSEATVYLPPVPVSKVVSTDEYVTRINIYYHAGSARLLTVGHPYYSIPKSDNPKKIVV PKVSGLQYRVFRVRLPDPNKFGFPDTSFYNPETQRLVWACVGLEVGRGQPLGVGISGHPL LNKFDDTENSNRYAGGPGTDNRECISMDYKQTQLCLLGCKPPIGEHWGKGSPCSNNAITP GDCPPLELKNSVIQDGDMVDTGFGAMDFTALQDTKSNVPLDICNSICKYPDYLKMVAEPY GDTLFFYLRREQMFVRHFFNRSGTVGESVPNDLYIKGSGSTATLANSTYFPTPSGSMVTS DAQIFNKPYWMQRAQGHNNGICWGNQLFVTVVDTTRSTNMSVCAAIANSDTTFKSSNFKE YLRHGEEFDLQFIFQLCKITLSADIMTYIHSMNPAILEDWNFGLTTPPSGSLEDTYRFVT SQAITCQKLYKTCKQAGTCPPDVIPPQKPKEDPFKDYVFWEVNLKEKFSADLDQFPLGRK FLLQAGYRAGPSFAAGAGSAPSASTTTPAGGSATGS 31L1h4.sub.428-431/dE-CS2 SEQIDNo.30 MRPSEATVYLPPVPVSKVVSTDEYVTRTNIYYHAGSARLLTVGHPYYSIPKSDNPKKIVV PKVSGLQYRVFRVRLPDPNKFGFPDTSFYNPETQRLVWACVGLEVGRGQPLGVGISGHPL LNKFDDTENSNRYAGGPGTDNRECISMDYKQTQLCLLGCKPPIGEHWGKGSPCSNNAITP GDCPPLELKNSVIQDGDMVDTGFGAMDFTALQDTKSNVPLDICNSICKYPDYLKMVAEPY GDTLFFYLRREQMFVRHFFNRSGTVGESVPNDLYIKGSGSTATLANSTYFPTPSGSMVTS DAQIFNKPYWMQRAQGHNNGICWGNQLFVTVVDTTRSTNMSVCAAIANSDTTFKSSNFKE YLRHGEEFDLQFIFQLCKITLSADIMTYIHSMNPAILEDWNFGLTTPPSGSLEDTYRFVT SQAITCQKLYKTCKQAGTCPPDVIPKVEGPQKPKEDPFKDYVFWEVNLKEKFSADLDQFP LGRKFLLQAGYGAGPSFAAGAGSAPSASTTTPAGGSATGS 31L1h4.sub.428-431/dES-CS2 SEQIDNo.31 MRPSEATVYLPPVPVSKVVSTDEYVTRTNIYYHAGSARLLTVGHPYYSIPKSDNPKKIVV PKVSGLQYRVFRVRLPDPNKFGFPDTSFYNPETQRLVWACVGLEVGRGQPLGVGISGHPL LNKFDDTENSNRYAGGPGTDNRECISMDYKQTQLCLLGCKPPIGEHWGKGSPCSNNAITP GDCPPLELKNSVIQDGDMVDTGFGAMDFTALQDTKSNVPLDICNSICKYPDYLKMVAEPY GDTLFFYLRREQMFVRHFFNRSGTVGESVPNDLYIKGSGSTATLANSTYFPTPSGSMVTS DAQIFNKPYWMQRAQGHNNGICWGNQLFVTVVDTTRSTNMSVCAAIANSDTTFKSSNFKE YLRHGEEFDLQFIFQLCKITLSADIMTYIHSMNPAILEDWNFGLTTPPSGSLEDTYRFVT SQAITCQKLYKTCKQAGTCPPDVIPPQKPKEDPFKDYVFWEVNLKEKFSADLDQFPLGRK FLLQAGYGAGPSFAAGAGSAPSASTTTPAGGSATGS 31L1h4.sub.428-431/dE-CS3 SEQIDNo.32 MRPSEATVYLPPVPVSKVVSTDEYVTRTNIYYHAGSARLLTVGHPYYSIPKSDNPKKIVV PKVSGLQYRVFRVRLPDPNKFGFPDTSFYNPETQRLVWACVGLEVGRGQPLGVGISGHPL LNKFDDTENSNRYAGGPGTDNRECISMDYKQTQLCLLGCKPPIGEHWGKGSPCSNNAITP GDCPPLELKNSVIQDGDMVDTGFGAMDFTALQDTKSNVPLDICNSICKYPDYLKMVAEPY GDTLFFYLRREQMFVRHFFNRSGTVGESVPNDLYIKGSGSTATLANSTYFPTPSGSMVTS DAQIFNKPYWMQRAQGHNNGICWGNQLFVTVVDTTRSTNMSVCAAIANSDTTFKSSNFKE YLRHGEEFDLQFIFQLCKITLSADIMTYIHSMNPAILEDWNFGLTTPPSGSLEDTYRFVT SQAITCQKLYKTCKQAGTCPPDVIPKVEGPQKPKEDPFKDYVFWEVNLKEKFSADLDQFP LGRKFLLQAGYRAGPSFKAGAGSAPSASTTTPAGGSATGS 31L1h4.sub.428-431/dES-CS3 SEQIDNo.33 MRPSEATVYLPPVPVSKVVSTDEYVTRINIYYHAGSARLLTVGHPYYSIPKSDNPKKIVV PKVSGLQYRVFRVRLPDPNKFGFPDTSFYNPETQRLVWACVGLEVGRGQPLGVGISGHPL LNKFDDTENSNRYAGGPGTDNRECISMDYKQTQLCLLGCKPPIGEHWGKGSPCSNNAITP GDCPPLELKNSVIQDGDMVDTGFGAMDFTALQDTKSNVPLDICNSICKYPDYLKMVAEPY GDTLFFYLRREQMFVRHFFNRSGTVGESVPNDLYIKGSGSTATLANSTYFPTPSGSMVTS DAQIFNKPYWMQRAQGHNNGICWGNQLFVTVVDTTRSTNMSVCAAIANSDTTFKSSNFKE YLRHGEEFDLQFIFQLCKITLSADIMTYIHSMNPAILEDWNFGLTTPPSGSLEDTYRFVT SQAITCQKLYKTCKQAGTCPPDVIPPQKPKEDPFKDYVFWEVNLKEKFSADLDQFPLGRK FLLQAGYRAGPSFKAGAGSAPSASTTTPAGGSATGS 31L1C29nt SEQIDNo.34 atgagcctgtggagaccatcagaggctacagtatatctgccacctgttcctgtaagcaaa gtggtttcaaccgatgagtacgtaacacgtaccaacatctactatcacgctggatctgcg cgcctcctgactgtcggtcacccatactactctattcccaagtcagacaatcccaagaaa atcgtggtacccaaagtgagcggactccagtatcgtgttttcagagtccgcttgccagat cccaacaagtttggcttcccagacacaagcttctacaatcctgaaacccaacgcctggta tgggcatgcgtgggactcgaggttggccgtggtcagcctctgggagtgggcatctcaggc cacccattgctcaacaaattcgatgacaccgagaattccaacagatacgcgggtggacca ggtacagataaccgcgaatgcatcagcatggactacaagcagacccaactgtgcctcttg ggctgcaagccaccaattggagagcactggggcaaaggctcaccttgctccaacaacgct atcacacctggagactgcccacccttggaactcaagaattctgtcattcaggatggtgac atggtggacactggctttggtgcaatggatttcaccgctcttcaagacaccaagtcaaac gtacctctggatatctgcaatagcatttgcaagtatccagactacctcaagatggttgct gagccttacggtgatacactgttcttctacctgagacgtgagcagatgtttgtgagacac ttcttcaaccgttccggcactgtcggagagtcagttcctacagacctctacatcaagggt tctggcagcacagcaactctggcgaactcaacctactttcctactccttccggatctatg gtcacgagcgatgctcagatcttcaacaagccctactggatgcaacgtgcccagggacac aacaatggcatttgctggggcaatcagctcttcgtcactgttgtggacactactcgctcc actaacatgtctgtctgcgctgccattgccaactccgataccactttcaaaagctctaac tttaaggaatatctgcgtcacggtgaggagttcgacttgcagttcatcttccaactctgc aagatcaccctgtccgctgatatcatgacctacattcacagcatgaatccagctatcctg gaagactggaacttcggtctgaccactccaccctctggtagcctggaggatacctacagg tttgttacatctcaagcaatcacttgccagaagactgccccacagaagcctaaagaggac cccttcaaagattacgtcttctgggaggtgaatctgaaggagaagttctctgctgatttg gatcagtttccactgggtcgtaagttcctgctccaagctggatactaag T.sub.274NC29nt SEQIDNo.35 atgagcctgtggagaccatcagaggctacagtatatctgccacctgttcctgtaagcaaa gtggtttcaaccgatgagtacgtaacacgtaccaacatctactatcacgctggatctgcg cgcctcctgactgtcggtcacccatactactctattcccaagtcagacaatcccaagaaa atcgtggtacccaaagtgagcggactccagtatcgtgttttcagagtccgcttgccagat cccaacaagtttggcttcccagacacaagcttctacaatcctgaaacccaacgcctggta tgggcatgcgtgggactcgaggttggccgtggtcagcctctgggagtgggcatctcaggc cacccattgctcaacaaattcgatgacaccgagaattccaacagatacgcgggtggacca ggtacagataaccgcgaatgcatcagcatggactacaagcagacccaactgtgcctcttg ggctgcaagccaccaattggagagcactggggcaaaggctcaccttgctccaacaacgct atcacacctggagactgcccacccttggaactcaagaattctgtcattcaggatggtgac atggtggacactggctttggtgcaatggatttcaccgctcttcaagacaccaagtcaaac gtacctctggatatctgcaatagcatttgcaagtatccagactacctcaagatggttgct gagccttacggtgatacactgttcttctacctgagacgtgagcagatgtttgtgagacac ttcttcaaccgttccggcactgtcggagagtcagttcctaacgacctctacatcaagggt tctggcagcacagcaactctggcgaactcaacctactttcctactccttccggatctatg gtcacgagcgatgctcagatcttcaacaagccctactggatgcaacgtgcccagggacac aacaatggcatttgctggggcaatcagctcttcgtcactgttgtggacactactcgctcc actaacatgtctgtctgcgctgccattgccaactccgataccactttcaaaagctctaac tttaaggaatatctgcgtcacggtgaggagttcgacttgcagttcatcttccaactctgc aagatcaccctgtccgctgatatcatgacctacattcacagcatgaatccagctatcctg gaagactggaacttcggtctgaccactccaccctctggtagcctggaggatacctacagg tttgttacatctcaagcaatcacttgccagaagactgccccacagaagcctaaagaggac cccttcaaagattacgtcttctgggaggtgaatctgaaggagaagttctctgctgatttg gatcagtttccactgggtcgtaagttcctgctccaagctggatactaag