HUMAN PAPILLOMAVIRUS TYPE 18 CHIMERIC PROTEIN AND USE THEREOF
20240317808 ยท 2024-09-26
Inventors
Cpc classification
C12N2800/22
CHEMISTRY; METALLURGY
A61K2039/58
HUMAN NECESSITIES
A61K39/00
HUMAN NECESSITIES
C12N2710/14143
CHEMISTRY; METALLURGY
C07K2319/735
CHEMISTRY; METALLURGY
C07K19/00
CHEMISTRY; METALLURGY
C07K2319/40
CHEMISTRY; METALLURGY
C12N2710/20022
CHEMISTRY; METALLURGY
C12N2710/20034
CHEMISTRY; METALLURGY
C12N15/70
CHEMISTRY; METALLURGY
C12N15/86
CHEMISTRY; METALLURGY
International classification
C12N15/70
CHEMISTRY; METALLURGY
Abstract
The present invention relates to a human papillomavirus type 18 chimeric protein and the use thereof. Specifically, the present invention relates to a papillomavirus chimeric protein, which comprises or consists of an HPV type 18 L1 protein or a mutant of the HPV type 18 L1 protein, and a polypeptide from a HPV type 59 L2 protein inserted into the surface region of the HPV type 18 L1 protein or the mutant of the HPV type 18 L1 protein, wherein the amino acid sequence of the HPV type 18 L1 protein is as shown in SEQ ID No. 1, and the amino acid sequence of the HPV type 59 L2 protein is as shown in SEQ ID No. 2.
Claims
1-10. (canceled)
11. A human papillomavirus chimeric protein comprising or consisting of a HPV type 18 L1 protein or a mutant of the HPV type 18 L1 protein, and a polypeptide from a HPV type 59 L2 protein inserted into the surface region of the HPV type 18 L1 protein or the mutant of the HPV type 18 L1 protein, wherein the amino acid sequence of the HPV type 18 L1 protein is as shown in SEQ ID No. 1, and the amino acid sequence of the HPV type 59 L2 protein is as shown in SEQ ID No. 2.
12. The human papillomavirus chimeric protein according to claim 11, wherein the mutant of the HPV type 18 L1 protein is selected from any one of the group consisting of: a mutant with a 32-amino acid truncation at C-terminus of the amino acid sequence as shown in SEQ ID No. 1; a mutant with amino acid substitutions of amino acids 477, 478, 484, 496, 499, 504, 506 to glycine (G) and amino acids 485, 500, 502 to serine (S) in the amino acid sequence as shown in SEQ ID No. 1; a mutant with amino acid substitutions of amino acids 477, 478, 485, 496, 499, 504, 506 to glycine (G) and amino acids 486, 500, 502 to serine (S) in the amino acid sequence as shown in SEQ ID No. 1; a mutant with amino acid substitutions of amino acids 477, 478, 484, 496, 499, 502, 506 to glycine (G), amino acids 485, 500 to serine (S) and amino acid 504 to aspartate (D) in the amino acid sequence as shown in SEQ ID No. 1; a mutant with amino acid substitutions of amino acids 477, 478, 485, 496, 502, 506 to glycine (G), amino acids 486, 500 to serine (S) and amino acids 499, 504 to aspartate (D) in the amino acid sequence as shown in SEQ ID No. 1; a mutant with amino acid substitutions of amino acids 477, 484, 496, 499, 504, 506 to glycine (G) and amino acids 485, 500, 502 to serine (S) in the amino acid sequence as shown in SEQ ID No. 1; and a mutant with amino acid substitutions of amino acids 477, 485, 496, 499, 504, 506 to glycine (G) and amino acids 486, 500, 502 to serine (S) in the amino acid sequence as shown in SEQ ID No. 1.
13. The human papillomavirus chimeric protein according to claim 11, wherein the polypeptide from HPV type 59 L2 protein is selected from any of the polypeptides as shown in SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5 and SEQ ID No. 6.
14. The human papillomavirus chimeric protein according to claim 11, wherein the polypeptide from the HPV type 59 L2 protein is inserted into the surface region of the HPV type 18 L1 protein or the mutant of the HPV type 18 L1 protein, and the surface region is a DE loop and/or h4 region.
15. The human papillomavirus chimeric protein according to claim 11, wherein the polypeptide from the HPV type 59 L2 protein is inserted between amino acids 137 and 138, between amino acids 432 and 433, or between amino acids 434 and 435 of the HPV type 18 L1 protein or the mutant of the HPV type 18 L1 protein by direct insertion, alternatively, the polypeptide from the HPV type 59 L2 protein is inserted into the region of amino acids 121 to 124, or the region of amino acids 131 to 138, or the region of amino acids 431 to 433, or the region of amino acids 432 to 435 of the HPV type 18 L1 protein or the mutant of the HPV type 18 L1 protein by non-isometric substitution.
16. The human papillomavirus chimeric protein according to claim 11, wherein the polypeptide from the HPV type 59 L2 protein comprises a linker of 1 to 3 amino acid residues at its N- and/or C-terminus, the linker comprises one or more amino acids selected from the group consisting of glycine, serine, alanine and proline.
17. The human papillomavirus chimeric protein according to claim 16, wherein the linker at N-terminus consists of glycine-proline, and the linker at C-terminus consists of proline.
18. The human papillomavirus chimeric protein according to claim 11, wherein the amino acid sequence of the human papillomavirus chimeric protein is as shown in any one of SEQ ID Nos. 7-32.
19. A polynucleotide encoding the human papillomavirus chimeric protein according to claim 11.
20. The polynucleotide according to claim 19, wherein the sequence of the polynucleotide is whole-gene optimized using E. coli codons or whole-gene optimized using insect cell codons.
21. The polynucleotide according to claim 19, wherein the sequence of the polynucleotide is as shown in any one of SEQ ID No. 33, SEQ ID No. 34, SEQ ID No. 35, SEQ ID No. 36, SEQ ID No. 37, SEQ ID No. 38, SEQ ID Any of No. 39, SEQ ID No. 40, SEQ ID No. 41, SEQ ID No. 42 and SEQ ID No. 43.
22. A vector comprising the polynucleotide according to claim 19.
23. A cell comprising the vector according to claim 22.
24. A polymer which is a chimeric pentamer or chimeric virus-like particle comprising the human papillomavirus chimeric protein according to claim 11, or formed by the human papillomavirus chimeric protein according to claim 11.
25. 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 11.
26. The method of claim 25, 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; and 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.
27. A vaccine for the prevention of papillomavirus infection and/or papillomavirus infection-induced diseases, comprising the human papillomavirus chimeric protein according to claim 11, an adjuvant, as well as an excipient or carrier for vaccines.
28. The vaccine for the prevention of papillomavirus infection and/or papillomavirus infection-induced diseases according to claim 27, further comprising at least one virus-like particle or chimeric virus-like particle of mucosa-tropic and/or skin-tropic HPVs.
29. The vaccine for the prevention of papillomavirus infection and/or papillomavirus infection-induced diseases according to claim 27, wherein the adjuvant is for use in human.
Description
DESCRIPTION OF THE DRAWINGS
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DETAILED DESCRIPTION OF THE INVENTION
[0072] The present invention will be further illustrated below by the following non-limiting examples. 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 embodiments 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.
[0073] 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: Detection of Immunoactivity of Different Types of RG-1 Epitope Peptides
[0074] HPV35, -39, -51, -53, -56, -59, -68, -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). 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.
[0075] Seventeen HPV pseudoviruses were used to detect the titers of neutralizing antibodies in the immune serum, and the results were shown in Table 2. The 59RG-1 epitope peptide had the best immune activity, and its antiserum could neutralize all 17 types used for detection, of which the titers of neutralizing antibodies of HPV45, -59, -16 were all above 10.sup.3, and the titers of neutralizing antibodies of HPV5, -31, -18, -39, -68, -57 were between 500 and 1000. It was worth noticing that the 59RG-1 epitope peptide antiserum had high levels of neutralizing antibodies against the five ?7-HPV used for detection.
[0076] Methods of polypeptide synthesis, pseudovirus preparation and pseudoviral neutralization experiments were all publicly available, for example, the patents CN 104418942A and 108676057A.
TABLE-US-00001 TABLE1 SequencesofdifferenttypesofRG-1epitope peptidessynthesized Type Sequenceofsyntheticpeptide SEQIDNo. HPV35 TQLYRTCKAAGTCPPDVIPKVEG 44 HPV39 STLYRTCKQSGTCPPDVVDKVEG 45 HPV51 TQLYSTCKAAGTCPPDVVNKVEG 46 HPV53 TQLYQTCKQSGTCPEDVINKIEH 47 HPV56 TQLYKTCKLSGTCPEDVVNKIEQ 48 HPV59 LYKTCKQAGTCPSDVINKVEGTT 49 HPV68 STLYKTCKQSGTCPPDVINKVEG 50 HPV82 TQLYSTCKAAGTCPPDVIPKVKG 51
TABLE-US-00002 TABLE 2 Titers of serum neutralizing antibodies induced by different RG1-KLH conjugated peptides in rabbits 35RG-1 39RG-1 51RG-1 53RG-1 56RG-1 59RG-1 68RG-1 82RG-1 Average ?7 HPV 18 ND* ND ND ND 50 603 25 100 titer subgenus HPV 39 ND 25 ND ND 100 575 100 400 of HPV 45 25 25 25 ND 1200 1850 1600 400 neutralizing HPV 59 ND ND ND 100 25 1800 ND ND antibodies HPV 68 ND ND ND ND 75 525 425 100 ?9 HPV 16 ND ND ND ND ND 1063 ND 50 subgenus HPV 31 ND ND ND ND ND 615 ND 25 HPV 33 ND ND ND ND ND 118 ND 25 HPV 35 25 ND ND ND ND 240 ND 100 HPV 52 ND ND ND ND ND 128 ND 50 HPV 58 ND ND ND ND 25 255 ND 100 ?10 HPV 6 ND ND ND ND 25 68 ND 50 subgenus HPV 11 ND 25 ND 25 ND 118 25 50 ?4 HPV 2 25 125 ND 50 ND 118 ND 50 subgenus HPV 27 50 50 25 50 50 140 50 25 HPV 57 75 50 50 50 75 515 50 125 ?1 HPV 5 50 50 25 ND 50 950 200 225 subgenus
Example 2: Synthesis of Chimeric L1 Protein Genes and Construction of Expression Vectors
[0077] There were 26 chimeric L1 proteins, namely: [0078] 1) Chimeric L1 protein 18L1DE.sub.137-138/59dES: the backbone was full-length HPV type 18 L1 protein (the sequence was as shown in SEQ ID No. 1), where the polypeptide of aa. 17-32 of HPV type 59 L2 protein (as shown in SEQ ID No. 4) was directly inserted at the aa. 137/138 site of its DE loop. The amino acid sequence of 18L1DE.sub.137-138/59dES chimeric protein was as shown in SEQ ID No. 7. The polynucleotide sequence encoding 18L1DE.sub.137-138/59dES was optimized with E. coli codons and constructed by whole-gene synthesis; [0079] 2) Chimeric L1 protein 18L1DE.sub.137-138/59dE: the backbone was full-length HPV type 18 L1 protein (the sequence was as shown in SEQ ID No. 1), where the polypeptide of aa. 16-35 of HPV type 59 L2 protein (as shown in SEQ ID No. 5) was directly inserted at the aa. 137/138 site of its DE loop. The amino acid sequence of 18L1DE.sub.137-138/59dE chimeric protein was as shown in SEQ ID No. 9. The polynucleotide sequence encoding 18L1DE.sub.137-138/59dE was optimized with E. coli codons and constructed by whole-gene synthesis; [0080] 3) Chimeric L1 protein 18L1h4.sub.432-433/59dE: the backbone was full-length HPV type 18 L1 protein (the sequence was as shown in SEQ ID No. 1), where the polypeptide of aa. 17-37 of HPV type 59 L2 protein (as shown in SEQ ID No. 3) was directly inserted at the aa. 432/433 site of its h4 region. The amino acid sequence of 18L1h4.sub.432-433/59dE chimeric protein was as shown in SEQ ID No. 11. The polynucleotide sequence encoding 18L1h4.sub.432-433/59dE was optimized with E. coli codons and constructed by whole-gene synthesis; [0081] 4) Chimeric L1 protein 18L1h4.sub.434-435/59dE: the backbone was full-length HPV type 18 L1 protein (the sequence was as shown in SEQ ID No. 1), where the polypeptide of aa. 17-37 of HPV type 59 L2 protein (as shown in SEQ ID No. 3) was directly inserted at the aa. 434/435 site of its h4 region. The amino acid sequence of 18L1h4.sub.434-435/59dE chimeric protein was as shown in SEQ ID No. 13. The polynucleotide sequence encoding 18L1h4.sub.434-435/59dE was optimized with E. coli codons and constructed by whole-gene synthesis; [0082] 5) Chimeric L1 protein 18L1DE.sub.134-135/59dES: the backbone was full-length HPV type 18 L1 protein (the sequence was as shown in SEQ ID No. 1), where the polypeptide of aa. 17-32 of HPV type 59 L2 protein containing a GP linker at N-terminus and a P linker at C-terminus (i.e., adding glycine-proline at N-terminus and adding proline at C-terminus of the sequence as shown in SEQ ID No. 4) was directly inserted at the aa. 134/135 site of its DE loop. The amino acid sequence of 18L1DE.sub.134-135/59dES chimeric protein was as shown in SEQ ID No. 15. The polynucleotide sequence encoding 18L1DE.sub.134-135/59dES was optimized with E. coli codons and constructed by whole-gene synthesis; [0083] 6) Chimeric L1 protein 18L1DE.sub.134-135/59dE: the backbone was full-length HPV type 18 L1 protein (the sequence was as shown in SEQ ID No. 1), where the polypeptide of aa. 16-37 of HPV type 59 L2 protein containing a GP linker at N-terminus and a P linker at C-terminus (i.e., adding glycine-proline at N-terminus and adding proline at C-terminus of the sequence as shown in SEQ ID No. 6) was directly inserted at the aa. 134/135 site of its DE loop. The amino acid sequence of 18L1DE.sub.134-135/59dE chimeric protein was as shown in SEQ ID No. 17. The polynucleotide sequence encoding 18L1DE.sub.134-135/59dES was optimized with E. coli codons and constructed by whole-gene synthesis; [0084] 7) Chimeric L1 protein 18L1DE.sub.131-138/59dE: the backbone was full-length HPV type 18 L1 protein (the sequence was as shown in SEQ ID No. 1), where the region of aa. 132-137 was deleted, and the polypeptide of aa. 17-37 of HPV type 59 L2 protein containing a GP linker at N-terminus was fused between aa. 131/138 (insertion at the region of aa. 132-137 of the HPV type 18 L1 protein by non-isometric substitution). The amino acid sequence of the inserted fragment was the sequence as shown in SEQ ID No. 3 with glycine-proline added at N-terminus, and the amino acid sequence of 18L1DE.sub.131-138/59dE chimeric protein was as shown in SEQ ID No. 19. The polynucleotide sequence encoding 18L1DE.sub.131-138/59dE was optimized with E. coli codons and constructed by whole-gene synthesis; [0085] 8) Chimeric L1 protein 18L1DE.sub.121-124/59dE: the backbone was full-length HPV type 18 L1 protein (the sequence was as shown in SEQ ID No. 1), where the region of aa. 122-123 was deleted, and the polypeptide of aa. 17-37 of HPV type 59 L2 protein was fused between aa. 121/124 (insertion at the region of aa. 122-133 of the HPV type 18 L1 protein by non-isometric substitution). The amino acid sequence of the inserted fragment was as shown in SEQ ID No. 3, and the amino acid sequence of 18L1DE.sub.121-124/59dE chimeric protein was as shown in SEQ ID No. 21. The polynucleotide sequence encoding 18L1DE.sub.121-124/59dE was optimized with E. coli codons and constructed by whole-gene synthesis; [0086] 9) Chimeric L1 protein 18L1h4.sub.431-433/59dE: the backbone was full-length HPV type 18 L1 protein (the sequence was as shown in SEQ ID No. 1), where aa. 432 was deleted, and the polypeptide of aa. 17-37 of HPV type 59 L2 protein was fused between aa. 431/433 (insertion at the region of aa. 431-433 of the HPV type 18 L1 protein by non-isometric substitution). The amino acid sequence of the inserted fragment was as shown in SEQ ID No. 3, and the amino acid sequence of 18L1h4.sub.431-433/59dE chimeric protein was as shown in SEQ ID No. 23. The polynucleotide sequence encoding 18L1h4.sub.431-433/59dE was optimized with E. coli codons and constructed by whole-gene synthesis; [0087] 10) Chimeric L1 protein 18L1h4.sub.432-435/59dE: the backbone was full-length HPV type 18 L1 protein (the sequence was as shown in SEQ ID No. 1), where the region of aa. 433-434 was deleted, and the polypeptide of aa. 17-37 of HPV type 59 L2 protein was fused between aa. 432/435 (insertion at the region of aa. 432-435 of the HPV type 18 L1 protein by non-isometric substitution). The amino acid sequence of the inserted fragment was as shown in SEQ ID No. 3, and the amino acid sequence of 18L1h4.sub.432-435/59dE chimeric protein was as shown in SEQ ID No. 25. The polynucleotide sequence encoding 18L1h4433-434/59dE was optimized with E. coli codons and constructed by whole-gene synthesis; [0088] 11) Chimeric L1 protein 18L1?CDE.sub.137-138/59dES: the backbone was the HPV type 18 L1 protein with 32-amino acid truncation at C-terminus (the sequence of SEQ ID No. 1 with 32-amino acid truncation at C-terminus), where the polypeptide of aa. 17-32 of HPV type 59 L2 protein (as shown in SEQ ID No. 4) was directly inserted at the aa. 137/138 site of its DE loop. The amino acid sequence of 18L1?CDE.sub.137-138/59dES chimeric protein was as shown in SEQ ID No. 8. The polynucleotide sequence encoding 18L1?CDE.sub.137-138/59dES was optimized with insect cell codons and constructed by whole-gene synthesis, and its nucleotide sequence was as shown in SEQ ID No. 33; [0089] 12) Chimeric L1 protein 18L1?CDE.sub.137-138/59dE: the backbone was the HPV type 18 L1 protein with 32-amino acid truncation at C-terminus (the sequence of SEQ ID No. 1 with 32-amino acid truncation at C-terminus), where the polypeptide of aa. 16-35 of HPV type 59 L2 protein (as shown in SEQ ID No. 5) was directly inserted at the aa. 137/138 site of its DE loop. The amino acid sequence of 18L1?CDE.sub.137-138/59dE chimeric protein was as shown in SEQ ID No. 10. The polynucleotide sequence encoding 18L1?CDE.sub.137-138/59dE was optimized with insect cell codons and constructed by whole-gene synthesis, and its nucleotide sequence was as shown in SEQ ID No. 34; [0090] 13) Chimeric L1 protein 18L1?Ch4.sub.432-433/59dE: the backbone was the HPV type 18 L1 protein with 32-amino acid truncation at C-terminus (the sequence of SEQ ID No. 1 with 32-amino acid truncation at C-terminus), where the polypeptide of aa. 17-37 of HPV type 59 L2 protein (as shown in SEQ ID No. 3) was directly inserted at the aa. 432/433 site of its h4 region. The amino acid sequence of 18L1?Ch4.sub.432-433/59dE chimeric protein was as shown in SEQ ID No. 12. The polynucleotide sequence encoding 18L1?Ch4.sub.432-433/59dE was optimized with insect cell codons and constructed by whole-gene synthesis; [0091] 14) Chimeric L1 protein 18L1?Ch4.sub.434-435/59dE: the backbone was the HPV type 18 L1 protein with 32-amino acid truncation at C-terminus (the sequence of SEQ ID No. 1 with 32-amino acid truncation at C-terminus), where the polypeptide of aa. 17-37 of HPV type 59 L2 protein (as shown in SEQ ID No. 3) was directly inserted at the aa. 434/435 site of its h4 region. The amino acid sequence of 18L1?Ch4.sub.434-435/59dE chimeric protein was as shown in SEQ ID No. 14. The polynucleotide sequence encoding 18L1?Ch4.sub.434-435/59dE was optimized with insect cell codons and constructed by whole-gene synthesis; [0092] 15) Chimeric L1 protein 18L1?CDE.sub.134-135/59dES: the backbone was the HPV type 18 L1 protein with 32-amino acid truncation at C-terminus (the sequence of SEQ ID No. 1 with 32-amino acid truncation at C-terminus), where the polypeptide of aa. 17-32 of HPV type 59 L2 protein containing a GP linker at N-terminus and a P linker at C-terminus (i.e., adding glycine-proline at N-terminus and adding proline at C-terminus of the sequence as shown in SEQ ID No. 4) was directly inserted at the aa. 134/135 site of its DE loop. The amino acid sequence of 18L1?CDE.sub.134-135/59dES chimeric protein was as shown in SEQ ID No. 16. The polynucleotide sequence encoding 18L1?CDE.sub.134-135/59dES was optimized with insect cell codons and constructed by whole-gene synthesis, and its nucleotide sequence was as shown in SEQ ID No. 35; [0093] 16) Chimeric L1 protein 18L1?CDE.sub.134-135/59dE: the backbone was the HPV type 18 L1 protein with 32-amino acid truncation at C-terminus (the sequence of SEQ ID No. 1 with 32-amino acid truncation at C-terminus), where the polypeptide of aa. 16-37 of HPV type 59 L2 protein containing a GP linker at N-terminus and a P linker at C-terminus (i.e., adding glycine-proline at N-terminus and adding proline at C-terminus of the sequence as shown in SEQ ID No. 6) was directly inserted at the aa. 134/135 site of its DE loop. The amino acid sequence of 18L1?CDE.sub.134-135/59dE chimeric protein was as shown in SEQ ID No. 18. The polynucleotide sequence encoding 18L1?CDE.sub.134-135/59dE was optimized with insect cell codons and constructed by whole-gene synthesis, and its nucleotide sequence was as shown in SEQ ID No. 36; [0094] 17) Chimeric L1 protein 18L1?CDE.sub.131-138/59dE: the backbone was the HPV type 18 L1 protein with 32-amino acid truncation at C-terminus (the sequence of SEQ ID No. 1 with 32-amino acid truncation at C-terminus), where the region of aa. 132-137 was deleted, and the polypeptide of aa. 17-37 of HPV type 59 L2 protein containing a GP linker at N-terminus was fused between aa. 131/138 (insertion at the region of aa. 132-137 of the HPV type 18 L1 protein by non-isometric substitution). The amino acid sequence of the inserted fragment was the sequence as shown in SEQ ID No. 3 with glycine-proline added at N-terminus, and the amino acid sequence of 18L1 DE.sub.131-138/59dE chimeric protein was as shown in SEQ ID No. 20. The polynucleotide sequence encoding 18L1DE.sub.131-138/59dE was optimized with insect cell codons and constructed by whole-gene synthesis, and its nucleotide sequence was as shown in SEQ ID No. 37; [0095] 18) Chimeric L1 protein 18L1?CDE.sub.121-124/59dE: the backbone was the HPV type 18 L1 protein with 32-amino acid truncation at C-terminus (the sequence of SEQ ID No. 1 with 32-amino acid truncation at C-terminus), where the region of aa. 122-123 was deleted, and the polypeptide of aa. 17-37 of HPV type 59 L2 protein was fused between aa. 121/124 (insertion at the region of aa. 122-133 of the HPV type 18 L1 protein by non-isometric substitution). The amino acid sequence of the inserted fragment was as shown in SEQ ID No. 3, and the amino acid sequence of 18L1?CDE.sub.121-124/59dE chimeric protein was as shown in SEQ ID No. 22. The polynucleotide sequence encoding 18L1?CDE.sub.121-124/59dE was optimized with insect cell codons and constructed by whole-gene synthesis; [0096] 19) Chimeric L1 protein 18L1?Ch4.sub.431-433/59dE: the backbone was the HPV type 18 L1 protein with 32-amino acid truncation at C-terminus (the sequence of SEQ ID No. 1 with 32-amino acid truncation at C-terminus), where aa. 432 was deleted, and the polypeptide of aa. 17-37 of HPV type 59 L2 protein was fused between aa. 431/433 (insertion at the region of aa. 431-433 of the HPV type 18 L1 protein by non-isometric substitution). The amino acid sequence of the inserted fragment was as shown in SEQ ID No. 3, and the amino acid sequence of 18L1?Ch4.sub.431-433/59dE chimeric protein was as shown in SEQ ID No. 24. The polynucleotide sequence encoding 18L1?Ch4.sub.431-433/59dE was optimized with insect cell codons and constructed by whole-gene synthesis: [0097] 20) Chimeric L1 protein 18L1?Ch4.sub.432-435/59dE: the backbone was the HPV type 18 L1 protein with 32-amino acid truncation at C-terminus (the sequence of SEQ ID No. 1 with 32-amino acid truncation at C-terminus), where the region of aa. 433-434 was deleted, and the polypeptide of aa. 17-37 of HPV type 59 L2 protein was fused between aa. 432/435 (insertion at the region of aa. 432-435 of the HPV type 18 L1 protein by non-isometric substitution). The amino acid sequence of the inserted fragment was as shown in SEQ ID No. 3, and the amino acid sequence of 18L1?Ch4.sub.432-435/59dE chimeric protein was as shown in SEQ ID No. 26. The polynucleotide sequence encoding 18L1?Ch4.sub.432-435/59dE was optimized with insect cell codons and constructed by whole-gene synthesis; [0098] 21) Chimeric L1 protein 18L1DE.sub.137-138/59dES-mut1: the backbone was the mutant mut1 of full-length HPV type 18 L1 protein (i.e., the mutant with amino acid substitutions of amino acids 477, 478, 484, 496, 499, 504, 506 to glycine (G) and amino acids 485, 500, 502 to serine (S) in the amino acid sequence as shown in SEQ ID No. 1), where the polypeptide of aa. 17-32 of HPV type 59 L2 protein (as shown in SEQ ID No. 4) was directly inserted at the aa. 137/138 site of its DE loop. The amino acid sequence of 18L1DE.sub.137-138/59dES-mut1 chimeric protein was as shown in SEQ ID No. 27. The polynucleotide sequence encoding 18L1DE.sub.137-138/59dES-mut1 was optimized with E. coli codons or insect cell codons and constructed by whole-gene synthesis, wherein the polynucleotide sequence encoding 18L1DE.sub.137-138/59dES-mut1 optimized with insect cell codons was as shown in SEQ ID No. 38; [0099] 22) Chimeric L1 protein 18L1DE.sub.137-138/59dES-mut2: the backbone was the mutant mut2 of full-length HPV type 18 L1 protein (i.e., the mutant with amino acid substitutions of amino acids 477, 478, 485, 496, 499, 504, 506 to glycine and amino acids 486, 500, 502 to serine in the amino acid sequence as shown in SEQ ID No. 1), where the polypeptide of aa. 17-32 of HPV type 59 L2 protein (as shown in SEQ ID No. 4) was directly inserted at the aa. 137/138 site of its DE loop. The amino acid sequence of 18L1DE.sub.137-138/59dES-mut2 chimeric protein was as shown in SEQ ID No. 28. The polynucleotide sequence encoding 18L1DE.sub.137-138/59dES-mut2 was optimized with E. coli codons or insect cell codons and constructed by whole-gene synthesis, wherein the polynucleotide sequence encoding 18L1DE.sub.137-138/59dES-mut2 optimized with insect cell codons was as shown in SEQ ID No. 39; [0100] 23) Chimeric L1 protein 18L1DE.sub.137-138/59dES-mut3: the backbone was the mutant mut3 of full-length HPV type 18 L1 protein (i.e., the mutant with amino acid substitutions of amino acids 477, 478, 484, 496, 499, 502, 506 to glycine, amino acids 485, 500 to serine and amino acid 504 to aspartate in the amino acid sequence as shown in SEQ ID No. 1), where the polypeptide of aa. 17-32 of HPV type 59 L2 protein (as shown in SEQ ID No. 4) was directly inserted at the aa. 137/138 site of its DE loop. The amino acid sequence of 18L1DE.sub.137-138/59dES-mut3 chimeric protein was as shown in SEQ ID No. 29. The polynucleotide sequence encoding 18L1DE.sub.137-138/59dES-mut3 was optimized with E. coli codons or insect cell codons and constructed by whole-gene synthesis, wherein the polynucleotide sequence encoding 18L1DE.sub.137-138/59dES-mut3 optimized with insect cell codons was as shown in SEQ ID No. 40; [0101] 24) Chimeric L1 protein 18L1DE.sub.137-138/59dES-mut4: the backbone was the mutant mut4 of full-length HPV type 18 L1 protein (i.e., the mutant with amino acid substitutions of amino acids 477, 478, 485, 496, 502, 506 to glycine, amino acids 486, 500 to serine and amino acids 499, 504 to aspartate in the amino acid sequence as shown in SEQ ID No. 1), where the polypeptide of aa. 17-32 of HPV type 59 L2 protein (as shown in SEQ ID No. 4) was directly inserted at the aa. 137/138 site of its DE loop. The amino acid sequence of 18L1DE.sub.137-138/59dES-mut4 chimeric protein was as shown in SEQ ID No. 30. The polynucleotide sequence encoding 18L1DE.sub.137-138/59dES-mut4 was optimized with E. coli codons or insect cell codons and constructed by whole-gene synthesis, wherein the polynucleotide sequence encoding 18L1DE.sub.137-138/59dES-mut4 optimized with insect cell codons was as shown in SEQ ID No. 41; [0102] 25) Chimeric L1 protein 18L1DE.sub.137-138/59dES-mut5: the backbone was the mutant mut5 of full-length HPV type 18 L1 protein (i.e., the mutant with amino acid substitutions of amino acids 477, 484, 496, 499, 504, 506 to glycine and amino acids 485, 500, 502 to serine in the amino acid sequence as shown in SEQ ID No. 1), where the polypeptide of aa. 17-32 of HPV type 59 L2 protein (as shown in SEQ ID No. 4) was directly inserted at the aa. 137/138 site of its DE loop. The amino acid sequence of 18L1DE.sub.137-138/59dES-mut5 chimeric protein was as shown in SEQ ID No. 31. The polynucleotide sequence encoding 18L1DE.sub.137-138/59dES-mut5 was optimized with E. coli codons or insect cell codons and constructed by whole-gene synthesis, wherein the polynucleotide sequence encoding 18L1DE.sub.137-138/59dES-mut5 optimized with insect cell codons was as shown in SEQ ID No. 42; [0103] 26) Chimeric L1 protein 18L1DE.sub.137-138/59dES-mut6: the backbone was the mutant mut6 of full-length HPV type 18 L1 protein (i.e., the mutant with amino acid substitutions of amino acids 477, 485, 496, 499, 504, 506 to glycine and amino acids 486, 500, 502 to serine in the amino acid sequence as shown in SEQ ID No. 1), where the polypeptide of aa. 17-32 of HPV type 59 L2 protein (as shown in SEQ ID No. 4) was directly inserted at the aa. 137/138 site of its DE loop. The amino acid sequence of 18L1DE.sub.137-138/59dES-mut6 chimeric protein was as shown in SEQ ID No. 32. The polynucleotide sequence encoding 18L1DE.sub.137-138/59dES-mut6 was optimized with E. coli codons or insect cell codons and constructed by whole-gene synthesis, wherein the polynucleotide sequence encoding 18L1DE.sub.137-138/59dES-mut5 optimized with insect cell codons was as shown in SEQ ID No. 43.