T.sub.274NN4C29 SEQIDNo.36 atgagaccatcagaggctacagtatatctgccacctgttcctgtaagcaaagtggtttca accgatgagtacgtaacacgtaccaacatctactatcacgctggatctgcgcgcctcctg actgtcggtcacccatactactctattcccaagtcagacaatcccaagaaaatcgtggta cccaaagtgagcggactccagtatcgtgttttcagagtccgcttgccagatcccaacaag tttggcttcccagacacaagcttctacaatcctgaaacccaacgcctggtatgggcatgc gtgggactcgaggttggccgtggtcagcctctgggagtgggcatctcaggccacccattg ctcaacaaattcgatgacaccgagaattccaacagatacgcgggtggaccaggtacagat aaccgcgaatgcatcagcatggactacaagcagacccaactgtgcctcttgggctgcaag ccaccaattggagagcactggggcaaaggctcaccttgctccaacaacgctatcacacct ggagactgcccacccttggaactcaagaattctgtcattcaggatggtgacatggtggac actggctttggtgcaatggatttcaccgctcttcaagacaccaagtcaaacgtacctctg gatatctgcaatagcatttgcaagtatccagactacctcaagatggttgctgagccttac ggtgatacactgttcttctacctgagacgtgagcagatgtttgtgagacacttcttcaac cgttccggcactgtcggagagtcagttcctaacgacctctacatcaagggttctggcagc acagcaactctggcgaactcaacctactttcctactccttccggatctatggtcacgagc gatgctcagatcttcaacaagccctactggatgcaacgtgcccagggacacaacaatggc atttgctggggcaatcagctcttcgtcactgttgtggacactactcgctccactaacatg tctgtctgcgctgccattgccaactccgataccactttcaaaagctctaactttaaggaa tatctgcgtcacggtgaggagttcgacttgcagttcatcttccaactctgcaagatcacc ctgtccgctgatatcatgacctacattcacagcatgaatccagctatcctggaagactgg aacttcggtctgaccactccaccctctggtagcctggaggatacctacaggtttgttaca tctcaagcaatcacttgccagaagactgccccacagaagcctaaagaggaccccttcaaa gattacgtcttctgggaggtgaatctgaaggagaagttctctgctgatttggatcagttt ccactgggtcgtaagttcctgctccaagctggatactaag 31L1DE.sub.132-136/dEnt SEQIDNo.37 atgagaccatcagaggctacagtatatctgccacctgttcctgtaagcaaagtggtttca accgatgagtacgtaacacgtaccaacatctactatcacgctggatctgcgcgcctcctg actgtcggtcacccatactactctattcccaagtcagacaatcccaagaaaatcgtggta cccaaagtgagcggactccagtatcgtgttttcagagtccgcttgccagatcccaacaag tttggcttcccagacacaagcttctacaatcctgaaacccaacgcctggtatgggcatgc gtgggactcgaggttggccgtggtcagcctctgggagtgggcatctcaggccacccattg ctcaacaaattcgatgacaccgagaattccaacagaggtcctcagctgtacaagacctgc aagcaggctggtacctgccctcctgacgtgatccctaaggtggagggaccaggtacagat aaccgcgaatgcatcagcatggactacaagcagacccaactgtgcctcttgggctgcaag ccaccaattggagagcactggggcaaaggctcaccttgctccaacaacgctatcacacct ggagactgcccacccttggaactcaagaattctgtcattcaggatggtgacatggtggac actggctttggtgcaatggatttcaccgctcttcaagacaccaagtcaaacgtacctctg gatatctgcaatagcatttgcaagtatccagactacctcaagatggttgctgagccttac ggtgatacactgttcttctacctgagacgtgagcagatgtttgtgagacacttcttcaac cgttccggcactgtcggagagtcagttcctaacgacctctacatcaagggttctggcagc acagcaactctggcgaactcaacctactttcctactccttccggatctatggtcacgagc gatgctcagatcttcaacaagccctactggatgcaacgtgcccagggacacaacaatggc atttgctggggcaatcagctcttcgtcactgttgtggacactactcgctccactaacatg tctgtctgcgctgccattgccaactccgataccactttcaaaagctctaactttaaggaa tatctgcgtcacggtgaggagttcgacttgcagttcatcttccaactctgcaagatcacc ctgtccgctgatatcatgacctacattcacagcatgaatccagctatcctggaagactgg aacttcggtctgaccactccaccctctggtagcctggaggatacctacaggtttgttaca tctcaagcaatcacttgccagaagactgccccacagaagcctaaagaggaccccttcaaa gattacgtcttctgggaggtgaatctgaaggagaagttctctgctgatttggatcagttt ccactgggtcgtaagttcctgctccaagctggatactaag 31L1DE.sub.132-136/dESnt SEQIDNo.38 atgagaccatcagaggctacagtatatctgccacctgttcctgtaagcaaagtggtttca accgatgagtacgtaacacgtaccaacatctactatcacgctggatctgcgcgcctcctg actgtcggtcacccatactactctattcccaagtcagacaatcccaagaaaatcgtggta cccaaagtgagcggactccagtatcgtgttttcagagtccgcttgccagatcccaacaag tttggcttcccagacacaagcttctacaatcctgaaacccaacgcctggtatgggcatgc gtgggactcgaggttggccgtggtcagcctctgggagtgggcatctcaggccacccattg ctcaacaaattcgatgacaccgagaattccaacagaggtcctctgtacaagacctgcaag caggctggtacctgccctcctgacgtgatccctggaccaggtacagataaccgcgaatgc atcagcatggactacaagcagacccaactgtgcctcttgggctgcaagccaccaattgga gagcactggggcaaaggctcaccttgctccaacaacgctatcacacctggagactgccca cccttggaactcaagaattctgtcattcaggatggtgacatggtggacactggctttggt gcaatggatttcaccgctcttcaagacaccaagtcaaacgtacctctggatatctgcaat agcatttgcaagtatccagactacctcaagatggttgctgagccttacggtgatacactg ttcttctacctgagacgtgagcagatgtttgtgagacacttcttcaaccgttccggcact gtcggagagtcagttcctaacgacctctacatcaagggttctggcagcacagcaactctg gcgaactcaacctactttcctactccttccggatctatggtcacgagcgatgctcagatc ttcaacaagccctactggatgcaacgtgcccagggacacaacaatggcatttgctggggc aatcagctcttcgtcactgttgtggacactactcgctccactaacatgtctgtctgcgct gccattgccaactccgataccactttcaaaagctctaactttaaggaatatctgcgtcac ggtgaggagttcgacttgcagttcatcttccaactctgcaagatcaccctgtccgctgat atcatgacctacattcacagcatgaatccagctatcctggaagactggaacttcggtctg accactccaccctctggtagcctggaggatacctacaggtttgttacatctcaagcaatc acttgccagaagactgccccacagaagcctaaagaggaccccttcaaagattacgtcttc tgggaggtgaatctgaaggagaagttctctgctgatttggatcagtttccactgggtcgt aagttcctgctccaagctggatactaag 31L1DE.sub.132-136/dE-CS1nt SEQIDNo.39 atgagaccatcagaggctacagtatatctgccacctgttcctgtaagcaaagtggtttca accgatgagtacgtaacacgtaccaacatctactatcacgctggatctgcgcgcctcctg actgtcggtcacccatactactctattcccaagtcagacaatcccaagaaaatcgtggta cccaaagtgagcggactccagtatcgtgttttcagagtccgcttgccagatcccaacaag tttggcttcccagacacaagcttctacaatcctgaaacccaacgcctggtatgggcatgc gtgggactcgaggttggccgtggtcagcctctgggagtgggcatctcaggccacccattg ctcaacaaattcgatgacaccgagaattccaacagaggtcctcagctgtacaagacctgc aagcaggctggtacctgccctcctgacgtgatccctaaggtggagggaccaggtacagat aaccgcgaatgcatcagcatggactacaagcagacccaactgtgcctcttgggctgcaag ccaccaattggagagcactggggcaaaggctcaccttgctccaacaacgctatcacacct ggagactgcccacccttggaactcaagaattctgtcattcaggatggtgacatggtggac actggctttggtgcaatggatttcaccgctcttcaagacaccaagtcaaacgtacctctg gatatctgcaatagcatttgcaagtatccagactacctcaagatggttgctgagccttac ggtgatacactgttcttctacctgagacgtgagcagatgtttgtgagacacttcttcaac cgttccggcactgtcggagagtcagttcctaacgacctctacatcaagggttctggcagc acagcaactctggcgaactcaacctactttcctactccttccggatctatggtcacgagc gatgctcagatcttcaacaagccctactggatgcaacgtgcccagggacacaacaatggc atttgctggggcaatcagctcttcgtcactgttgtggacactactcgctccactaacatg tctgtctgcgctgccattgccaactccgataccactttcaaaagctctaactttaaggaa tatctgcgtcacggtgaggagttcgacttgcagttcatcttccaactctgcaagatcacc ctgtccgctgatatcatgacctacattcacagcatgaatccagctatcctggaagactgg aacttcggtctgaccactccaccctctggtagcctggaggatacctacaggtttgttaca tctcaagcaatcacttgccagaagactgccccacagaagcctaaagaggaccccttcaaa gattacgtcttctgggaggtgaatctgaaggagaagttctctgctgatttggatcagttt ccactgggtcgtaagttcctgctccaagctggataccgtgctggtccttcgtttgccgct ggcgcgggttcggctcctagcgcctcgactaccacgccggctggcggttcggccacgggc agctaag 31L1DE.sub.132-136/dES-CS1nt SEQIDNo.40 atgagaccatcagaggctacagtatatctgccacctgttcctgtaagcaaagtggtttca accgatgagtacgtaacacgtaccaacatctactatcacgctggatctgcgcgcctcctg actgtcggtcacccatactactctattcccaagtcagacaatcccaagaaaatcgtggta cccaaagtgagcggactccagtatcgtgttttcagagtccgcttgccagatcccaacaag tttggcttcccagacacaagcttctacaatcctgaaacccaacgcctggtatgggcatgc gtgggactcgaggttggccgtggtcagcctctgggagtgggcatctcaggccacccattg ctcaacaaattcgatgacaccgagaattccaacagaggtcctctgtacaagacctgcaag caggctggtacctgccctcctgacgtgatccctggaccaggtacagataaccgcgaatgc atcagcatggactacaagcagacccaactgtgcctcttgggctgcaagccaccaattgga gagcactggggcaaaggctcaccttgctccaacaacgctatcacacctggagactgccca cccttggaactcaagaattctgtcattcaggatggtgacatggtggacactggctttggt gcaatggatttcaccgctcttcaagacaccaagtcaaacgtacctctggatatctgcaat agcatttgcaagtatccagactacctcaagatggttgctgagccttacggtgatacactg ttcttctacctgagacgtgagcagatgtttgtgagacacttcttcaaccgttccggcact gtcggagagtcagttcctaacgacctctacatcaagggttctggcagcacagcaactctg gcgaactcaacctactttcctactccttccggatctatggtcacgagcgatgctcagatc ttcaacaagccctactggatgcaacgtgcccagggacacaacaatggcatttgctggggc aatcagctcttcgtcactgttgtggacactactcgctccactaacatgtctgtctgcgct gccattgccaactccgataccactttcaaaagctctaactttaaggaatatctgcgtcac ggtgaggagttcgacttgcagttcatcttccaactctgcaagatcaccctgtccgctgat atcatgacctacattcacagcatgaatccagctatcctggaagactggaacttcggtctg accactccaccctctggtagcctggaggatacctacaggtttgttacatctcaagcaatc acttgccagaagactgccccacagaagcctaaagaggaccccttcaaagattacgtcttc tgggaggtgaatctgaaggagaagttctctgctgatttggatcagtttccactgggtcgt aagttcctgctccaagctggataccgtgctggtccttcgtttgccgct ggcgcgggttcggctcctagcgcctcgactaccacgccggctggcggttcggccacgggc agctaag 31L1DE.sub.132-136/dE-CS2nt SEQIDNo.41 atgagaccatcagaggctacagtatatctgccacctgttcctgtaagcaaagtggtttca accgatgagtacgtaacacgtaccaacatctactatcacgctggatctgcgcgcctcctg actgtcggtcacccatactactctattcccaagtcagacaatcccaagaaaatcgtggta cccaaagtgagcggactccagtatcgtgttttcagagtccgcttgccagatcccaacaag tttggcttcccagacacaagcttctacaatcctgaaacccaacgcctggtatgggcatgc gtgggactcgaggttggccgtggtcagcctctgggagtgggcatctcaggccacccattg ctcaacaaattcgatgacaccgagaattccaacagaggtcctcagctgtacaagacctgc aagcaggctggtacctgccctcctgacgtgatccctaaggtggagggaccaggtacagat aaccgcgaatgcatcagcatggactacaagcagacccaactgtgcctcttgggctgcaag ccaccaattggagagcactggggcaaaggctcaccttgctccaacaacgctatcacacct ggagactgcccacccttggaactcaagaattctgtcattcaggatggtgacatggtggac actggctttggtgcaatggatttcaccgctcttcaagacaccaagtcaaacgtacctctg gatatctgcaatagcatttgcaagtatccagactacctcaagatggttgctgagccttac ggtgatacactgttcttctacctgagacgtgagcagatgtttgtgagacacttcttcaac cgttccggcactgtcggagagtcagttcctaacgacctctacatcaagggttctggcagc acagcaactctggcgaactcaacctactttcctactccttccggatctatggtcacgagc gatgctcagatcttcaacaagccctactggatgcaacgtgcccagggacacaacaatggc atttgctggggcaatcagctcttcgtcactgttgtggacactactcgctccactaacatg tctgtctgcgctgccattgccaactccgataccactttcaaaagctctaactttaaggaa tatctgcgtcacggtgaggagttcgacttgcagttcatcttccaactctgcaagatcacc ctgtccgctgatatcatgacctacattcacagcatgaatccagctatcctggaagactgg aacttcggtctgaccactccaccctctggtagcctggaggatacctacaggtttgttaca tctcaagcaatcacttgccagaagactgccccacagaagcctaaagaggaccccttcaaa gattacgtcttctgggaggtgaatctgaaggagaagttctctgctgatttggatcagttt ccactgggtcgtaagttcctgctccaagctggatacggcgctggtccttcgtttgccgct ggcgcgggttcggctcctagcgcctcgactaccacgccggctggcggttcggccacgggc agctaag 31L1DE.sub.132-136/dES-CS2nt SEQIDNo.42 atgagaccatcagaggctacagtatatctgccacctgttcctgtaagcaaagtggtttca accgatgagtacgtaacacgtaccaacatctactatcacgctggatctgcgcgcctcctg actgtcggtcacccatactactctattcccaagtcagacaatcccaagaaaatcgtggta cccaaagtgagcggactccagtatcgtgttttcagagtccgcttgccagatcccaacaag tttggcttcccagacacaagcttctacaatcctgaaacccaacgcctggtatgggcatgc gtgggactcgaggttggccgtggtcagcctctgggagtgggcatctcaggccacccattg ctcaacaaattcgatgacaccgagaattccaacagaggtcctctgtacaagacctgcaag caggctggtacctgccctcctgacgtgatccctggaccaggtacagataaccgcgaatgc atcagcatggactacaagcagacccaactgtgcctcttgggctgcaagccaccaattgga gagcactggggcaaaggctcaccttgctccaacaacgctatcacacctggagactgccca cccttggaactcaagaattctgtcattcaggatggtgacatggtggacactggctttggt gcaatggatttcaccgctcttcaagacaccaagtcaaacgtacctctggatatctgcaat agcatttgcaagtatccagactacctcaagatggttgctgagccttacggtgatacactg ttcttctacctgagacgtgagcagatgtttgtgagacacttcttcaaccgttccggcact gtcggagagtcagttcctaacgacctctacatcaagggttctggcagcacagcaactctg gcgaactcaacctactttcctactccttccggatctatggtcacgagcgatgctcagatc ttcaacaagccctactggatgcaacgtgcccagggacacaacaatggcatttgctggggc aatcagctcttcgtcactgttgtggacactactcgctccactaacatgtctgtctgcgct gccattgccaactccgataccactttcaaaagctctaactttaaggaatatctgcgtcac ggtgaggagttcgacttgcagttcatcttccaactctgcaagatcaccctgtccgctgat atcatgacctacattcacagcatgaatccagctatcctggaagactggaacttcggtctg accactccaccctctggtagcctggaggatacctacaggtttgttacatctcaagcaatc acttgccagaagactgccccacagaagcctaaagaggaccccttcaaagattacgtcttc tgggaggtgaatctgaaggagaagttctctgctgatttggatcagtttccactgggtcgt aagttcctgctccaagctggatacggcgctggtccttcgtttgccgctggcgcgggtt cggctcctagcgcctcgactaccacgccggctggcggttcggccacgggcagctaag 31L1DE.sub.132-136/dE-CS3nt SEQIDNo.43 atgagaccatcagaggctacagtatatctgccacctgttcctgtaagcaaagtggtttca accgatgagtacgtaacacgtaccaacatctactatcacgctggatctgcgcgcctcctg actgtcggtcacccatactactctattcccaagtcagacaatcccaagaaaatcgtggta cccaaagtgagcggactccagtatcgtgttttcagagtccgcttgccagatcccaacaag tttggcttcccagacacaagcttctacaatcctgaaacccaacgcctggtatgggcatgc gtgggactcgaggttggccgtggtcagcctctgggagtgggcatctcaggccacccattg