[0104] The chimeric protein genes optimized with E. coli codons were digested by NdeI/XhoI and inserted into the commercial expression vector pET22b (produced by Novagen), respectively. The chimeric protein genes optimized with insect cell codons were digested by BamHI/EcoRI and inserted into the commercial expression vector pFastBac1 (produced by Invitrogen), respectively. Expression vectors comprising the chimeric protein genes were obtained. There were a total of 16 expression vectors for E. coli, namely: pET22b-18L1DE.sub.137-138/59dES, pET22b-18L1DE.sub.137-138/59dE, pET22b-18L1h4.sub.432-433/59dE, pET22b-18L1h4.sub.434-435/59dE, pET22b-18L1DE.sub.134-135/59dES, pET22b-18L1DE.sub.134-135/59dE, pET22b-18L1DE.sub.131-138/59dE, pET22b-18L1DE.sub.121-124/59dE, pET22b-18L1h4.sub.431-433/59dE, pET22b-18L1h4.sub.432-435/59dE, pET22b-18L1DE.sub.137-138/59dES-mut1, pET22b-18L1DE.sub.137-138/59dES-mut3, pET22b-18L1DE.sub.137-138/59dES-mut5, pET22b-18L1DE.sub.137-138/59dES-mut2, pET22b-18L1DE.sub.137-138/59dES-mut4, pET22b-18L1DE.sub.137-138/59dES-mut6. There were a total of 16 expression vectors for insect cells, namely: pFastBac1-18L1?CDE.sub.137-138/59dES, pFastBac1-18L1?CDE.sub.137-138/59dE, pFastBac1-18L1?Ch4.sub.432-433/59dE, pFastBac1-18L1?Ch4.sub.434-435/59dE, pFastBac1-18L1?CDE.sub.134-135/59dE, pFastBac1-18L1?CDE.sub.134-135/59dES, pFastBac1-18L1?CDE.sub.121-124/59dE, pFastBac1-18L1?CDE.sub.131-138/59dE, pFastBac1-18L1?Ch4.sub.432-435/59dE, pFastBac1-18L1?Ch4.sub.431-433/59dE, pFastBac1-18L1DE.sub.137-138/59dES-mut2, pFastBac1-18L1DE.sub.137-138/59dES-mut1, pFastBac1-18L1DE.sub.137-138/59dES-mut3, pFastBac1-18L1DE.sub.137-138/59dES-mut4, pFastBac1-18L1DE.sub.137-138/59dES-mut5, pFastBac1-18L1DE.sub.137-138/59dES-mut6. The above methods of enzyme digestion, ligation and construction of clones were all well known, for example, the patent CN 101293918 B.
[0105] The amino acid sequences of the polypeptides used in the present invention were shown as follows:
TABLE-US-00003 Full-lengthaminoacidsofHPVtype18L1 SEQIDNO.1 MALWRPSDNTVYLPPPSVARVVNTDDYVTRTSIFYHAGSSRLLTVGNPYF RVPAGGGNKQDIPKVSAYQYRVFRVQLPDPNKFGLPDTSIYNPETQRLVW ACAGVEIGRGQPLGVGLSGHPFYNKLDDTESSHAATSNVSEDVRDNVSVD YKQTQLCILGCAPAIGEHWAKGTACKSRPLSQGDCPPLELKNTVLEDGDM VDTGYGAMDFSTLQDTKCEVPLDICQSICKYPDYLQMSADPYGDSMFFCL RREQLFARHFWNRAGTMGDTVPQSLYIKGTGMRASPGSCVYSPSPSGSIV TSDSQLFNKPYWLHKAQGHNNGVCWHNQLFVTVVDTTRSTNLTICASTQS PVPGQYDATKFKQYSRHVEEYDLQFIFQLCTITLTADVMSYIHSMNSSIL EDWNFGVPPPPTTSLVDTYRFVQSVAITCQKDAAPAENKDPYDKLKFWNV DLKEKFSLDLDQYPLGRKFLVQAGLRRKPTIGPRKRSAPSATTSSKPAKR VRVRARK Full-lengthaminoacidsequenceofHPVtype59L2 SEQIDNO.2 MVSHRAARRKRASATDLYKTCKQAGTCPSDVINKVEGTTLADKILQWTSL GIFLGGLGIGTGSGTGGRTGYIPLGGRTNTIVDVSPAKPPVVIEPVGPTD PSIVTLVEDSSVITSGAPAPTFTGTSGFEISTSSTTTPAVLDITPTSSVQ ISSSSFINPAFTDPSVIEVPQTGEISGNILISTPTSGAHGYEEIPMQTFA TEGTGLEPISSTPNPTVRRVAGPRLYSRANQQVRVSDANFLTRPSTFVTY DNPAYDPIDTTLTFDPSSEVPDPDFMDIVRLHRPALTSRRSTVRFSRLGQ RATMFTRSGKQIGARVHFYHDISPIPHAENIELQPLVSSQAATDDIYDIY ADITDEAPTSTANTAFTIPKSSFQSLSLTRSASSTESNVTVPLATAWDVP VNTGPDIVLPNTNIVGPTYSTTPFTTIQSINIEGTNYFLWPIYYFLPRKR KRVPYFFTDGSMAF aa.17-37ofHPVtype59L2 SEQIDNO.3 LYKTCKQAGTCPSDVINKVEG aa.17-32ofHPVtype59L2 SEQIDNO.4 LYKTCKQAGTCPSDVI aa.16-35ofHPVtype59L2 SEQIDNO.5 DLYKTCKQAGTCPSDVINKV aa.16-37ofHPVtype59L2 SEQIDNO.6 DLYKTCKQAGTCPSDVINKVEG 18L1DE.sub.137-138/59dES SEQIDNO.7 MALWRPSDNTVYLPPPSVARVVNTDDYVTRTSIFYHAGSSRLLTVGNPYFRVPAGGGNKQ DIPKVSAYQYRVFRVQLPDPNKFGLPDTSIYNPETQRLVWACAGVEIGRGQPLGVGLSGH PFYNKLDDTESSHAATSLYKTCKQAGTCPSDVINVSEDVRDNVSVDYKQTQLCILGCAPA IGEHWAKGTACKSRPLSQGDCPPLELKNTVLEDGDMVDTGYGAMDFSTLQDTKCEVPLDI CQSICKYPDYLQMSADPYGDSMFFCLRREQLFARHFWNRAGTMGDTVPQSLYIKGTGMRA SPGSCVYSPSPSGSIVTSDSQLFNKPYWLHKAQGHNNGVCWHNQLFVTVVDTTRSTNLTI CASTQSPVPGQYDATKFKQYSRHVEEYDLQFIFQLCTITLTADVMSYIHSMNSSILEDWN FGVPPPPTTSLVDTYRFVQSVAITCQKDAAPAENKDPYDKLKFWNVDLKEKFSLDLDQYP LGRKFLVQAGLRRKPTIGPRKRSAPSATTSSKPAKRVRVRARK 18L1?CDE.sub.137-138/59dES SEQIDNo.8 MALWRPSDNTVYLPPPSVARVVNTDDYVTRTSIFYHAGSSRLLTVGNPYFRVPAGGGNKQ DIPKVSAYQYRVFRVQLPDPNKFGLPDTSIYNPETQRLVWACAGVEIGRGQPLGVGLSGH PFYNKLDDTESSHAATSLYKTCKQAGTCPSDVINVSEDVRDNVSVDYKQTQLCILGCAPA IGEHWAKGTACKSRPLSQGDCPPLELKNTVLEDGDMVDTGYGAMDFSTLQDTKCEVPLDI CQSICKYPDYLQMSADPYGDSMFFCLRREQLFARHFWNRAGTMGDTVPQSLYIKGTGMRA SPGSCVYSPSPSGSIVTSDSQLFNKPYWLHKAQGHNNGVCWHNQLFVTVVDTTRSTNLTI CASTQSPVPGQYDATKFKQYSRHVEEYDLQFIFQLCTITLTADVMSYIHSMNSSILEDWN FGVPPPPTTSLVDTYRFVQSVAITCQKDAAPAENKDPYDKLKFWNVDLKEKFSLDLDQYP LGRKFLVQAGL 18L1DE.sub.137-138/59dE SEQIDNo.9 MALWRPSDNTVYLPPPSVARVVNTDDYVTRTSIFYHAGSSRLLTVGNPYFRVPAGGGNKQ DIPKVSAYQYRVFRVQLPDPNKFGLPDTSIYNPETQRLVWACAGVEIGRGQPLGVGLSGH PFYNKLDDTESSHAATSDLYKTCKQAGTCPSDVINKVNVSEDVRDNVSVDYKQTQLCILG CAPAIGEHWAKGTACKSRPLSQGDCPPLELKNTVLEDGDMVDTGYGAMDFSTLQDTKCEV PLDICQSICKYPDYLQMSADPYGDSMFFCLRREQLFARHFWNRAGTMGDTVPQSLYIKGT GMRASPGSCVYSPSPSGSIVTSDSQLFNKPYWLHKAQGHNNGVCWHNQLFVTVVDTTRST NLTICASTQSPVPGQYDATKFKQYSRHVEEYDLQFIFQLCTITLTADVMSYIHSMNSSIL EDWNFGVPPPPTTSLVDTYRFVQSVAITCQKDAAPAENKDPYDKLKFWNVDLKEKFSLDL DQYPLGRKFLVQAGLRRKPTIGPRKRSAPSATTSSKPAKRVRVRARK 18L1?CDE.sub.137-138/59dE SEQIDNo.10 MALWRPSDNTVYLPPPSVARVVNTDDYVTRTSIFYHAGSSRLLTVGNPYFRVPAGGGNKQ DIPKVSAYQYRVFRVQLPDPNKFGLPDTSIYNPETQRLVWACAGVEIGRGQPLGVGLSGH PFYNKLDDTESSHAATSDLYKTCKQAGTCPSDVINKVNVSEDVRDNVSVDYKQTQLCILG CAPAIGEHWAKGTACKSRPLSQGDCPPLELKNTVLEDGDMVDTGYGAMDFSTLQDTKCEV PLDICQSICKYPDYLQMSADPYGDSMFFCLRREQLFARHFWNRAGTMGDTVPQSLYIKGT GMRASPGSCVYSPSPSGSIVTSDSQLFNKPYWLHKAQGHNNGVCWHNQLFVTVVDTTRST NLTICASTQSPVPGQYDATKFKQYSRHVEEYDLQFIFQLCTITLTADVMSYIHSMNSSIL EDWNFGVPPPPTTSLVDTYRFVQSVAITCQKDAAPAENKDPYDKLKFWNVDLKEKFSLDL DQYPLGRKFLVQAGL 18L1h4.sub.432-433/59dE SEQIDNo.11 MALWRPSDNTVYLPPPSVARVVNTDDYVTRTSIFYHAGSSRLLTVGNPYF RVPAGGGNKQDIPKVSAYQYRVFRVQLPDPNKFGLPDTSIYNPETQRLVW ACAGVEIGRGQPLGVGLSGHPFYNKLDDTESSHAATSNVSEDVRDNVSVD YKQTQLCILGCAPAIGEHWAKGTACKSRPLSQGDCPPLELKNTVLEDGDM VDTGYGAMDFSTLQDTKCEVPLDICQSICKYPDYLQMSADPYGDSMFFCL RREQLFARHFWNRAGTMGDTVPQSLYIKGTGMRASPGSCVYSPSPSGSIV TSDSQLFNKPYWLHKAQGHNNGVCWHNQLFVTVVDTTRSTNLTICASTQS PVPGQYDATKFKQYSRHVEEYDLQFIFQLCTITLTADVMSYIHSMNSSIL EDWNFGVPPPPTTSLVDTYRFVQSVAITCQKDLYKTCKQAGTCPSDVINK VEGAAPAENKDPYDKLKFWNVDLKEKFSLDLDQYPLGRKFLVQAGLRRKP TIGPRKRSAPSATTSSKPAKRVRVRARK 18L1?Ch4.sub.432-433/59dE SEQIDNo.12 MALWRPSDNTVYLPPPSVARVVNTDDYVTRTSIFYHAGSSRLLTVGNPYF RVPAGGGNKQDIPKVSAYQYRVFRVQLPDPNKFGLPDTSIYNPETQRLVW ACAGVEIGRGQPLGVGLSGHPFYNKLDDTESSHAATSNVSEDVRDNVSVD YKQTQLCILGCAPAIGEHWAKGTACKSRPLSQGDCPPLELKNTVLEDGDM VDTGYGAMDFSTLQDTKCEVPLDICQSICKYPDYLQMSADPYGDSMFFCL RREQLFARHFWNRAGTMGDTVPQSLYIKGTGMRASPGSCVYSPSPSGSIV TSDSQLFNKPYWLHKAQGHNNGVCWHNQLFVTVVDTTRSTNLTICASTQS PVPGQYDATKFKQYSRHVEEYDLQFIFQLCTITLTADVMSYIHSMNSSIL EDWNFGVPPPPTTSLVDTYRFVQSVAITCQKDLYKTCKQAGTCPSDVINK VEGAAPAENKDPYDKLKFWNVDLKEKFSLDLDQYPLGRKFLVQAGL 18L1h4.sub.434-435/59dE SEQIDNo.13 MALWRPSDNTVYLPPPSVARVVNTDDYVTRTSIFYHAGSSRLLTVGNPYF RVPAGGGNKQDIPKVSAYQYRVFRVQLPDPNKFGLPDTSIYNPETQRLVW ACAGVEIGRGQPLGVGLSGHPFYNKLDDTESSHAATSNVSEDVRDNVSVD YKQTQLCILGCAPAIGEHWAKGTACKSRPLSQGDCPPLELKNTVLEDGDM VDTGYGAMDFSTLQDTKCEVPLDICQSICKYPDYLQMSADPYGDSMFFCL RREQLFARHFWNRAGTMGDTVPQSLYIKGTGMRASPGSCVYSPSPSGSIV TSDSQLFNKPYWLHKAQGHNNGVCWHNQLFVTVVDTTRSTNLTICASTQS