ctcaacaaattcgatgacaccgagaattccaacagaggtcctcagctgtacaagacctgc aagcaggctggtacctgccctcctgacgtgatccctaaggtggagggaccaggtacagat aaccgcgaatgcatcagcatggactacaagcagacccaactgtgcctcttgggctgcaag ccaccaattggagagcactggggcaaaggctcaccttgctccaacaacgctatcacacct ggagactgcccacccttggaactcaagaattctgtcattcaggatggtgacatggtggac actggctttggtgcaatggatttcaccgctcttcaagacaccaagtcaaacgtacctctg gatatctgcaatagcatttgcaagtatccagactacctcaagatggttgctgagccttac ggtgatacactgttcttctacctgagacgtgagcagatgtttgtgagacacttcttcaac cgttccggcactgtcggagagtcagttcctaacgacctctacatcaagggttctggcagc acagcaactctggcgaactcaacctactttcctactccttccggatctatggtcacgagc gatgctcagatcttcaacaagccctactggatgcaacgtgcccagggacacaacaatggc atttgctggggcaatcagctcttcgtcactgttgtggacactactcgctccactaacatg tctgtctgcgctgccattgccaactccgataccactttcaaaagctctaactttaaggaa tatctgcgtcacggtgaggagttcgacttgcagttcatcttccaactctgcaagatcacc ctgtccgctgatatcatgacctacattcacagcatgaatccagctatcctggaagactgg aacttcggtctgaccactccaccctctggtagcctggaggatacctacaggtttgttaca tctcaagcaatcacttgccagaagactgccccacagaagcctaaagaggaccccttcaaa gattacgtcttctgggaggtgaatctgaaggagaagttctctgctgatttggatcagttt ccactgggtcgtaagttcctgctccaagctggataccgtgctggtccttcgtttaaagc tggcgcgggttcggctcctagcgcctcgactaccacgccggctggcggttcggccacggg cagctaag 31L1DE.sub.132-136/dES-CS3nt SEQIDNo.44 atgagaccatcagaggctacagtatatctgccacctgttcctgtaagcaaagtggtttca accgatgagtacgtaacacgtaccaacatctactatcacgctggatctgcgcgcctcctg actgtcggtcacccatactactctattcccaagtcagacaatcccaagaaaatcgtggta cccaaagtgagcggactccagtatcgtgttttcagagtccgcttgccagatcccaacaag tttggcttcccagacacaagcttctacaatcctgaaacccaacgcctggtatgggcatgc gtgggactcgaggttggccgtggtcagcctctgggagtgggcatctcaggccacccattg ctcaacaaattcgatgacaccgagaattccaacagaggtcctctgtacaagacctgcaag caggctggtacctgccctcctgacgtgatccctggaccaggtacagataaccgcgaatgc atcagcatggactacaagcagacccaactgtgcctcttgggctgcaagccaccaattgga gagcactggggcaaaggctcaccttgctccaacaacgctatcacacctggagactgccca cccttggaactcaagaattctgtcattcaggatggtgacatggtggacactggctttggt gcaatggatttcaccgctcttcaagacaccaagtcaaacgtacctctggatatctgcaat agcatttgcaagtatccagactacctcaagatggttgctgagccttacggtgatacactg ttcttctacctgagacgtgagcagatgtttgtgagacacttcttcaaccgttccggcact gtcggagagtcagttcctaacgacctctacatcaagggttctggcagcacagcaactctg gcgaactcaacctactttcctactccttccggatctatggtcacgagcgatgctcagatc ttcaacaagccctactggatgcaacgtgcccagggacacaacaatggcatttgctggggc aatcagctcttcgtcactgttgtggacactactcgctccactaacatgtctgtctgcgct gccattgccaactccgataccactttcaaaagctctaactttaaggaatatctgcgtcac ggtgaggagttcgacttgcagttcatcttccaactctgcaagatcaccctgtccgctgat atcatgacctacattcacagcatgaatccagctatcctggaagactggaacttcggtctg accactccaccctctggtagcctggaggatacctacaggtttgttacatctcaagcaatc acttgccagaagactgccccacagaagcctaaagaggaccccttcaaagattacgtcttc tgggaggtgaatctgaaggagaagttctctgctgatttggatcagtttccactgggtcgt aagttcctgctccaagctggataccgtgctggtccttcgtttaaagc tggcgcgggttcggctcctagcgcctcgactaccacgccggctggcggttcggccacggg cagctaag 31L1h4.sub.428-431/dEnt SEQIDNo.45 atgagaccatcagaggctacagtatatctgccacctgttcctgtaagcaaagtggtttca accgatgagtacgtaacacgtaccaacatctactatcacgctggatctgcgcgcctcctg actgtcggtcacccatactactctattcccaagtcagacaatcccaagaaaatcgtggta cccaaagtgagcggactccagtatcgtgttttcagagtccgcttgccagatcccaacaag tttggcttcccagacacaagcttctacaatcctgaaacccaacgcctggtatgggcatgc gtgggactcgaggttggccgtggtcagcctctgggagtgggcatctcaggccacccattg ctcaacaaattcgatgacaccgagaattccaacagatacgcgggtggaccaggtacagat aaccgcgaatgcatcagcatggactacaagcagacccaactgtgcctcttgggctgcaag ccaccaattggagagcactggggcaaaggctcaccttgctccaacaacgctatcacacct ggagactgcccacccttggaactcaagaattctgtcattcaggatggtgacatggtggac actggctttggtgcaatggatttcaccgctcttcaagacaccaagtcaaacgtacctctg gatatctgcaatagcatttgcaagtatccagactacctcaagatggttgctgagccttac ggtgatacactgttcttctacctgagacgtgagcagatgtttgtgagacacttcttcaac cgttccggcactgtcggagagtcagttcctaacgacctctacatcaagggttctggcagc acagcaactctggcgaactcaacctactttcctactccttccggatctatggtcacgagc gatgctcagatcttcaacaagccctactggatgcaacgtgcccagggacacaacaatggc atttgctggggcaatcagctcttcgtcactgttgtggacactactcgctccactaacatg tctgtctgcgctgccattgccaactccgataccactttcaaaagctctaactttaaggaa tatctgcgtcacggtgaggagttcgacttgcagttcatcttccaactctgcaagatcacc ctgtccgctgatatcatgacctacattcacagcatgaatccagctatcctggaagactgg aacttcggtctgaccactccaccctctggtagcctggaggatacctacaggtttgttaca tctcaagcaatcacttgccagaagctgtacaagacctgcaagcaggctggtacctgccct cctgacgtgatccctaaggtggagggtccacagaagcctaaagaggaccccttcaaagat tacgtcttctgggaggtgaatctgaaggagaagttctctgctgatttggatcagtttcca ctgggtcgtaagttcctgctccaagctggatactaag 31L1h4.sub.428-431/dESnt SEQIDNo.46 atgagaccatcagaggctacagtatatctgccacctgttcctgtaagcaaagtggtttca accgatgagtacgtaacacgtaccaacatctactatcacgctggatctgcgcgcctcctg actgtcggtcacccatactactctattcccaagtcagacaatcccaagaaaatcgtggta cccaaagtgagcggactccagtatcgtgttttcagagtccgcttgccagatcccaacaag tttggcttcccagacacaagcttctacaatcctgaaacccaacgcctggtatgggcatgc gtgggactcgaggttggccgtggtcagcctctgggagtgggcatctcaggccacccattg ctcaacaaattcgatgacaccgagaattccaacagatacgcgggtggaccaggtacagat aaccgcgaatgcatcagcatggactacaagcagacccaactgtgcctcttgggctgcaag ccaccaattggagagcactggggcaaaggctcaccttgctccaacaacgctatcacacct ggagactgcccacccttggaactcaagaattctgtcattcaggatggtgacatggtggac actggctttggtgcaatggatttcaccgctcttcaagacaccaagtcaaacgtacctctg gatatctgcaatagcatttgcaagtatccagactacctcaagatggttgctgagccttac ggtgatacactgttcttctacctgagacgtgagcagatgtttgtgagacacttcttcaac cgttccggcactgtcggagagtcagttcctaacgacctctacatcaagggttctggcagc acagcaactctggcgaactcaacctactttcctactccttccggatctatggtcacgagc gatgctcagatcttcaacaagccctactggatgcaacgtgcccagggacacaacaatggc atttgctggggcaatcagctcttcgtcactgttgtggacactactcgctccactaacatg tctgtctgcgctgccattgccaactccgataccactttcaaaagctctaactttaaggaa tatctgcgtcacggtgaggagttcgacttgcagttcatcttccaactctgcaagatcacc ctgtccgctgatatcatgacctacattcacagcatgaatccagctatcctggaagactgg aacttcggtctgaccactccaccctctggtagcctggaggatacctacaggtttgttaca