PVPGQYDATKFKQYSRHVEEYDLQFIFQLCTITLTADVMSYIHSMNSSIL EDWNFGVPPPPTTSLVDTYRFVQSVAITCQKDAALYKTCKQAGTCPSDVI NKVEGPAENKDPYDKLKFWNVDLKEKFSLDLDQYPLGRKFLVQAGLRRKP TIGPRKRSAPSATTSSKPAKRVRVRARK 18L1?Ch4.sub.434-435/59dE SEQIDNo.14 MALWRPSDNTVYLPPPSVARVVNTDDYVTRTSIFYHAGSSRLLTVGNPYF RVPAGGGNKQDIPKVSAYQYRVFRVQLPDPNKFGLPDTSIYNPETQRLVW ACAGVEIGRGQPLGVGLSGHPFYNKLDDTESSHAATSNVSEDVRDNVSVD YKQTQLCILGCAPAIGEHWAKGTACKSRPLSQGDCPPLELKNTVLEDGDM VDTGYGAMDFSTLQDTKCEVPLDICQSICKYPDYLQMSADPYGDSMFFCL RREQLFARHFWNRAGTMGDTVPQSLYIKGTGMRASPGSCVYSPSPSGSIV TSDSQLFNKPYWLHKAQGHNNGVCWHNQLFVTVVDTTRSTNLTICASTQS PVPGQYDATKFKQYSRHVEEYDLQFIFQLCTITLTADVMSYIHSMNSSIL EDWNFGVPPPPTTSLVDTYRFVQSVAITCQKDAALYKTCKQAGTCPSDVI NKVEGPAENKDPYDKLKFWNVDLKEKFSLDLDQYPLGRKFLVQAGL 18L1DE.sub.134-135/59dES SEQIDNo.15 MALWRPSDNTVYLPPPSVARVVNTDDYVTRTSIFYHAGSSRLLTVGNPYFRVPAGGGNKQ DIPKVSAYQYRVFRVQLPDPNKFGLPDTSIYNPETQRLVWACAGVEIGRGQPLGVGLSGH PFYNKLDDTESSHAGPLYKTCKQAGTCPSDVIPATSNVSEDVRDNVSVDYKQTQLCILGC APAIGEHWAKGTACKSRPLSQGDCPPLELKNTVLEDGDMVDTGYGAMDFSTLQDTKCEVP LDICQSICKYPDYLQMSADPYGDSMFFCLRREQLFARHFWNRAGTMGDTVPQSLYIKGTG MRASPGSCVYSPSPSGSIVTSDSQLFNKPYWLHKAQGHNNGVCWHNQLFVTVVDTTRSTN LTICASTQSPVPGQYDATKFKQYSRHVEEYDLQFIFQLCTITLTADVMSYIHSMNSSILE DWNFGVPPPPTTSLVDTYRFVQSVAITCQKDAAPAENKDPYDKLKFWNVDLKEKFSLDLD QYPLGRKFLVQAGLRRKPTIGPRKRSAPSATTSSKPAKRVRVRARK 18L1?CDE.sub.134-135/59dES SEQIDNo.16 MALWRPSDNTVYLPPPSVARVVNTDDYVTRTSIFYHAGSSRLLTVGNPYFRVPAGGGNKQ DIPKVSAYQYRVFRVQLPDPNKFGLPDTSIYNPETQRLVWACAGVEIGRGQPLGVGLSGH PFYNKLDDTESSHAGPLYKTCKQAGTCPSDVIPATSNVSEDVRDNVSVDYKQTQLCILGC APAIGEHWAKGTACKSRPLSQGDCPPLELKNTVLEDGDMVDTGYGAMDFSTLQDTKCEVP LDICQSICKYPDYLQMSADPYGDSMFFCLRREQLFARHEWNRAGTMGDTVPQSLYIKGTG MRASPGSCVYSPSPSGSIVTSDSQLFNKPYWLHKAQGHNNGVCWHNQLFVTVVDTTRSTN LTICASTQSPVPGQYDATKFKQYSRHVEEYDLQFIFQLCTITLTADVMSYIHSMNSSILE DWNFGVPPPPTTSLVDTYRFVQSVAITCQKDAAPAENKDPYDKLKFWNVDLKEKFSLDLD QYPLGRKFLVQAGL 18L1DE.sub.134-135/59dE SEQIDNo.17 MALWRPSDNTVYLPPPSVARVVNTDDYVTRTSIFYHAGSSRLLTVGNPYFRVPAGGGNKQ DIPKVSAYQYRVFRVQLPDPNKFGLPDTSIYNPETQRLVWACAGVEIGRGQPLGVGLSGH PFYNKLDDTESSHAGPDLYKTCKQAGTCPSDVINKVEGPATSNVSEDVRDNVSVDYKQTQ LCILGCAPAIGEHWAKGTACKSRPLSQGDCPPLELKNTVLEDGDMVDTGYGAMDFSTLQD TKCEVPLDICQSICKYPDYLQMSADPYGDSMFFCLRREQLFARHFWNRAGTMGDTVPQSL YIKGTGMRASPGSCVYSPSPSGSIVTSDSQLFNKPYWLHKAQGHNNGVCWHNQLFVTVVD TTRSTNLTICASTQSPVPGQYDATKFKQYSRHVEEYDLQFIFQLCTITLTADVMSYIHSM NSSILEDWNFGVPPPPTTSLVDTYRFVQSVAITCQKDAAPAENKDPYDKLKFWNVDLKEK FSLDLDQYPLGRKFLVQAGLRRKPTIGPRKRSAPSATTSSKPAKRVRVRARK 18L1?CDE.sub.134-135/59dE SEQIDNo.18 MALWRPSDNTVYLPPPSVARVVNTDDYVTRTSIFYHAGSSRLLTVGNPYFRVPAGGGNKQ DIPKVSAYQYRVFRVQLPDPNKFGLPDTSIYNPETQRLVWACAGVEIGRGQPLGVGLSGH PFYNKLDDTESSHAGPDLYKTCKQAGTCPSDVINKVEGPATSNVSEDVRDNVSVDYKQTQ LCILGCAPAIGEHWAKGTACKSRPLSQGDCPPLELKNTVLEDGDMVDTGYGAMDFSTLQD TKCEVPLDICQSICKYPDYLQMSADPYGDSMFFCLRREQLFARHFWNRAGTMGDTVPQSL YIKGTGMRASPGSCVYSPSPSGSIVTSDSQLFNKPYWLHKAQGHNNGVCWHNQLFVTVVD TTRSTNLTICASTQSPVPGQYDATKFKQYSRHVEEYDLQFIFQLCTITLTADVMSYIHSM NSSILEDWNFGVPPPPTTSLVDTYRFVQSVAITCQKDAAPAENKDPYDKLKFWNVDLKEK FSLDLDQYPLGRKFLVQAGL 18L1DE.sub.131-138/59dE SEQIDNo.19 MALWRPSDNTVYLPPPSVARVVNTDDYVTRTSIFYHAGSSRLLTVGNPYFRVPAGGGNKQ DIPKVSAYQYRVFRVQLPDPNKFGLPDTSIYNPETQRLVWACAGVEIGRGQPLGVGLSGH PFYNKLDDTESGPLYKTCKQAGTCPSDVINKVEGNVSEDVRDNVSVDYKQTQLCILGCAP AIGEHWAKGTACKSRPLSQGDCPPLELKNTVLEDGDMVDTGYGAMDFSTLQDTKCEVPLD ICQSICKYPDYLQMSADPYGDSMFFCLRREQLFARHFWNRAGTMGDTVPQSLYIKGTGMR ASPGSCVYSPSPSGSIVTSDSQLFNKPYWLHKAQGHNNGVCWHNQLFVTVVDTTRSTNLT ICASTQSPVPGQYDATKFKQYSRHVEEYDLQFIFQLCTITLTADVMSYIHSMNSSILEDW NFGVPPPPTTSLVDTYRFVQSVAITCQKDAAPAENKDPYDKLKFWNVDLKEKFSLDLDQY PLGRKFLVQAGLRRKPTIGPRKRSAPSATTSSKPAKRVRVRARK 18L1?CDE.sub.131-138/59dE SEQIDNo.20 MALWRPSDNTVYLPPPSVARVVNTDDYVTRTSIFYHAGSSRLLTVGNPYFRVPAGGGNKQ DIPKVSAYQYRVFRVQLPDPNKFGLPDTSIYNPETQRLVWACAGVEIGRGQPLGVGLSGH PFYNKLDDTESGPLYKTCKQAGTCPSDVINKVEGNVSEDVRDNVSVDYKQTQLCILGCAP AIGEHWAKGTACKSRPLSQGDCPPLELKNTVLEDGDMVDTGYGAMDFSTLQDTKCEVPLD ICQSICKYPDYLQMSADPYGDSMFFCLRREQLFARHFWNRAGTMGDTVPQSLYIKGTGMR ASPGSCVYSPSPSGSIVTSDSQLFNKPYWLHKAQGHNNGVCWHNQLFVTVVDTTRSTNLT ICASTQSPVPGQYDATKFKQYSRHVEEYDLQFIFQLCTITLTADVMSYIHSMNSSILEDW NFGVPPPPTTSLVDTYRFVQSVAITCQKDAAPAENKDPYDKLKFWNVDLKEKFSLDLDQY PLGRKFLVQAGL 18L1DE.sub.121-124/59dE SEQIDNo.21 MALWRPSDNTVYLPPPSVARVVNTDDYVTRTSIFYHAGSSRLLTVGNPYF RVPAGGGNKQDIPKVSAYQYRVFRVQLPDPNKFGLPDTSIYNPETQRLVW ACAGVEIGRGQPLGVGLSGHPLYKTCKQAGTCPSDVINKVEGNKLDDTES SHAATSNVSEDVRDNVSVDYKQTQLCILGCAPAIGEHWAKGTACKSRPLS QGDCPPLELKNTVLEDGDMVDTGYGAMDFSTLQDTKCEVPLDICQSICKY PDYLQMSADPYGDSMFFCLRREQLFARHFWNRAGTMGDTVPQSLYIKGTG MRASPGSCVYSPSPSGSIVTSDSQLFNKPYWLHKAQGHNNGVCWHNQLFV TVVDTTRSTNLTICASTQSPVPGQYDATKFKQYSRHVEEYDLQFIFQLCT ITLTADVMSYIHSMNSSILEDWNFGVPPPPTTSLVDTYRFVQSVAITCQK DAAPAENKDPYDKLKFWNVDLKEKFSLDLDQYPLGRKFLVQAGLRRKPTI GPRKRSAPSATTSSKPAKRVRVRARK 18L1?CDE.sub.121-124/59dE SEQIDNo.22 MALWRPSDNTVYLPPPSVARVVNTDDYVTRTSIFYHAGSSRLLTVGNPYF RVPAGGGNKQDIPKVSAYQYRVFRVQLPDPNKFGLPDTSIYNPETQRLVW ACAGVEIGRGQPLGVGLSGHPLYKTCKQAGTCPSDVINKVEGNKLDDTES SHAATSNVSEDVRDNVSVDYKQTQLCILGCAPAIGEHWAKGTACKSRPLS QGDCPPLELKNTVLEDGDMVDTGYGAMDFSTLQDTKCEVPLDICQSICKY PDYLQMSADPYGDSMFFCLRREQLFARHFWNRAGTMGDTVPQSLYIKGTG MRASPGSCVYSPSPSGSIVTSDSQLFNKPYWLHKAQGHNNGVCWHNQLFV TVVDTTRSTNLTICASTQSPVPGQYDATKFKQYSRHVEEYDLQFIFQLCT ITLTADVMSYIHSMNSSILEDWNFGVPPPPTTSLVDTYRFVQSVAITCQK DAAPAENKDPYDKLKFWNVDLKEKFSLDLDQYPLGRKFLVQAGL 18L1h4.sub.431-433/59dE SEQIDNo.23 MALWRPSDNTVYLPPPSVARVVNTDDYVTRTSIFYHAGSSRLLTVGNPYF RVPAGGGNKQDIPKVSAYQYRVFRVQLPDPNKFGLPDTSIYNPETQRLVW ACAGVEIGRGQPLGVGLSGHPFYNKLDDTESSHAATSNVSEDVRDNVSVD YKQTQLCILGCAPAIGEHWAKGTACKSRPLSQGDCPPLELKNTVLEDGDM VDTGYGAMDFSTLQDTKCEVPLDICQSICKYPDYLQMSADPYGDSMFFCL RREQLFARHFWNRAGTMGDTVPQSLYIKGTGMRASPGSCVYSPSPSGSIV TSDSQLFNKPYWLHKAQGHNNGVCWHNQLFVTVVDTTRSTNLTICASTQS PVPGQYDATKFKQYSRHVEEYDLQFIFQLCTITLTADVMSYIHSMNSSIL EDWNFGVPPPPTTSLVDTYRFVQSVAITCQKLYKTCKQAGTCPSDVINKV EGAAPAENKDPYDKLKFWNVDLKEKFSLDLDQYPLGRKFLVQAGLRRKPT IGPRKRSAPSATTSSKPAKRVRVRARK 18L1?Ch4.sub.431-433/59dE SEQIDNo.24 MALWRPSDNTVYLPPPSVARVVNTDDYVTRTSIFYHAGSSRLLTVGNPYF RVPAGGGNKQDIPKVSAYQYRVFRVQLPDPNKFGLPDTSIYNPETQRLVW ACAGVEIGRGQPLGVGLSGHPFYNKLDDTESSHAATSNVSEDVRDNVSVD YKQTQLCILGCAPAIGEHWAKGTACKSRPLSQGDCPPLELKNTVLEDGDM VDTGYGAMDFSTLQDTKCEVPLDICQSICKYPDYLQMSADPYGDSMFFCL RREQLFARHFWNRAGTMGDTVPQSLYIKGTGMRASPGSCVYSPSPSGSIV TSDSQLFNKPYWLHKAQGHNNGVCWHNQLFVTVVDTTRSTNLTICASTQS PVPGQYDATKFKQYSRHVEEYDLQFIFQLCTITLTADVMSYIHSMNSSIL EDWNFGVPPPPTTSLVDTYRFVQSVAITCQKLYKTCKQAGTCPSDVINKV EGAAPAENKDPYDKLKFWNVDLKEKFSLDLDQYPLGRKFLVQAGL 18L1h4.sub.432-435/59dE SEQIDNo.25 MALWRPSDNTVYLPPPSVARVVNTDDYVTRTSIFYHAGSSRLLTVGNPYF RVPAGGGNKQDIPKVSAYQYRVFRVQLPDPNKFGLPDTSIYNPETQRLVW ACAGVEIGRGQPLGVGLSGHPFYNKLDDTESSHAATSNVSEDVRDNVSVD YKQTQLCILGCAPAIGEHWAKGTACKSRPLSQGDCPPLELKNTVLEDGDM VDTGYGAMDFSTLQDTKCEVPLDICQSICKYPDYLQMSADPYGDSMFFCL RREQLFARHFWNRAGTMGDTVPQSLYIKGTGMRASPGSCVYSPSPSGSIV TSDSQLFNKPYWLHKAQGHNNGVCWHNQLFVTVVDTTRSTNLTICASTQS PVPGQYDATKFKQYSRHVEEYDLQFIFQLCTITLTADVMSYIHSMNSSIL EDWNFGVPPPPTTSLVDTYRFVQSVAITCQKDLYKTCKQAGTCPSDVINK VEGPAENKDPYDKLKFWNVDLKEKFSLDLDQYPLGRKFLVQAGLRRKPTI GPRKRSAPSATTSSKPAKRVRVRARK 18L1?Ch4.sub.432-435/59dE SEQIDNo.26 MALWRPSDNTVYLPPPSVARVVNTDDYVTRTSIFYHAGSSRLLTVGNPYF RVPAGGGNKQDIPKVSAYQYRVFRVQLPDPNKFGLPDTSIYNPETQRLVW ACAGVEIGRGQPLGVGLSGHPFYNKLDDTESSHAATSNVSEDVRDNVSVD YKQTQLCILGCAPAIGEHWAKGTACKSRPLSQGDCPPLELKNTVLEDGDM VDTGYGAMDFSTLQDTKCEVPLDICQSICKYPDYLQMSADPYGDSMFFCL RREQLFARHFWNRAGTMGDTVPQSLYIKGTGMRASPGSCVYSPSPSGSIV TSDSQLFNKPYWLHKAQGHNNGVCWHNQLFVTVVDTTRSTNLTICASTQS PVPGQYDATKFKQYSRHVEEYDLQFIFQLCTITLTADVMSYIHSMNSSIL EDWNFGVPPPPTTSLVDTYRFVQSVAITCQKDLYKTCKQAGTCPSDVINK VEGPAENKDPYDKLKFWNVDLKEKFSLDLDQYPLGRKFLVQAGL 18L1DE.sub.137-138/59dES-mut1 SEQIDNo.27 MALWRPSDNTVYLPPPSVARVVNTDDYVTRTSIFYHAGSSRLLTVGNPYF RVPAGGGNKQDIPKVSAYQYRVFRVQLPDPNKFGLPDTSIYNPETQRLVW ACAGVEIGRGQPLGVGLSGHPFYNKLDDTESSHAATSLYKTCKQAGTCPS DVINVSEDVRDNVSVDYKQTQLCILGCAPAIGEHWAKGTACKSRPLSQGD CPPLELKNTVLEDGDMVDTGYGAMDFSTLQDTKCEVPLDICQSICKYPDY LQMSADPYGDSMFFCLRREQLFARHFWNRAGTMGDTVPQSLYIKGTGMRA SPGSCVYSPSPSGSIVTSDSQLFNKPYWLHKAQGHNNGVCWHNQLFVTVV DTTRSTNLTICASTQSPVPGQYDATKFKQYSRHVEEYDLQFIFQLCTITL TADVMSYIHSMNSSILEDWNFGVPPPPTTSLVDTYRFVQSVAITCQKDAA PAENKDPYDKLKFWNVDLKEKFSLDLDQYPLGRKFLVQAGLRGGPTIGPG SRSAPSATTSSGPAGSVSVGAGK 18L1DE.sub.137-138/59dES-mut2 SEQIDNo.28 MALWRPSDNTVYLPPPSVARVVNTDDYVTRTSIFYHAGSSRLLTVGNPYF RVPAGGGNKQDIPKVSAYQYRVFRVQLPDPNKFGLPDTSIYNPETQRLVW ACAGVEIGRGQPLGVGLSGHPFYNKLDDTESSHAATSLYKTCKQAGTCPS DVINVSEDVRDNVSVDYKQTQLCILGCAPAIGEHWAKGTACKSRPLSQGD CPPLELKNTVLEDGDMVDTGYGAMDFSTLQDTKCEVPLDICQSICKYPDY LQMSADPYGDSMFFCLRREQLFARHFWNRAGTMGDTVPQSLYIKGTGMRA SPGSCVYSPSPSGSIVTSDSQLFNKPYWLHKAQGHNNGVCWHNQLFVTVV DTTRSTNLTICASTQSPVPGQYDATKFKQYSRHVEEYDLQFIFQLCTITL TADVMSYIHSMNSSILEDWNFGVPPPPTTSLVDTYRFVQSVAITCQKDAA PAENKDPYDKLKFWNVDLKEKFSLDLDQYPLGRKFLVQAGLRGGPTIGPR GSSAPSATTSSGPAGSVSVGAGK 18L1DE.sub.137-138/59dES-mut3 SEQIDNo.29 MALWRPSDNTVYLPPPSVARVVNTDDYVTRTSIFYHAGSSRLLTVGNPYFRVPAGGGNKQ DIPKVSAYQYRVFRVQLPDPNKFGLPDTSIYNPETQRLVWACAGVEIGRGQPLGVGLSGH PFYNKLDDTESSHAATSLYKTCKQAGTCPSDVINVSEDVRDNVSVDYKQTQLCILGCAPA IGEHWAKGTACKSRPLSQGDCPPLELKNTVLEDGDMVDTGYGAMDFSTLQDTKCEVPLDI CQSICKYPDYLQMSADPYGDSMFFCLRREQLFARHFWNRAGTMGDTVPQSLYIKGTGMRA SPGSCVYSPSPSGSIVTSDSQLFNKPYWLHKAQGHNNGVCWHNQLFVTVVDTTRSTNLTI CASTQSPVPGQYDATKFKQYSRHVEEYDLQFIFQLCTITLTADVMSYIHSMNSSILEDWN FGVPPPPTTSLVDTYRFVQSVAITCQKDAAPAENKDPYDKLKFWNVDLKEKFSLDLDQYP LGRKFLVQAGLRGGPTIGPGSRSAPSATTSSGPAGSVGVDAGK 18L1DE.sub.137-138/59dES-mut4 SEQIDNo.30 MALWRPSDNTVYLPPPSVARVVNTDDYVTRTSIFYHAGSSRLLTVGNPYFRVPAGGGNKQ DIPKVSAYQYRVFRVQLPDPNKFGLPDTSIYNPETQRLVWACAGVEIGRGQPLGVGLSGH PFYNKLDDTESSHAATSLYKTCKQAGTCPSDVINVSEDVRDNVSVDYKQTQLCILGCAPA IGEHWAKGTACKSRPLSQGDCPPLELKNTVLEDGDMVDTGYGAMDFSTLQDTKCEVPLDI CQSICKYPDYLQMSADPYGDSMFFCLRREQLFARHFWNRAGTMGDTVPQSLYIKGTGMRA SPGSCVYSPSPSGSIVTSDSQLFNKPYWLHKAQGHNNGVCWHNQLFVTVVDTTRSTNLTI CASTQSPVPGQYDATKFKQYSRHVEEYDLQFIFQLCTITLTADVMSYIHSMNSSILEDWN FGVPPPPTTSLVDTYRFVQSVAITCQKDAAPAENKDPYDKLKFWNVDLKEKFSLDLDQYP LGRKFLVQAGLRGGPTIGPRGSSAPSATTSSGPADSVGVDAGK 18L1DE.sub.137-138/59dES-mut5 SEQIDNo.31 MALWRPSDNTVYLPPPSVARVVNTDDYVTRTSIFYHAGSSRLLTVGNPYFRVPAGGGNKQ DIPKVSAYQYRVFRVQLPDPNKFGLPDTSIYNPETQRLVWACAGVEIGRGQPLGVGLSGH PFYNKLDDTESSHAATSLYKTCKQAGTCPSDVINVSEDVRDNVSVDYKQTQLCILGCAPA IGEHWAKGTACKSRPLSQGDCPPLELKNTVLEDGDMVDTGYGAMDFSTLQDTKCEVPLDI CQSICKYPDYLQMSADPYGDSMFFCLRREQLFARHFWNRAGTMGDTVPQSLYIKGTGMRA SPGSCVYSPSPSGSIVTSDSQLFNKPYWLHKAQGHNNGVCWHNQLFVTVVDTTRSTNLTI CASTQSPVPGQYDATKFKQYSRHVEEYDLQFIFQLCTITLTADVMSYIHSMNSSILEDWN FGVPPPPTTSLVDTYRFVQSVAITCQKDAAPAENKDPYDKLKFWNVDLKEKFSLDLDQYP LGRKFLVQAGLRGKPTIGPGSRSAPSATTSSGPAGSVSVGAGK 18L1DE.sub.137-138/59dES-mut6 SEQIDNo.32 MALWRPSDNTVYLPPPSVARVVNTDDYVTRTSIFYHAGSSRLLTVGNPYFRVPAGGGNKQ DIPKVSAYQYRVFRVQLPDPNKFGLPDTSIYNPETQRLVWACAGVEIGRGQPLGVGLSGH PFYNKLDDTESSHAATSLYKTCKQAGTCPSDVINVSEDVRDNVSVDYKQTQLCILGCAPA IGEHWAKGTACKSRPLSQGDCPPLELKNTVLEDGDMVDTGYGAMDFSTLQDTKCEVPLDI CQSICKYPDYLQMSADPYGDSMFFCLRREQLFARHFWNRAGTMGDTVPQSLYIKGTGMRA SPGSCVYSPSPSGSIVTSDSQLFNKPYWLHKAQGHNNGVCWHNQLFVTVVDTTRSTNLTI CASTQSPVPGQYDATKFKQYSRHVEEYDLQFIFQLCTITLTADVMSYIHSMNSSILEDWN FGVPPPPTTSLVDTYRFVQSVAITCQKDAAPAENKDPYDKLKFWNVDLKEKFSLDLDQYP LGRKFLVQAGLRGKPTIGPRGSSAPSATTSSGPAGSVSVGAGK
[0106] The nucleotide sequences encoding the chimeric proteins of the present invention were shown as follows:
TABLE-US-00004 18L1?CDE.sub.137-138/59dESnt SEQIDNo.33 ATGGCTCTCTGGAGACCCTCCGATAACACAGTGTAC TTGCCCCCCCCCAGCGTCGCCCGCGTCGTGAACACAGACGACTAC GTCACCAGGACCTCAATCTTCTACCACGCCGGTTCAAGCCGCCTG CTGACCGTCGGCAACCCCTACTTCCGCGTCCCCGCCGGTGGCGGT AACAAACAAGACATCCCCAAAGTCAGCGCCTATCAGTACCGCGTG TTCCGCGTCCAACTGCCCGATCCCAACAAGTTCGGCCTGCCCGAC ACCTCCATCTACAACCCCGAGACCCAGAGGCTGGTCTGGGCATGC GCCGGCGTCGAGATCGGTAGGGGCCAACCCCTGGGCGTCGGTTTG TCCGGCCACCCCTTCTACAACAAGCTGGACGATACCGAGTCCTCC CACGCAGCAACCAGCCTGTACAAGACCTGCAAGCAGGCCGGTACC TGCCCCTCCGACGTCATCAACGTCAGCGAAGATGTCCGCGATAAC GTCAGCGTGGACTACAAACAAACCCAACTGTGCATCCTCGGTTGC GCACCCGCCATCGGCGAGCATTGGGCCAAGGGTACCGCCTGCAAG AGCAGGCCCCTGAGCCAAGGTGACTGTCCACCCCTGGAGTTGAAG AATACCGTCCTCGAGGACGGCGACATGGTGGACACCGGCTACGGC GCAATGGATTTCTCCACCCTGCAGGACACCAAGTGCGAAGTGCCC CTCGACATCTGCCAAAGCATCTGCAAGTACCCCGACTACCTGCAG ATGAGCGCCGACCCCTACGGCGACTCCATGTTCTTCTGTCTGAGA AGGGAACAATTGTTCGCCCGCCACTTCTGGAACCGCGCCGGCACC ATGGGCGATACCGTCCCCCAGTCCCTGTACATCAAGGGTACCGGC ATGAGGGCCAGCCCCGGTTCATGCGTCTACAGCCCAAGCCCCTCC GGTAGCATCGTCACAAGCGATTCCCAACTCTTCAACAAGCCCTAC TGGCTGCACAAAGCCCAAGGCCACAATAACGGCGTCTGTTGGCAC AACCAGCTGTTCGTCACCGTCGTGGACACAACCAGGTCCACAAAC CTGACCATCTGCGCCAGCACCCAAAGCCCCGTGCCCGGCCAGTAC GACGCCACAAAGTTCAAACAATACTCACGCCACGTCGAAGAGTAC GACCTCCAATTCATCTTCCAACTCTGCACCATCACCCTGACCGCC GACGTCATGTCCTACATCCACTCCATGAACTCATCCATCCTGGAA GACTGGAATTTCGGCGTCCCACCACCCCCCACCACCTCCCTCGTC GACACCTACAGGTTCGTGCAGAGCGTCGCCATCACATGCCAGAAA GACGCCGCCCCCGCCGAGAACAAAGACCCATACGACAAACTGAAA TTCTGGAACGTCGACCTGAAAGAGAAATTCAGCCTGGATCTGGAC CAGTACCCATTGGGCAGGAAGTTCCTCGTCCAGGCGGGTCTCTAA T 18L1?CDE.sub.137-138/59dEnt SEQIDNo.34 ATGGCTCTCTGGAGACCCTCCGATAACACAGTGTACTTG CCCCCCCCCAGCGTCGCCCGCGTCGTGAACACAGACGACTACGTCACC AGGACCTCAATCTTCTACCACGCCGGTTCAAGCCGCCTGCTGACCGTC GGCAACCCCTACTTCCGCGTCCCCGCCGGTGGCGGTAACAAACAAGAC ATCCCCAAAGTCAGCGCCTATCAGTACCGCGTGTTCCGCGTCCAACTG CCCGATCCCAACAAGTTCGGCCTGCCCGACACCTCCATCTACAACCCC GAGACCCAGAGGCTGGTCTGGGCATGCGCCGGCGTCGAGATCGGTAGG GGCCAACCCCTGGGCGTCGGTTTGTCCGGCCACCCCTTCTACAACAAG CTGGACGATACCGAGTCCTCCCACGCAGCAACCAGCGACCTGTACAAG ACCTGCAAGCAGGCCGGTACCTGCCCCTCCGACGTCATCAACAAGGTC AACGTCAGCGAAGATGTCCGCGATAACGTCAGCGTGGACTACAAACAA ACCCAACTGTGCATCCTCGGTTGCGCACCCGCCATCGGCGAGCATTGG GCCAAGGGTACCGCCTGCAAGAGCAGGCCCCTGAGCCAAGGTGACTGT CCACCCCTGGAGTTGAAGAATACCGTCCTCGAGGACGGCGACATGGTG GACACCGGCTACGGCGCAATGGATTTCTCCACCCTGCAGGACACCAAG TGCGAAGTGCCCCTCGACATCTGCCAAAGCATCTGCAAGTACCCCGAC TACCTGCAGATGAGCGCCGACCCCTACGGCGACTCCATGTTCTTCTGT CTGAGAAGGGAACAATTGTTCGCCCGCCACTTCTGGAACCGCGCCGGC ACCATGGGCGATACCGTCCCCCAGTCCCTGTACATCAAGGGTACCGGC ATGAGGGCCAGCCCCGGTTCATGCGTCTACAGCCCAAGCCCCTCCGGT AGCATCGTCACAAGCGATTCCCAACTCTTCAACAAGCCCTACTGGCTG CACAAAGCCCAAGGCCACAATAACGGCGTCTGTTGGCACAACCAGCTG TTCGTCACCGTCGTGGACACAACCAGGTCCACAAACCTGACCATCTGC GCCAGCACCCAAAGCCCCGTGCCCGGCCAGTACGACGCCACAAAGTTC AAACAATACTCACGCCACGTCGAAGAGTACGACCTCCAATTCATCTTC CAACTCTGCACCATCACCCTGACCGCCGACGTCATGTCCTACATCCAC TCCATGAACTCATCCATCCTGGAAGACTGGAATTTCGGCGTCCCACCA CCCCCCACCACCTCCCTCGTCGACACCTACAGGTTCGTGCAGAGCGTC GCCATCACATGCCAGAAAGACGCCGCCCCCGCCGAGAACAAAGACCCA TACGACAAACTGAAATTCTGGAACGTCGACCTGAAAGAGAAATTCAGC CTGGATCTGGACCAGTACCCATTGGGCAGGAAGTTCCTCGTCCAGGCG GGTCTCTAAT 18L1?CDE.sub.134-135/59dESnt