tctcaagcaatcacttgccagaagctgtacaagacctgcaagcaggctggtacctgccct cctgacgtgatccctccacagaagcctaaagaggaccccttcaaagattacgtcttctgg gaggtgaatctgaaggagaagttctctgctgatttggatcagtttccactgggtcgtaag ttcctgctccaagctggatactaag 31L1h4.sub.428-431/dE-CS1nt SEQIDNo.47 atgagaccatcagaggctacagtatatctgccacctgttcctgtaagcaaagtggtttca accgatgagtacgtaacacgtaccaacatctactatcacgctggatctgcgcgcctcctg actgtcggtcacccatactactctattcccaagtcagacaatcccaagaaaatcgtggta cccaaagtgagcggactccagtatcgtgttttcagagtccgcttgccagatcccaacaag tttggcttcccagacacaagcttctacaatcctgaaacccaacgcctggtatgggcatgc gtgggactcgaggttggccgtggtcagcctctgggagtgggcatctcaggccacccattg ctcaacaaattcgatgacaccgagaattccaacagatacgcgggtggaccaggtacagat aaccgcgaatgcatcagcatggactacaagcagacccaactgtgcctcttgggctgcaag ccaccaattggagagcactggggcaaaggctcaccttgctccaacaacgctatcacacct ggagactgcccacccttggaactcaagaattctgtcattcaggatggtgacatggtggac actggctttggtgcaatggatttcaccgctcttcaagacaccaagtcaaacgtacctctg gatatctgcaatagcatttgcaagtatccagactacctcaagatggttgctgagccttac ggtgatacactgttcttctacctgagacgtgagcagatgtttgtgagacacttcttcaac cgttccggcactgtcggagagtcagttcctaacgacctctacatcaagggttctggcagc acagcaactctggcgaactcaacctactttcctactccttccggatctatggtcacgagc gatgctcagatcttcaacaagccctactggatgcaacgtgcccagggacacaacaatggc atttgctggggcaatcagctcttcgtcactgttgtggacactactcgctccactaacatg tctgtctgcgctgccattgccaactccgataccactttcaaaagctctaactttaaggaa tatctgcgtcacggtgaggagttcgacttgcagttcatcttccaactctgcaagatcacc ctgtccgctgatatcatgacctacattcacagcatgaatccagctatcctggaagactgg aacttcggtctgaccactccaccctctggtagcctggaggatacctacaggtttgttaca tctcaagcaatcacttgccagaagctgtacaagacctgcaagcaggctggtacctgccct cctgacgtgatccctaaggtggagggtccacagaagcctaaagaggaccccttcaaagat tacgtcttctgggaggtgaatctgaaggagaagttctctgctgatttggatcagtttcca ctgggtcgtaagttcctgctccaagctggataccgtgctggtccttcgtttgccgct ggcgcgggttcggctcctagcgcctcgactaccacgccggctggcggttcggccacgggc agctaag 31L1h4.sub.428-431/dES-CS1nt SEQIDNo.48 atgagaccatcagaggctacagtatatctgccacctgttcctgtaagcaaagtggtttca accgatgagtacgtaacacgtaccaacatctactatcacgctggatctgcgcgcctcctg actgtcggtcacccatactactctattcccaagtcagacaatcccaagaaaatcgtggta cccaaagtgagcggactccagtatcgtgttttcagagtccgcttgccagatcccaacaag tttggcttcccagacacaagcttctacaatcctgaaacccaacgcctggtatgggcatgc gtgggactcgaggttggccgtggtcagcctctgggagtgggcatctcaggccacccattg ctcaacaaattcgatgacaccgagaattccaacagatacgcgggtggaccaggtacagat aaccgcgaatgcatcagcatggactacaagcagacccaactgtgcctcttgggctgcaag ccaccaattggagagcactggggcaaaggctcaccttgctccaacaacgctatcacacct ggagactgcccacccttggaactcaagaattctgtcattcaggatggtgacatggtggac actggctttggtgcaatggatttcaccgctcttcaagacaccaagtcaaacgtacctctg gatatctgcaatagcatttgcaagtatccagactacctcaagatggttgctgagccttac ggtgatacactgttcttctacctgagacgtgagcagatgtttgtgagacacttcttcaac cgttccggcactgtcggagagtcagttcctaacgacctctacatcaagggttctggcagc acagcaactctggcgaactcaacctactttcctactccttccggatctatggtcacgagc gatgctcagatcttcaacaagccctactggatgcaacgtgcccagggacacaacaatggc atttgctggggcaatcagctcttcgtcactgttgtggacactactcgctccactaacatg tctgtctgcgctgccattgccaactccgataccactttcaaaagctctaactttaaggaa tatctgcgtcacggtgaggagttcgacttgcagttcatcttccaactctgcaagatcacc ctgtccgctgatatcatgacctacattcacagcatgaatccagctatcctggaagactgg aacttcggtctgaccactccaccctctggtagcctggaggatacctacaggtttgttaca tctcaagcaatcacttgccagaagctgtacaagacctgcaagcaggctggtacctgccct cctgacgtgatccctccacagaagcctaaagaggaccccttcaaagattacgtcttctgg gaggtgaatctgaaggagaagttctctgctgatttggatcagtttccactgggtcgtaag ttcctgctccaagctggataccgtgctggtccttcgtttgccgct ggcgcgggttcggctcctagcgcctcgactaccacgccggctggcggttcggccacgggc agctaag 31L1h4.sub.428-431/dE-CS2nt SEQIDNo.49 atgagaccatcagaggctacagtatatctgccacctgttcctgtaagcaaagtggtttca accgatgagtacgtaacacgtaccaacatctactatcacgctggatctgcgcgcctcctg actgtcggtcacccatactactctattcccaagtcagacaatcccaagaaaatcgtggta cccaaagtgagcggactccagtatcgtgttttcagagtccgcttgccagatcccaacaag tttggcttcccagacacaagcttctacaatcctgaaacccaacgcctggtatgggcatgc gtgggactcgaggttggccgtggtcagcctctgggagtgggcatctcaggccacccattg ctcaacaaattcgatgacaccgagaattccaacagatacgcgggtggaccaggtacagat aaccgcgaatgcatcagcatggactacaagcagacccaactgtgcctcttgggctgcaag ccaccaattggagagcactggggcaaaggctcaccttgctccaacaacgctatcacacct ggagactgcccacccttggaactcaagaattctgtcattcaggatggtgacatggtggac actggctttggtgcaatggatttcaccgctcttcaagacaccaagtcaaacgtacctctg gatatctgcaatagcatttgcaagtatccagactacctcaagatggttgctgagccttac ggtgatacactgttcttctacctgagacgtgagcagatgtttgtgagacacttcttcaac cgttccggcactgtcggagagtcagttcctaacgacctctacatcaagggttctggcagc acagcaactctggcgaactcaacctactttcctactccttccggatctatggtcacgagc gatgctcagatcttcaacaagccctactggatgcaacgtgcccagggacacaacaatggc atttgctggggcaatcagctcttcgtcactgttgtggacactactcgctccactaacatg tctgtctgcgctgccattgccaactccgataccactttcaaaagctctaactttaaggaa tatctgcgtcacggtgaggagttcgacttgcagttcatcttccaactctgcaagatcacc ctgtccgctgatatcatgacctacattcacagcatgaatccagctatcctggaagactgg aacttcggtctgaccactccaccctctggtagcctggaggatacctacaggtttgttaca tctcaagcaatcacttgccagaagctgtacaagacctgcaagcaggctggtacctgccct cctgacgtgatccctaaggtggagggtccacagaagcctaaagaggaccccttcaaagat tacgtcttctgggaggtgaatctgaaggagaagttctctgctgatttggatcagtttcca ctgggtcgtaagttcctgctccaagctggatacggcgctggtccttcgtttgccgct ggcgcgggttcggctcctagcgcctcgactaccacgccggctggcggttcggccacgggc agctaag 31L1h4.sub.428-431/dES-CS2nt SEQIDNo.50 atgagaccatcagaggctacagtatatctgccacctgttcctgtaagcaaagtggtttca accgatgagtacgtaacacgtaccaacatctactatcacgctggatctgcgcgcctcctg actgtcggtcacccatactactctattcccaagtcagacaatcccaagaaaatcgtggta cccaaagtgagcggactccagtatcgtgttttcagagtccgcttgccagatcccaacaag tttggcttcccagacacaagcttctacaatcctgaaacccaacgcctggtatgggcatgc gtgggactcgaggttggccgtggtcagcctctgggagtgggcatctcaggccacccattg ctcaacaaattcgatgacaccgagaattccaacagatacgcgggtggaccaggtacagat aaccgcgaatgcatcagcatggactacaagcagacccaactgtgcctcttgggctgcaag ccaccaattggagagcactggggcaaaggctcaccttgctccaacaacgctatcacacct ggagactgcccacccttggaactcaagaattctgtcattcaggatggtgacatggtggac actggctttggtgcaatggatttcaccgctcttcaagacaccaagtcaaacgtacctctg gatatctgcaatagcatttgcaagtatccagactacctcaagatggttgctgagccttac ggtgatacactgttcttctacctgagacgtgagcagatgtttgtgagacacttcttcaac cgttccggcactgtcggagagtcagttcctaacgacctctacatcaagggttctggcagc acagcaactctggcgaactcaacctactttcctactccttccggatctatggtcacgagc gatgctcagatcttcaacaagccctactggatgcaacgtgcccagggacacaacaatggc atttgctggggcaatcagctcttcgtcactgttgtggacactactcgctccactaacatg tctgtctgcgctgccattgccaactccgataccactttcaaaagctctaactttaaggaa tatctgcgtcacggtgaggagttcgacttgcagttcatcttccaactctgcaagatcacc ctgtccgctgatatcatgacctacattcacagcatgaatccagctatcctggaagactgg aacttcggtctgaccactccaccctctggtagcctggaggatacctacaggtttgttaca tctcaagcaatcacttgccagaagctgtacaagacctgcaagcaggctggtacctgccct cctgacgtgatccctccacagaagcctaaagaggaccccttcaaagattacgtcttctgg gaggtgaatctgaaggagaagttctctgctgatttggatcagtttccactgggtcgtaag ttcctgctccaagctggatacggcgctggtccttcgtttgccgct ggcgcgggttcggctcctagcgcctcgactaccacgccggctggcggttcggccacgggc agctaag 31L1h4.sub.428-431/dE-CS3nt SEQIDNo.51 atgagaccatcagaggctacagtatatctgccacctgttcctgtaagcaaagtggtttca accgatgagtacgtaacacgtaccaacatctactatcacgctggatctgcgcgcctcctg actgtcggtcacccatactactctattcccaagtcagacaatcccaagaaaatcgtggta cccaaagtgagcggactccagtatcgtgttttcagagtccgcttgccagatcccaacaag tttggcttcccagacacaagcttctacaatcctgaaacccaacgcctggtatgggcatgc gtgggactcgaggttggccgtggtcagcctctgggagtgggcatctcaggccacccattg ctcaacaaattcgatgacaccgagaattccaacagatacgcgggtggaccaggtacagat aaccgcgaatgcatcagcatggactacaagcagacccaactgtgcctcttgggctgcaag ccaccaattggagagcactggggcaaaggctcaccttgctccaacaacgctatcacacct ggagactgcccacccttggaactcaagaattctgtcattcaggatggtgacatggtggac actggctttggtgcaatggatttcaccgctcttcaagacaccaagtcaaacgtacctctg gatatctgcaatagcatttgcaagtatccagactacctcaagatggttgctgagccttac ggtgatacactgttcttctacctgagacgtgagcagatgtttgtgagacacttcttcaac cgttccggcactgtcggagagtcagttcctaacgacctctacatcaagggttctggcagc acagcaactctggcgaactcaacctactttcctactccttccggatctatggtcacgagc gatgctcagatcttcaacaagccctactggatgcaacgtgcccagggacacaacaatggc atttgctggggcaatcagctcttcgtcactgttgtggacactactcgctccactaacatg tctgtctgcgctgccattgccaactccgataccactttcaaaagctctaactttaaggaa tatctgcgtcacggtgaggagttcgacttgcagttcatcttccaactctgcaagatcacc ctgtccgctgatatcatgacctacattcacagcatgaatccagctatcctggaagactgg aacttcggtctgaccactccaccctctggtagcctggaggatacctacaggtttgttaca tctcaagcaatcacttgccagaagctgtacaagacctgcaagcaggctggtacctgccct cctgacgtgatccctaaggtggagggtccacagaagcctaaagaggaccccttcaaagat tacgtcttctgggaggtgaatctgaaggagaagttctctgctgatttggatcagtttcca ctgggtcgtaagttcctgctccaagctggataccgtgctggtccttcgtttaaagc tggcgcgggttcggctcctagcgcctcgactaccacgccggctggcggttcggccacggg cagctaag 31L1h4.sub.428-431/dES-CS3nt SEQIDNo.52 atgagaccatcagaggctacagtatatctgccacctgttcctgtaagcaaagtggtttca accgatgagtacgtaacacgtaccaacatctactatcacgctggatctgcgcgcctcctg actgtcggtcacccatactactctattcccaagtcagacaatcccaagaaaatcgtggta cccaaagtgagcggactccagtatcgtgttttcagagtccgcttgccagatcccaacaag tttggcttcccagacacaagcttctacaatcctgaaacccaacgcctggtatgggcatgc gtgggactcgaggttggccgtggtcagcctctgggagtgggcatctcaggccacccattg ctcaacaaattcgatgacaccgagaattccaacagatacgcgggtggaccaggtacagat aaccgcgaatgcatcagcatggactacaagcagacccaactgtgcctcttgggctgcaag ccaccaattggagagcactggggcaaaggctcaccttgctccaacaacgctatcacacct ggagactgcccacccttggaactcaagaattctgtcattcaggatggtgacatggtggac actggctttggtgcaatggatttcaccgctcttcaagacaccaagtcaaacgtacctctg gatatctgcaatagcatttgcaagtatccagactacctcaagatggttgctgagccttac ggtgatacactgttcttctacctgagacgtgagcagatgtttgtgagacacttcttcaac cgttccggcactgtcggagagtcagttcctaacgacctctacatcaagggttctggcagc acagcaactctggcgaactcaacctactttcctactccttccggatctatggtcacgagc gatgctcagatcttcaacaagccctactggatgcaacgtgcccagggacacaacaatggc atttgctggggcaatcagctcttcgtcactgttgtggacactactcgctccactaacatg tctgtctgcgctgccattgccaactccgataccactttcaaaagctctaactttaaggaa tatctgcgtcacggtgaggagttcgacttgcagttcatcttccaactctgcaagatcacc ctgtccgctgatatcatgacctacattcacagcatgaatccagctatcctggaagactgg aacttcggtctgaccactccaccctctggtagcctggaggatacctacaggtttgttaca tctcaagcaatcacttgccagaagctgtacaagacctgcaagcaggctggtacctgccct cctgacgtgatccctccacagaagcctaaagaggaccccttcaaagattacgtcttctgg gaggtgaatctgaaggagaagttctctgctgatttggatcagtttccactgggtcgtaag ttcctgctccaagctggataccgtgctggtccttcgtttaaagc tggcgcgggttcggctcctagcgcctcgactaccacgccggctggcggttcggccacggg cagctaag
Example 5: Construction of Recombinant Bacmids and Recombinant Baculoviruses of Genes of L1 Proteins and Chimeric L1 Proteins
[0116] The recombinant expression vectors comprising L1 genes, namely pFastBac1-31L1, pFastBac1-T.sub.274N, pFastBac1-31L1MC, pFastBac1-T.sub.274NC, pFastBac1-T.sub.267AC, pFastBac1-T.sub.267AT.sub.274NC, pFastBac1-T.sub.274NN2C, pFastBac1-T.sub.274NN4C, pFastBac1-T.sub.274NN5C, pFastBac1-T.sub.274NN8C, and pFastBac1-T.sub.274NN10C; or the recombinant expression vectors of chimeric L1 genes, pFastBac1-31L1DE.sub.132-136/dE, pFastBac1-31L1DE.sub.132-136/dES, pFastBac1-31L1DE.sub.132-136/dE-CS1, pFastBac1-31L1DE.sub.132-136/dES-CS1, pFastBac1-31L1DE.sub.132-136/dE-CS2, pFastBac1-31L1DE.sub.132-136/dES-CS2, pFastBac1-31L1DE.sub.132-136/dE-CS3, pFastBac1-31L1DE.sub.132-136/dES-CS3, pFastBac1-31L1h4.sub.428-431/dE, pFastBac1-31L1h4.sub.428-431/dES, pFastBac1-31L1h4.sub.428-431/dE-CS1, pFastBac1-31L1h4.sub.428-431/dES-CS1, pFastBac1-31L1h4.sub.428-431/dE-CS2, pFastBac1-31L1h4.sub.428-431/dES-CS2, pFastBac1-31L1h4.sub.428-431/dE-CS3, and pFastBac1-31L1h4.sub.428-431/dES-CS3, were used to transform E. coli DH10Bac competent cells, which were screened to obtain recombinant Bacmids. Then the recombinant Bacmids were used to transfect Sf9 insect cells so as to amplify recombinant baculoviruses within the Sf9 cells. Methods of screening of recombinant Bacmids and amplification of recombinant baculoviruses were all well known, for example, the patent CN 101148661 B.