SEQIDNo.35 ATGGCTCTCTGGAGACCCTCCGATAACACAGTGTAC TTGCCCCCCCCCAGCGTCGCCCGCGTCGTGAACACAGACGACTAC GTCACCAGGACCTCAATCTTCTACCACGCCGGTTCAAGCCGCCTG CTGACCGTCGGCAACCCCTACTTCCGCGTCCCCGCCGGTGGCGGT AACAAACAAGACATCCCCAAAGTCAGCGCCTATCAGTACCGCGTG TTCCGCGTCCAACTGCCCGATCCCAACAAGTTCGGCCTGCCCGAC ACCTCCATCTACAACCCCGAGACCCAGAGGCTGGTCTGGGCATGC GCCGGCGTCGAGATCGGTAGGGGCCAACCCCTGGGCGTCGGTTTG TCCGGCCACCCCTTCTACAACAAGCTGGACGATACCGAGTCCTCC CACGCAGGACCACTGTACAAGACCTGCAAGCAGGCCGGTACCTGC CCCTCCGACGTCATCCCAGCAACCAGCAACGTCAGCGAAGATGTC CGCGATAACGTCAGCGTGGACTACAAACAAACCCAACTGTGCATC CTCGGTTGCGCACCCGCCATCGGCGAGCATTGGGCCAAGGGTACC GCCTGCAAGAGCAGGCCCCTGAGCCAAGGTGACTGTCCACCCCTG GAGTTGAAGAATACCGTCCTCGAGGACGGCGACATGGTGGACACC GGCTACGGCGCAATGGATTTCTCCACCCTGCAGGACACCAAGTGC GAAGTGCCCCTCGACATCTGCCAAAGCATCTGCAAGTACCCCGAC TACCTGCAGATGAGCGCCGACCCCTACGGCGACTCCATGTTCTTC TGTCTGAGAAGGGAACAATTGTTCGCCCGCCACTTCTGGAACCGC GCCGGCACCATGGGCGATACCGTCCCCCAGTCCCTGTACATCAAG GGTACCGGCATGAGGGCCAGCCCCGGTTCATGCGTCTACAGCCCA AGCCCCTCCGGTAGCATCGTCACAAGCGATTCCCAACTCTTCAAC AAGCCCTACTGGCTGCACAAAGCCCAAGGCCACAATAACGGCGTC TGTTGGCACAACCAGCTGTTCGTCACCGTCGTGGACACAACCAGG TCCACAAACCTGACCATCTGCGCCAGCACCCAAAGCCCCGTGCCC GGCCAGTACGACGCCACAAAGTTCAAACAATACTCACGCCACGTC GAAGAGTACGACCTCCAATTCATCTTCCAACTCTGCACCATCACC CTGACCGCCGACGTCATGTCCTACATCCACTCCATGAACTCATCC ATCCTGGAAGACTGGAATTTCGGCGTCCCACCACCCCCCACCACC TCCCTCGTCGACACCTACAGGTTCGTGCAGAGCGTCGCCATCACA TGCCAGAAAGACGCCGCCCCCGCCGAGAACAAAGACCCATACGAC AAACTGAAATTCTGGAACGTCGACCTGAAAGAGAAATTCAGCCTG GATCTGGACCAGTACCCATTGGGCAGGAAGTTCCTCGTCCAGGCG GGTCTCTAAT 18L1?CDE.sub.134-135/59dEnt SEQIDNO.36 ATGGCTCTCTGGAGACCCTCCGATAACACAGTGTACTTG CCCCCCCCCAGCGTCGCCCGCGTCGTGAACACAGACGACTACGTCACC AGGACCTCAATCTTCTACCACGCCGGTTCAAGCCGCCTGCTGACCGTC GGCAACCCCTACTTCCGCGTCCCCGCCGGTGGCGGTAACAAACAAGAC ATCCCCAAAGTCAGCGCCTATCAGTACCGCGTGTTCCGCGTCCAACTG CCCGATCCCAACAAGTTCGGCCTGCCCGACACCTCCATCTACAACCCC GAGACCCAGAGGCTGGTCTGGGCATGCGCCGGCGTCGAGATCGGTAGG GGCCAACCCCTGGGCGTCGGTTTGTCCGGCCACCCCTTCTACAACAGG CTGGACGATACCGAGTCCTCCCACGCAGGACCAGACCTGTACAAGACC TGCAAGCAGGCCGGTACCTGCCCCTCCGACGTCATCAACAAGGTCGAA GGACCAGCAACCAGCAACGTCAGCGAAGATGTCCGCGATAACGTCAGC GTGGACTACAAACAAACCCAACTGTGCATCCTCGGTTGCGCACCCGCC ATCGGCGAGCATTGGGCCAAGGGTACCGCCTGCAAGAGCAGGCCCCTG AGCCAAGGTGACTGTCCACCCCTGGAGTTGAAGAATACCGTCCTCGAG GACGGCGACATGGTGGACACCGGCTACGGCGCAATGGATTTCTCCACC CTGCAGGACACCAAGTGCGAAGTGCCCCTCGACATCTGCCAAAGCATC TGCAAGTACCCCGACTACCTGCAGATGAGCGCCGACCCCTACGGCGAC TCCATGTTCTTCTGTCTGAGAAGGGAACAATTGTTCGCCCGCCACTTC TGGAACCGCGCCGGCACCATGGGCGATACCGTCCCCCAGTCCCTGTAC ATCAAGGGTACCGGCATGAGGGCCAGCCCCGGTTCATGCGTCTACAGC CCAAGCCCCTCCGGTAGCATCGTCACAAGCGATTCCCAACTCTTCAAC AAGCCCTACTGGCTGCACAAAGCCCAAGGCCACAATAACGGCGTCTGT TGGCACAACCAGCTGTTCGTCACCGTCGTGGACACAACCAGGTCCACA AACCTGACCATCTGCGCCAGCACCCAAAGCCCCGTGCCCGGCCAGTAC GACGCCACAAAGTTCAAACAATACTCACGCCACGTCGAAGAGTACGAC CTCCAATTCATCTTCCAACTCTGCACCATCACCCTGACCGCCGACGTC ATGTCCTACATCCACTCCATGAACTCATCCATCCTGGAAGACTGGAAT TTCGGTGTCCCACCACCCCCCACCACCTCCCTCGTCGACACCTACAGG TTCGTGCAGAGCGTCGCCATCACATGCCAGAAAGACGCCGCCCCCGCC GAGAACAAAGACCCATACGACAAACTGAAATTCTGGAACGTCGACCTG AAAGAGAAATTCAGCCTGGATCTGGACCAGTACCCATTGGGCAGGAAG TTCCTCGTCCAGGCGGGTCTCTAAT 15L1?CDE.sub.131-138/59dEnt SEQIDNo.37 ATGGCTCTCTGGAGACCCTCCGATAACACAGTGTACTTGCCCCCCCCC AGCGTCGCCCGCGTCGTGAACACAGACGACTACGTCACCAGGACCTCA ATCTTCTACCACGCCGGTTCAAGCCGCCTGCTGACCGTCGGCAACCCC TACTTCCGCGTCCCCGCCGGTGGCGGTAACAAACAAGACATCCCCAAA GTCAGCGCCTATCAGTACCGCGTGTTCCGCGTCCAACTGCCCGATCCC AACAAGTTCGGCCTGCCCGACACCTCCATCTACAACCCCGAGACCCAG AGGCTGGTCTGGGCATGCGCCGGCGTCGAGATCGGTAGGGGCCAACCC CTGGGCGTCGGTTTGTCCGGCCACCCCTTCTACAACAAGCTGGACGAT ACCGAGTCCGGTCCCCTGTACAAGACCTGCAAGCAGGCCGGTACCTGC CCCTCCGACGTCATCAACAAGGTCGAAGGAAACGTCAGCGAAGATGTC CGCGATAACGTCAGCGTGGACTACAAACAAACCCAACTGTGCATCCTC GGTTGCGCACCCGCCATCGGCGAGCATTGGGCCAAGGGTACCGCCTGC AAGAGCAGGCCCCTGAGCCAAGGTGACTGTCCACCCCTGGAGTTGAAG AATACCGTCCTCGAGGACGGCGACATGGTGGACACCGGCTACGGCGCA ATGGATTTCTCCACCCTGCAGGACACCAAGTGCGAAGTGCCCCTCGAC ATCTGCCAAAGCATCTGCAAGTACCCCGACTACCTGCAGATGAGCGCC GACCCCTACGGCGACTCCATGTTCTTCTGTCTGAGAAGGGAACAATTG TTCGCCCGCCACTTCTGGAACCGCGCCGGCACCATGGGCGATACCGTC CCCCAGTCCCTGTACATCAAGGGTACCGGCATGAGGGCCAGCCCCGGT TCATGCGTCTACAGCCCAAGCCCCTCCGGTAGCATCGTCACAAGCGAT TCCCAACTCTTCAACAAGCCCTACTGGCTGCACAAAGCCCAAGGCCAC AATAACGGCGTCTGTTGGCACAACCAGCTGTTCGTCACCGTCGTGGAC ACAACCAGGTCCACAAACCTGACCATCTGCGCCAGCACCCAAAGCCCC GTGCCCGGCCAGTACGACGCCACAAAGTTCAAACAATACTCACGCCAC GTCGAAGAGTACGACCTCCAATTCATCTTCCAACTCTGCACCATCACC CTGACCGCCGACGTCATGTCCTACATCCACTCCATGAACTCATCCATC CTGGAAGACTGGAATTTCGGCGTCCCACCACCCCCCACCACCTCCCTC GTCGACACCTACAGGTTCGTGCAGAGCGTCGCCATCACATGCCAGAAA GACGCCGCCCCCGCCGAGAACAAAGACCCATACGACAAACTGAAATTC TGGAACGTCGACCTGAAAGAGAAATTCAGCCTGGATCTGGACCAGTAC CCATTGGGCAGGAAGTTCCTCGTCCAGGCGGGTCTCTAAT 18L1DE.sub.137-138/59dES-mut1nt SEQIDNo.38 ATGGCTCTCTGGAGACCCTCCGATAACACAGTGTACTTGCCCCCCCCCAGCGTCGCCCGC GTCGTGAACACAGACGACTACGTCACCAGGACCTCAATCTTCTACCACGCCGGTTCAAGC CGCCTGCTGACCGTCGGCAACCCCTACTTCCGCGTCCCCGCCGGTGGCGGTAACAAACAA GACATCCCCAAAGTCAGCGCCTATCAGTACCGCGTGTTCCGCGTCCAACTGCCCGATCCC AACAAGTTCGGCCTGCCCGACACCTCCATCTACAACCCCGAGACCCAGAGGCTGGTCTGG GCATGCGCCGGCGTCGAGATCGGTAGGGGCCAACCCCTGGGCGTCGGTTTGTCCGGCCAC CCCTTCTACAACAAGCTGGACGATACCGAGTCCTCCCACGCAGCAACCAGCCTGTACAAG ACCTGCAAGCAGGCCGGTACCTGCCCCTCCGACGTCATCAACGTCAGCGAAGATGTCCGC GATAACGTCAGCGTGGACTACAAACAAACCCAACTGTGCATCCTCGGTTGCGCACCCGCC ATCGGCGAGCATTGGGCCAAGGGTACCGCCTGCAAGAGCAGGCCCCTGAGCCAAGGTGAC TGTCCACCCCTGGAGTTGAAGAATACCGTCCTCGAGGACGGCGACATGGTGGACACCGGC TACGGCGCAATGGATTTCTCCACCCTGCAGGACACCAAGTGCGAAGTGCCCCTCGACATC TGCCAAAGCATCTGCAAGTACCCCGACTACCTGCAGATGAGCGCCGACCCCTACGGCGAC TCCATGTTCTTCTGTCTGAGAAGGGAACAATTGTTCGCCCGCCACTTCTGGAACCGCGCC GGCACCATGGGCGATACCGTCCCCCAGTCCCTGTACATCAAGGGTACCGGCATGAGGGCC AGCCCCGGTTCATGCGTCTACAGCCCAAGCCCCTCCGGTAGCATCGTCACAAGCGATTCC CAACTCTTCAACAAGCCCTACTGGCTGCACAAAGCCCAAGGCCACAATAACGGCGTCTGT TGGCACAACCAGCTGTTCGTCACCGTCGTGGACACAACCAGGTCCACAAACCTGACCATC TGCGCCAGCACCCAAAGCCCCGTGCCCGGCCAGTACGACGCCACAAAGTTCAAACAATAC TCACGCCACGTCGAAGAGTACGACCTCCAATTCATCTTCCAACTCTGCACCATCACCCTG ACCGCCGACGTCATGTCCTACATCCACTCCATGAACTCATCCATCCTGGAAGACTGGAAT TTCGGCGTCCCACCACCCCCCACCACCTCCCTCGTCGACACCTACAGGTTCGTGCAGAGC GTCGCCATCACATGCCAGAAAGACGCCGCCCCCGCCGAGAACAAAGACCCATACGACAAA CTGAAATTCTGGAACGTCGACCTGAAAGAGAAATTCAGCCTGGATCTGGACCAGTACCCA TTGGGCAGGAAGTTCCTCGTCCAGGCGGGTCTCCGTGGCGGTCCGACGATTGGCCCTGGC TCTCGTTCTGCCCCGTCGGCCACGACCAGCAGCGGCCCTGCCGGTAGCGTGAGCGTGGGC GCTGGCAAATAAT 18L1DE.sub.137-138/dES-mut2nt SEQIDNo.39 ATGGCTCTCTGGAGACCCTCCGATAACACAGTGTACTTGCCCCCCCCCAGCGTCGCCCGC GTCGTGAACACAGACGACTACGTCACCAGGACCTCAATCTTCTACCACGCCGGTTCAAGC CGCCTGCTGACCGTCGGCAACCCCTACTTCCGCGTCCCCGCCGGTGGCGGTAACAAACAA GACATCCCCAAAGTCAGCGCCTATCAGTACCGCGTGTTCCGCGTCCAACTGCCCGATCCC AACAAGTTCGGCCTGCCCGACACCTCCATCTACAACCCCGAGACCCAGAGGCTGGTCTGG GCATGCGCCGGCGTCGAGATCGGTAGGGGCCAACCCCTGGGCGTCGGTTTGTCCGGCCAC CCCTTCTACAACAAGCTGGACGATACCGAGTCCTCCCACGCAGCAACCAGCCTGTACAAG ACCTGCAAGCAGGCCGGTACCTGCCCCTCCGACGTCATCAACGTCAGCGAAGATGTCCGC GATAACGTCAGCGTGGACTACAAACAAACCCAACTGTGCATCCTCGGTTGCGCACCCGCC ATCGGCGAGCATTGGGCCAAGGGTACCGCCTGCAAGAGCAGGCCCCTGAGCCAAGGTGAC TGTCCACCCCTGGAGTTGAAGAATACCGTCCTCGAGGACGGCGACATGGTGGACACCGGC