Example 6: Identification of the Expression of Genes of L1 Proteins and Chimeric L1 Proteins
[0117] Sf9 cells were inoculated with the 11 types of recombinant baculoviruses containing the genes of 31L1 protein or mutants or the 16 types of recombinant baculoviruses containing the chimeric L1 genes, respectively, to express the proteins. After incubation at 27 C. for about 88 h, the fermentation broth was collected and centrifuged at 3,000 rpm for 15 min. The supernatant was discarded, and the cells were washed with PBS for use in expression identification and purification. Methods of infection and expression were publicly available, for example, the patent CN 101148661 B.
Example 7: Identification of the Expression of L1 Proteins and Chimeric L1 Proteins
[0118] For each of cells expressing the different L1 proteins or chimeric L1 proteins described in Example 6, 110.sup.6 cells were collected and resuspended in 200 l PBS solution. 50 l of 6Loading Buffer was added and the samples were denatured at 75 C. for 8 minutes. 10 l of sample was used for SDS-PAGE electrophoresis and Western blot identification, respectively. The results were as shown in
Example 8: Comparison of the Expression Amounts of L1 Proteins and Chimeric L1 Proteins in Insect Cells
[0119] For each of cells expressing the different recombinant proteins described in Example 6, 110.sup.6 cells were collected and resuspended in 200 l PBS solution. The cells were disrupted by ultrasonic disruption (Ningbo Scientz Ultrasonic Cell Disruptor, 2 #probe, 100 W, ultrasound 5 s, interval 7 s, total time 3 min) and centrifuged at a high speed of 12,000 rpm for 10 minutes. The lysed supernatant was collected and the L1 content in the supernatant was detected by sandwich ELISA, which was well known, for example, the patent CN104513826A.
[0120] Microtiter plates were coated with HPV31L1 monoclonal antibodies prepared by the inventor at 80 ng/well by overnight incubation at 4 C. The plate was blocked with 5% BSA-PBST at room temperature for 2 h and washed for 3 times with PBST. The lysed supernatant was subjected to 2-fold serial dilution with PBS. The HPV31L1VLP standard was also subjected to serial dilution from a concentration of 2 /ml to 0.0625 g/ml. The diluted samples were added to the plate respectively at 100 l per well and incubated at 37 C. for 1 h. The plate was washed for 3 times with PBST, and 1:3000 diluted HPV31L1 rabbit polyclonal antibody was added at 100 l per well and incubated at 37 C. for 1 h. The plate was washed for 3 times with PBST, and 1:3000 diluted HRP-labeled goat anti-mouse IgG (1:3000 dilution, ZSGB-Bio Corporation) was added and incubated at 37 C. for 45 minutes. The plate was washed for 5 times with PBST, and 100 l of OPD substrate (Sigma) was added to each well for chromogenic reaction at 37 C. for 5 minutes. The reaction was stopped with 50 l of 2 M sulfuric acid, and the absorbance at 490 nm was determined. The concentrations of the 31L1 protein, mutants of the 31L1 protein or chimeric L1 proteins in the lysed supernatant were calculated according to the standard curve.
[0121] As shown in Table 4, the expression amount of the 31L1 mutant protein with a 29-amino acid truncation at the C-terminus of the present invention (31L1MC) was significantly higher than that of the HPV31L1 full-length protein. The expression amounts of the mutant proteins obtained by point mutation of the 31L1 protein also varied, among which the expression amount of the T.sub.274N mutant was significantly higher than that of the original HPV31L1 protein, and the expression amount of the T.sub.274NC mutant protein was further increased than that of the 31L1MC protein, indicating that the mutation of threonine at position 274 to asparagine could increase the expression amount of the 31L1 protein. Different N-terminus truncations were performed on the basis of T.sub.274NC, and it was found that different truncations had different effects on the expression amount, among which the expression amounts of truncation mutations obtained by a 4-amino acid truncation at the N-terminus (T.sub.274NN4C) or an 8-amino acid truncation at the N-terminus (T.sub.274NN8C) were 2 folds and 1.28 folds that of T.sub.274NC, respectively. The expression amounts of the chimeric proteins (31L1DE.sub.132-136/dE, 31L1DE.sub.132-136/dES, 31L1h4.sub.428-431/dE, 31L1h4.sub.428-431/dES) constructed on the basis of T.sub.274NN4C were all comparable to that of their backbone T.sub.274NN4C. In addition, the expression amounts of 12 types of chimeric proteins with the 31L1 mutant with C-terminus substitutions as the backbone were all higher than that of the corresponding chimeric protein with C-terminus truncation.
TABLE-US-00005 TABLE 4 Analysis of the expression amounts of 31L1 protein, 31L1 protein mutants and chimeric L1 proteins Expression amount (mg/L) Protein name Batch 1 Batch 2 Batch 3 Average HPV31L1 19 25 15 19.7 T.sub.274N 38 40 36 38 31L1MC 35 38 30 34.3 T.sub.274NC 59 59 52 56.7 T.sub.267AC 22 28 27 25.7 T.sub.267AT.sub.274NC 14 12 18 14.6 T.sub.274NN2C 29 27 26 27.3 T.sub.274NN4C 115 108 120 114.3 T.sub.274NN5C 30 32 33 31.7 T.sub.274NN8C 70 75 73 72.7 T.sub.274NN10C 11 15 12 12.7 31L1DE.sub.132-136/dE 102 125 119 115.3 31L1DE.sub.132-136/dES 128 133 122 127.6 31L1DE.sub.132-136/dE-CS1 154 152 160 155.3 31L1DE.sub.132-136/dES-CS1 173 141 139 151 31L1DE.sub.132-136/dE-CS2 158 162 155 158.3 31L1DE.sub.132-136dES-CS2 157 143 140 146.7 31L1DE.sub.132-136/dE-CS3 163 182 145 163.3 31L1DE.sub.132-136/dES-CS3 171 157 166 164.7 31L1h4.sub.428-431/dE 112 118 117 115.7 31L1h4.sub.428-431/dES 115 123 125 121 31L1h4.sub.428-431/dE-CS1 182 130 155 155.7 31L1h4.sub.428-431/dES-CS1 173 148 170 163.7 31L1h4.sub.428-431/dES-CS2 149 162 151 154 31L1h4.sub.428-431/dE-CS2 154 158 150 154 31L1h4.sub.428-431/dE-CS3 162 143 148 151 31L1h4.sub.428-431/dES-CS3 151 160 154 155
Example 9: Purification and Dynamic Light Scattering Particle Size Analysis of L1 Proteins and Chimeric L1 Proteins
[0122] An appropriate amount of cell fermentation broth of the above recombinant proteins was collected and the cells were resuspended with 10 ml PBS. PMSF was added to a final concentration of 1 mg/ml. The cells were ultrasonically disrupted (Ningbo Scientz Ultrasonic Cell Disruptor, 6 #probe, 200 W, ultrasound 5 s, interval 7 s, total time 10 min) and the disrupted supernatant was collected for purification. The purification steps were carried out at room temperature. 4% -mercaptoethanol (w/w) was added to the lysate to disaggregate VLP. Then the samples were filtered with 0.22 m filters, followed by successive purification with DMAE anion exchange chromatography or CM cation exchange chromatography (20 mM Tris, 180 mM NaCl, 4% -ME, elution at pH 7.9), TMAE anion exchange chromatography or Q cation exchange chromatography (20 mM Tris, 180 mM NaCl, 4% -ME, elution at pH 7.9) and hydroxyapatite chromatography (100 mM NaH.sub.2PO.sub.4, 30 mM NaCl, 4% -ME, elution at pH 6.0). The purified product was concentrated and buffer (20 mM NaH.sub.2PO.sub.4, 500 mM NaCl, pH 6.0) exchange was performed using Planova ultrafiltration system to prompt VLP assembly. The above purification methods were all publicly available, for example the patents CN101293918B, CN1976718A, etc.
[0123] The purified HPV31L1 protein, 31L1 mutant proteins and chimeric L1 proteins could all be effectively assembled. The solutions of the assembled proteins were subjected to DLS particle size analysis (Zetasizer Nano ZS 90 Dynamic Light Scattering Analyzer, Malvern), and the results were as shown in Table 5. Among them, the DLS analysis plots of 31L1MC, T.sub.274NC, T.sub.274NN4C, 31L1DE.sub.132-136/dE, and 31L1h4.sub.428-431/dE were as shown in
TABLE-US-00006 TABLE 5 DLS analysis of L1 proteins and chimeric L1 proteins Hydraulic Protein name diameter (nm) PDI HPV31L1 102.5 0.128 T.sub.274N 104.2 0.192 31L1MC 103.3 0.190 T.sub.274NC 99.78 0.169 T.sub.267AC 101.4 0.187 T.sub.267AT.sub.274NC 98.2 0.188 T.sub.274NN2C 105.8 0.166 T.sub.274NN4C 106.8 0.172 T.sub.274NN5C 102.4 0.127 T.sub.274NN8C 100.9 0.153 T.sub.274NN10C 97.55 0.132 31L1DE.sub.132-136/dE 104.59 0.192 31L1DE.sub.132-136/dES 108.5 0.183 31L1DE.sub.132-136/dE-CS1 109.4 0.112 31L1DE.sub.132-136/dES-CS1 108.8 0.146 31L1DE.sub.132-136/dE-CS2 103.2 0.159 31L1DE.sub.132-136/dES-CS2 105.7 0.182 31L1DE.sub.132-136/dE-CS3 117.4 0.193 31L1DE.sub.132-136/dES-CS3 116.2 0.162 31L1h4.sub.428-431/dE 47.8 0.267 31L1h4.sub.428-431/dES 39.6 0.201 31L1h4.sub.428-431/dE-CS1 42.4 0.196 31L1h4.sub.428-431/dES-CS1 36.7 0.175 31L1h4.sub.428-431/dES-CS2 45.3 0.173 31L1h4.sub.428-431/dE-CS2 49.2 0.211 31L1h4.sub.428-431/dE-CS3 46.1 0.156 31L1h4.sub.428-431/dES-CS3 38.4 0.133
Example 10: Transmission Electron Microscopy Observation of VLPs and Chimeric VLPs
[0124] The recombinant proteins were purified separately according to the chromatographic purification method described in Example 9. The assembled chimeras were prepared on copper mesh, stained with 1% uranium acetate, fully dried and then observed using JEM-1400 electron microscope (Olympus). The results showed that the HPV31L1, T.sub.274N, 31L1MC, T.sub.274NC, T.sub.267AC and T.sub.267AT.sub.274NC proteins expressed by insect cells could all be assembled into VLPs with a diameter of about 50-60 nm. The mutants of the 31L1 protein with N-terminal truncation in combination with C-terminal truncation could be assembled into VLPs with a diameter of 17-35 nm. The chimeric proteins with insertion of 73L2 polypeptide in the surface region of the DE loop could be assembled into cVLPs of 30-50 nm. The chimeric proteins with insertion of 73L2 polypeptide in the h4 region could be assembled into cVLPs with a diameter of approximately 17-30 nm. The electron microscopy images of VLPs or cVLPs of 31L1MC, T.sub.274NN4C, 31L1DE.sub.132-136/dE, 31L1h4.sub.428-431/dE, and 31L1h4.sub.428-431/dE-CS1 were as shown in
Example 11: Immunization of Mice with HPV31L1 or Mutant VLPs and Determination of Neutralizing Antibody Titers
[0125] 4-6 weeks old BALB/c mice were randomly divided into groups, 5 mice per group, and immunized with 0.1 g VLP. VLP was subcutaneously injected at Week 0 and Week 2 for a total of 2 doses. Tail vein blood was collected 2 weeks after the second immunization and serum was isolated.
[0126] The neutralizing antibody titers of immune serum were detected using HPV31 pseudovirus, and the VLP-immunized mice of various 31L1 mutants showed that the levels of HPV31-specific neutralizing antibodies were comparable to those of the prototype. The immunization results of 31L1MC, T274NC, and T274NN4C are shown in
Example 12: Immunization of Mice with Chimeric VLPs and Determination of Neutralizing Antibody Titers
[0127] 4-6 weeks old BALB/c mice were randomly divided into groups, 5 mice in each group, and 10 g cVLP in combination with 50 g Al(OH).sub.3 and 5 g MPL adjuvant were used to immunize the mice by subcutaneous injection at Weeks 0, 4, 7, and 10, for a total of 4 times. Tail vein blood was collected 2 weeks after the 4th immunization and serum was isolated.
[0128] 17 types of HPV pseudoviruses were used to detect the neutralizing antibody titers of the antiserum. The results showed that after immunizing mice with various cVLPs, the levels and neutralization range of the induced cross-neutralizing antibodies were different with each other. Among them, the neutralizing antibody titers of the backbone type HPV type 31 induced by cVLPs with chimeric epitopes in the surface region of h4 were comparable to that of HPV31L1VLP, and their antiserum had high titers of cross-neutralizing antibodies, which could neutralize the 17 types of pseudoviruses used for detection. The neutralizing antibody titers of HPV type 31 induced by cVLPs with chimeric epitopes in the surface region of DE loop were reduced by 1 order of magnitude compared with that of 31L1VLP, and the cross-neutralization spectrum of their immune serum was relatively narrow. The cross-neutralization activities of some cVLP immune serum were as shown in Table 5, in which 31L1h4.sub.428-431/dE, 31L1h4.sub.428-431/dES and 31L1h4.sub.428-431/dE-CS1 antiserum could neutralize at least 17 types of pseudoviruses, and 31L1DE.sub.132-136/dE and 31L1DE.sub.132-136/dES antiserum only neutralized 10 and 8 types of pseudoviruses, respectively. It was worth mentioning that the neutralizing titers of 31L1h4.sub.428-431/dE, 31L1h4.sub.428-431/dES and 31L1h4.sub.428-431/dE-CS1 antiserum against HPV types 16, -18 and -45 were all greater than 10.sup.3, and the neutralizing antibody titer of 31L1h4.sub.428-431/dE-CS1 antiserum against HPV type 73 was also greater than 10.sup.3.
[0129] In addition, in the present invention, after immunizing mice with the above strategy using the cVLPs constructed with the 31L1 mutant with C-terminus substitutions, the levels and neutralization ranges of the induced cross-neutralizing antibodies were consistent with the corresponding C-terminus truncated cVLPs.
[0130] Methods of pseudovirus preparation and pseudoviral neutralization experiments were all publicly available, for example, the patent CN 104418942A.
TABLE-US-00007 TABLE 6 Neutralizing antibody titers induced by different cVLPs in mice T.sub.274NN 31L1DE.sub.132-136/ 31L1DE.sub.132-136/ 31L1h4.sub.428-431/ 31L1h4.sub.428-431/ 31L1h4.sub.428-431/ 4C dE dES dE dES dE-CS1 Average 9 HPV 31 800000 28000 22500 608000 440000 480000 titer subgenus HPV 16 ND 400 200 1080 1020 2900 of HPV 35 ND 15 ND 440 400 162.5 neutralizing HPV 52 ND ND ND 165 125 125 antibodies HPV 58 ND 125 155 600 525 237.5 7 HPV 18 ND 165 215 1160 1050 1400 subgenus HPV 39 ND 34 66 535 550 950 HPV 45 ND 200 440 2880 1890 1150 HPV 59 ND ND ND 66 80 165 HPV 68 ND 25 ND 120 175 650 11 HPV 73 ND 200 115 440 480 2000 subgenus 10 HPV 6 ND ND ND 55 25 37.5 subgenus HPV 11 ND ND 25 115 75 78 4 HPV 2 ND ND ND 90 78 55 subgenus HPV 27 ND ND ND 34 25 25 HPV 57 ND 155 ND 530 600 480 1 HPV 5 ND ND ND 155 125 115 subgenus