TACGGCGCAATGGATTTCTCCACCCTGCAGGACACCAAGTGCGAAGTGCCCCTCGACATC TGCCAAAGCATCTGCAAGTACCCCGACTACCTGCAGATGAGCGCCGACCCCTACGGCGAC TCCATGTTCTTCTGTCTGAGAAGGGAACAATTGTTCGCCCGCCACTTCTGGAACCGCGCC GGCACCATGGGCGATACCGTCCCCCAGTCCCTGTACATCAAGGGTACCGGCATGAGGGCC AGCCCCGGTTCATGCGTCTACAGCCCAAGCCCCTCCGGTAGCATCGTCACAAGCGATTCC CAACTCTTCAACAAGCCCTACTGGCTGCACAAAGCCCAAGGCCACAATAACGGCGTCTGT TGGCACAACCAGCTGTTCGTCACCGTCGTGGACACAACCAGGTCCACAAACCTGACCATC TGCGCCAGCACCCAAAGCCCCGTGCCCGGCCAGTACGACGCCACAAAGTTCAAACAATAC TCACGCCACGTCGAAGAGTACGACCTCCAATTCATCTTCCAACTCTGCACCATCACCCTG ACCGCCGACGTCATGTCCTACATCCACTCCATGAACTCATCCATCCTGGAAGACTGGAAT TTCGGCGTCCCACCACCCCCCACCACCTCCCTCGTCGACACCTACAGGTTCGTGCAGAGC GTCGCCATCACATGCCAGAAAGACGCCGCCCCCGCCGAGAACAAAGACCCATACGACAAA CTGAAATTCTGGAACGTCGACCTGAAAGAGAAATTCAGCCTGGATCTGGACCAGTACCCA TTGGGCAGGAAGTTCCTCGTCCAGGCGGGTCTCCGTGGCGGTCCGACGATTGGCCCTCGT GGCTCTTCTGCCCCGTCGGCCACGACCAGCAGCGGCCCTGCCGGTAGCGTGAGCGTGGGC GCTGGCAAATAAT 18L1DE.sub.137-138/59dES-mut3nt SEQIDNo.40 ATGGCTCTCTGGAGACCCTCCGATAACACAGTGTACTTGCCCCCCCCCAGCGTCGCCCGC GTCGTGAACACAGACGACTACGTCACCAGGACCTCAATCTTCTACCACGCCGGTTCAAGC CGCCTGCTGACCGTCGGCAACCCCTACTTCCGCGTCCCCGCCGGTGGCGGTAACAAACAA GACATCCCCAAAGTCAGCGCCTATCAGTACCGCGTGTTCCGCGTCCAACTGCCCGATCCC AACAAGTTCGGCCTGCCCGACACCTCCATCTACAACCCCGAGACCCAGAGGCTGGTCTGG GCATGCGCCGGCGTCGAGATCGGTAGGGGCCAACCCCTGGGCGTCGGTTTGTCCGGCCAC CCCTTCTACAACAAGCTGGACGATACCGAGTCCTCCCACGCAGCAACCAGCCTGTACAAG ACCTGCAAGCAGGCCGGTACCTGCCCCTCCGACGTCATCAACGTCAGCGAAGATGTCCGC GATAACGTCAGCGTGGACTACAAACAAACCCAACTGTGCATCCTCGGTTGCGCACCCGCC ATCGGCGAGCATTGGGCCAAGGGTACCGCCTGCAAGAGCAGGCCCCTGAGCCAAGGTGAC TGTCCACCCCTGGAGTTGAAGAATACCGTCCTCGAGGACGGCGACATGGTGGACACCGGC TACGGCGCAATGGATTTCTCCACCCTGCAGGACACCAAGTGCGAAGTGCCCCTCGACATC TGCCAAAGCATCTGCAAGTACCCCGACTACCTGCAGATGAGCGCCGACCCCTACGGCGAC TCCATGTTCTTCTGTCTGAGAAGGGAACAATTGTTCGCCCGCCACTTCTGGAACCGCGCC GGCACCATGGGCGATACCGTCCCCCAGTCCCTGTACATCAAGGGTACCGGCATGAGGGCC AGCCCCGGTTCATGCGTCTACAGCCCAAGCCCCTCCGGTAGCATCGTCACAAGCGATTCC CAACTCTTCAACAAGCCCTACTGGCTGCACAAAGCCCAAGGCCACAATAACGGCGTCTGT TGGCACAACCAGCTGTTCGTCACCGTCGTGGACACAACCAGGTCCACAAACCTGACCATC TGCGCCAGCACCCAAAGCCCCGTGCCCGGCCAGTACGACGCCACAAAGTTCAAACAATAC TCACGCCACGTCGAAGAGTACGACCTCCAATTCATCTTCCAACTCTGCACCATCACCCTG ACCGCCGACGTCATGTCCTACATCCACTCCATGAACTCATCCATCCTGGAAGACTGGAAT TTCGGCGTCCCACCACCCCCCACCACCTCCCTCGTCGACACCTACAGGTTCGTGCAGAGC GTCGCCATCACATGCCAGAAAGACGCCGCCCCCGCCGAGAACAAAGACCCATACGACAAA CTGAAATTCTGGAACGTCGACCTGAAAGAGAAATTCAGCCTGGATCTGGACCAGTACCCA TTGGGCAGGAAGTTCCTCGTCCAGGCGGGTCTCCGTGGCGGTCCGACGATTGGCCCTGGC TCTCGTTCTGCCCCGTCGGCCACGACCAGCAGCGGCCCTGCCGGTAGCGTGGGCGTGGAC GCTGGCAAATAAT 18L1DE.sub.137-138/59dES-mut4nt SEQIDNo.41 ATGGCTCTCTGGAGACCCTCCGATAACACAGTGTACTTGCCCCCCCCCAGCGTCGCCCGC GTCGTGAACACAGACGACTACGTCACCAGGACCTCAATCTTCTACCACGCCGGTTCAAGC CGCCTGCTGACCGTCGGCAACCCCTACTTCCGCGTCCCCGCCGGTGGCGGTAACAAACAA GACATCCCCAAAGTCAGCGCCTATCAGTACCGCGTGTTCCGCGTCCAACTGCCCGATCCC AACAAGTTCGGCCTGCCCGACACCTCCATCTACAACCCCGAGACCCAGAGGCTGGTCTGG GCATGCGCCGGCGTCGAGATCGGTAGGGGCCAACCCCTGGGCGTCGGTTTGTCCGGCCAC CCCTTCTACAACAAGCTGGACGATACCGAGTCCTCCCACGCAGCAACCAGCCTGTACAAG ACCTGCAAGCAGGCCGGTACCTGCCCCTCCGACGTCATCAACGTCAGCGAAGATGTCCGC GATAACGTCAGCGTGGACTACAAACAAACCCAACTGTGCATCCTCGGTTGCGCACCCGCC ATCGGCGAGCATTGGGCCAAGGGTACCGCCTGCAAGAGCAGGCCCCTGAGCCAAGGTGAC TGTCCACCCCTGGAGTTGAAGAATACCGTCCTCGAGGACGGCGACATGGTGGACACCGGC TACGGCGCAATGGATTTCTCCACCCTGCAGGACACCAAGTGCGAAGTGCCCCTCGACATC TGCCAAAGCATCTGCAAGTACCCCGACTACCTGCAGATGAGCGCCGACCCCTACGGCGAC TCCATGTTCTTCTGTCTGAGAAGGGAACAATTGTTCGCCCGCCACTTCTGGAACCGCGCC GGCACCATGGGCGATACCGTCCCCCAGTCCCTGTACATCAAGGGTACCGGCATGAGGGCC AGCCCCGGTTCATGCGTCTACAGCCCAAGCCCCTCCGGTAGCATCGTCACAAGCGATTCC CAACTCTTCAACAAGCCCTACTGGCTGCACAAAGCCCAAGGCCACAATAACGGCGTCTGT TGGCACAACCAGCTGTTCGTCACCGTCGTGGACACAACCAGGTCCACAAACCTGACCATC TGCGCCAGCACCCAAAGCCCCGTGCCCGGCCAGTACGACGCCACAAAGTTCAAACAATAC TCACGCCACGTCGAAGAGTACGACCTCCAATTCATCTTCCAACTCTGCACCATCACCCTG ACCGCCGACGTCATGTCCTACATCCACTCCATGAACTCATCCATCCTGGAAGACTGGAAT TTCGGCGTCCCACCACCCCCCACCACCTCCCTCGTCGACACCTACAGGTTCGTGCAGAGC GTCGCCATCACATGCCAGAAAGACGCCGCCCCCGCCGAGAACAAAGACCCATACGACAAA CTGAAATTCTGGAACGTCGACCTGAAAGAGAAATTCAGCCTGGATCTGGACCAGTACCCA TTGGGCAGGAAGTTCCTCGTCCAGGCGGGTCTCCGTGGCGGTCCGACGATTGGCCCTCGT GGCTCTTCTGCCCCGTCGGCCACGACCAGCAGCGGCCCTGCCGACAGCGTGGGCGTGGAC GCTGGCAAATAAT 18L1DE.sub.137-138/59dES-mut5nt SEQIDNo.42 ATGGCTCTCTGGAGACCCTCCGATAACACAGTGTACTTGCCCCCCCCCAGCGTCGCCCGC GTCGTGAACACAGACGACTACGTCACCAGGACCTCAATCTTCTACCACGCCGGTTCAAGC CGCCTGCTGACCGTCGGCAACCCCTACTTCCGCGTCCCCGCCGGTGGCGGTAACAAACAA GACATCCCCAAAGTCAGCGCCTATCAGTACCGCGTGTTCCGCGTCCAACTGCCCGATCCC AACAAGTTCGGCCTGCCCGACACCTCCATCTACAACCCCGAGACCCAGAGGCTGGTCTGG GCATGCGCCGGCGTCGAGATCGGTAGGGGCCAACCCCTGGGCGTCGGTTTGTCCGGCCAC CCCTTCTACAACAAGCTGGACGATACCGAGTCCTCCCACGCAGCAACCAGCCTGTACAAG ACCTGCAAGCAGGCCGGTACCTGCCCCTCCGACGTCATCAACGTCAGCGAAGATGTCCGC GATAACGTCAGCGTGGACTACAAACAAACCCAACTGTGCATCCTCGGTTGCGCACCCGCC ATCGGCGAGCATTGGGCCAAGGGTACCGCCTGCAAGAGCAGGCCCCTGAGCCAAGGTGAC TGTCCACCCCTGGAGTTGAAGAATACCGTCCTCGAGGACGGCGACATGGTGGACACCGGC TACGGCGCAATGGATTTCTCCACCCTGCAGGACACCAAGTGCGAAGTGCCCCTCGACATC TGCCAAAGCATCTGCAAGTACCCCGACTACCTGCAGATGAGCGCCGACCCCTACGGCGAC TCCATGTTCTTCTGTCTGAGAAGGGAACAATTGTTCGCCCGCCACTTCTGGAACCGCGCC GGCACCATGGGCGATACCGTCCCCCAGTCCCTGTACATCAAGGGTACCGGCATGAGGGCC AGCCCCGGTTCATGCGTCTACAGCCCAAGCCCCTCCGGTAGCATCGTCACAAGCGATTCC CAACTCTTCAACAAGCCCTACTGGCTGCACAAAGCCCAAGGCCACAATAACGGCGTCTGT TGGCACAACCAGCTGTTCGTCACCGTCGTGGACACAACCAGGTCCACAAACCTGACCATC TGCGCCAGCACCCAAAGCCCCGTGCCCGGCCAGTACGACGCCACAAAGTTCAAACAATAC TCACGCCACGTCGAAGAGTACGACCTCCAATTCATCTTCCAACTCTGCACCATCACCCTG ACCGCCGACGTCATGTCCTACATCCACTCCATGAACTCATCCATCCTGGAAGACTGGAAT TTCGGCGTCCCACCACCCCCCACCACCTCCCTCGTCGACACCTACAGGTTCGTGCAGAGC GTCGCCATCACATGCCAGAAAGACGCCGCCCCCGCCGAGAACAAAGACCCATACGACAAA CTGAAATTCTGGAACGTCGACCTGAAAGAGAAATTCAGCCTGGATCTGGACCAGTACCCA TTGGGCAGGAAGTTCCTCGTCCAGGCGGGTCTCCGTGGCAAACCGACGATTGGCCCTGGC TCTCGTTCTGCCCCGTCGGCCACGACCAGCAGCGGCCCTGCCGGTAGCGTGAGCGTGGGC GCTGGCAAATAAT 18L1DE.sub.137-138/59dES-mut6nt SEQIDNo.43 ATGGCTCTCTGGAGACCCTCCGATAACACAGTGTACTTGCCCCCCCCCAGCGTCGCCCGC GTCGTGAACACAGACGACTACGTCACCAGGACCTCAATCTTCTACCACGCCGGTTCAAGC CGCCTGCTGACCGTCGGCAACCCCTACTTCCGCGTCCCCGCCGGTGGCGGTAACAAACAA GACATCCCCAAAGTCAGCGCCTATCAGTACCGCGTGTTCCGCGTCCAACTGCCCGATCCC AACAAGTTCGGCCTGCCCGACACCTCCATCTACAACCCCGAGACCCAGAGGCTGGTCTGG GCATGCGCCGGCGTCGAGATCGGTAGGGGCCAACCCCTGGGCGTCGGTTTGTCCGGCCAC CCCTTCTACAACAAGCTGGACGATACCGAGTCCTCCCACGCAGCAACCAGCCTGTACAAG ACCTGCAAGCAGGCCGGTACCTGCCCCTCCGACGTCATCAACGTCAGCGAAGATGTCCGC GATAACGTCAGCGTGGACTACAAACAAACCCAACTGTGCATCCTCGGTTGCGCACCCGCC ATCGGCGAGCATTGGGCCAAGGGTACCGCCTGCAAGAGCAGGCCCCTGAGCCAAGGTGAC TGTCCACCCCTGGAGTTGAAGAATACCGTCCTCGAGGACGGCGACATGGTGGACACCGGC TACGGCGCAATGGATTTCTCCACCCTGCAGGACACCAAGTGCGAAGTGCCCCTCGACATC TGCCAAAGCATCTGCAAGTACCCCGACTACCTGCAGATGAGCGCCGACCCCTACGGCGAC TCCATGTTCTTCTGTCTGAGAAGGGAACAATTGTTCGCCCGCCACTTCTGGAACCGCGCC GGCACCATGGGCGATACCGTCCCCCAGTCCCTGTACATCAAGGGTACCGGCATGAGGGCC AGCCCCGGTTCATGCGTCTACAGCCCAAGCCCCTCCGGTAGCATCGTCACAAGCGATTCC CAACTCTTCAACAAGCCCTACTGGCTGCACAAAGCCCAAGGCCACAATAACGGCGTCTGT TGGCACAACCAGCTGTTCGTCACCGTCGTGGACACAACCAGGTCCACAAACCTGACCATC TGCGCCAGCACCCAAAGCCCCGTGCCCGGCCAGTACGACGCCACAAAGTTCAAACAATAC TCACGCCACGTCGAAGAGTACGACCTCCAATTCATCTTCCAACTCTGCACCATCACCCTG ACCGCCGACGTCATGTCCTACATCCACTCCATGAACTCATCCATCCTGGAAGACTGGAAT TTCGGCGTCCCACCACCCCCCACCACCTCCCTCGTCGACACCTACAGGTTCGTGCAGAGC GTCGCCATCACATGCCAGAAAGACGCCGCCCCCGCCGAGAACAAAGACCCATACGACAAA CTGAAATTCTGGAACGTCGACCTGAAAGAGAAATTCAGCCTGGATCTGGACCAGTACCCA TTGGGCAGGAAGTTCCTCGTCCAGGCGGGTCTCCGTGGCAAACCGACGATTGGCCCTCGT GGCTCTTCTGCCCCGTCGGCCACGACCAGCAGCGGCCCTGCCGGTAGCGTGAGCGTGGGC GCTGGCAAATAAT
Example 3: Construction of Recombinant Bacmids and Recombinant Baculoviruses of the Chimeric L1 Protein Genes
[0107] Recombinant expression vectors comprising the chimeric L1 genes, namely pFastBac1-18L1?CDE.sub.137-138/59dE, pFastBac1-18L1?Ch4.sub.434-435/59dE, pFastBac1-18L1?CDE.sub.134-135/59dE, pFastBac1-18L1?CDE.sub.121-124/59dE, pFastBac1-18L1?Ch4.sub.432-435/59dE, pFastBac1-18L1DE.sub.137-138/59dES-mut1, pFastBac1-18L1DE.sub.137-138/59dES-mut2, pFastBac1-18L1DE.sub.137-138/59dES-mut3, pFastBac1-18L1DE.sub.137-138/59dES-mut4, pFastBac1-18L1DE.sub.137-138/59dES-mut5, pFastBac1-18L1DE.sub.137-138/59dES-mut6, 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 pFastBac1-18L1?CDE.sub.137-138/59dES, pFastBac1-18L?Ch4.sub.432-433/59dE, pFastBac1-18L1?CDE.sub.134-135/59dES, pFastBac1-18L1?CDE.sub.131-138/59dE, pFastBac1-18L1?Ch4.sub.431-433/59dE, 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 4: Expression of Chimeric L1 Protein Genes in Sf9 Cells
[0108] Sf9 cells were inoculated with the recombinant baculoviruses of the 16 chimeric L1 genes to express the chimeric L1 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 5: Expression of Chimeric L1 Protein Genes in E. coli
[0109] Recombinant expression vectors comprising the chimeric L1 genes, namely pET22b-18L1DE.sub.137-138/59dES, pET22b-18L1DE.sub.137-138/59dE, pET22b-18L1h4.sub.432-433/59dE, pET22b-18L1h4.sub.434-435/59dE, pET22b-18L1DE.sub.134-135/59dES, pET22b-18L1DE.sub.134-135/59dE, pET22b-18L1DE.sub.131-138/59dE, pET22b-18L1DE.sub.121-124/59dE, pET22b-18L1h4.sub.431-433/59dE, pET22b-18L1h4.sub.432-435/59dE, pET22b-18L1DE.sub.137-138/59dES-mut1, pET22b-18L1DE.sub.137-138/59dES-mut2, pET22b-18L1DE.sub.137-138/59dES-mut3, pET22b-18L1DE.sub.137-138/59dES-mut4, pET22b-18L1DE.sub.137-138/59dES-mut5, pET22b-18L1DE.sub.137-138/59dES-mut6, were used to transform E. coli BL21 (DE3).
[0110] Single clones were picked and inoculated into 3 ml of LB medium containing ampicillin and incubated at 37? C. overnight. The bacterial fluid cultured overnight was added to LB medium at a ratio of 1:100 and incubated at 37? C. for about 3 h. When the OD600 reached between 0.8-1.0, IPTG was added to a final concentration of 0.5 ?M, and the bacterial fluid was incubated at 16? C. for about 12 h and collected.
Example 6: Identification of the Expression of the Chimeric L1 Proteins
[0111] 1?10.sup.6 cells expressing the different chimeric L1 proteins described in Examples 4 and 5 respectively were collected and resuspended in 200 ?l of PBS solution. 50 ?l of 6? loading 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 7: Comparison of Expression Amount of Chimeric L1 Proteins in Insect Cells
[0112] 1?10.sup.6 cells expressing the 18L1 backbone protein with 32-amino acid truncation at C-terminus, as well as cells expressing the chimeric L1 proteins with the 18L1 with 32-amino acid truncation at C-terminus as backbone, or with the 6 types of 18L1 mutants as backbone, respectively, were collected and resuspended in 200 ?l of 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.
[0113] Microtiter plates were coated with HPV18 L1 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 3 times with PBST. The lysed supernatant was subjected to 2-fold serial dilution with PBS. The HPV18L1 VLP standard was also subjected to serial dilution from a concentration of 2 ?g/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 3 times with PBST, and 1:3000 diluted HPV18L1 rabbit polyclonal antibody was added at 100 ?l per well and incubated at 37? C. for 1 h. The plate was washed 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 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 HPV18L1 protein and 18L1 chimeric proteins in the lysed supernatant were calculated according to the standard curve.
[0114] The results were as shown in Table 3. The expression amounts of 18L1?CDE.sub.134-135/59dES, 18L1?CDE.sub.137-138/59dES, 18L1?Ch4.sub.431-433/59dE and 18L1?Ch4.sub.432-435/59dE of the present invention were very high, comparable to that of the HPV18L1 backbone. In addition, the expression amounts of the chimeric proteins with the 18L1 mutants with amino acid substitutions at C-terminus as backbone, namely 18L1DE.sub.137-138/59dES-mut1, 18L1DE.sub.137-138/59dES-mut4, 18L1DE.sub.137-138/59dES-mut5, were all higher than that of the HPV18L1 backbone and the corresponding C-terminal truncated chimeric protein.
TABLE-US-00005 TABLE 3 Analysis of expression amounts of chimeric L1 proteins Expression amount (mg/L) Protein name Batch 1 Batch 2 Batch 3 Average HPV18L1 55 52 59 55.3 18L1?CDE.sub.137-138/59dES 59 51 54 54.7 18L1?CDE.sub.137-138/59dE 43 41 46 43.3 18L1?Ch4.sub.432-433/59dE 29 29 30 29.3 18L1?Ch4.sub.434-435/59dE 46 44 44 44.7 18L1?CDE.sub.134-135/59dES 56 58 53 55.7 18L1?CDE.sub.134-135/59dE 43 43 41 42.3 18L1?CDE.sub.131-138/59dE 41 39 40 40 18L1?CDE.sub.121-124/59dE 45 42 39 42 18L1?Ch4.sub.431-433/59dE 52 59 57 56 18L1?Ch4.sub.432-435/59dE 54 55 51 53.3 18L1DE.sub.137-138/59dES-mut1 75 72 77 74.7 18L1DE.sub.137-138/59dES-mut2 42 39 35 38.7 18L1DE.sub.137-138/59dES-mut3 32 28 36 32 18L1DE.sub.137-138/59dES-mut4 78 80 76 78 18L1DE.sub.137-138/59dES-mut5 81 79 83 81 18L1DE.sub.137-138/59dES-mut6 50 55 52 52.3
Example 8: Purification and Dynamic Light Scattering Particle Size Analysis of the Chimeric L1 Proteins
[0115] An appropriate amount of cell fermentation broth of chimeric L1 was collected and the cells were resuspended with 10 ml of 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 sequential 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 CN 101293918 B, CN 1976718 A, etc.
[0116] Severe aggregation was found for 18L1h4.sub.431-433/59dE, 18L1h4.sub.432-435/59dE, 18L1?Ch4.sub.431-433/59dE and 18L1?Ch4.sub.432-435/59dE during the assembly of pure chimeric proteins, while aggregation was not observed for other chimeric proteins after assembly. The assembled chimeric protein solutions were subjected to DLS particle size analysis (Zetasizer Nano ZS 90 Dynamic Light Scattering Analyzer, Malvern), and the results were as shown in Table 4. Among them, the DLS analysis plots of 18L1?CDE.sub.134-135/59dE, 18L1?CDE.sub.134-135/59dES, 18L1?CDE.sub.137-138/59dE, 18L1?CDE.sub.137-138/59dES were as shown in
TABLE-US-00006 TABLE 4 DLS analysis of chimeric L1 proteins Protein name Hydraulic diameter (nm) PDI 18L1DE.sub.137-138/59dES 115.2 0.182 18L1DE.sub.137-138/59dE 119.1 0.172 18L1h4.sub.432-433/59dE 125.4 0.163 18L1h4.sub.434-435/59dE 114.2 0.177 18L1DE.sub.134-135/59dES 104.3 0.193 18L1DE.sub.134-135/59dE 106.8 0.169 18L1DE.sub.131-138/59dE 113.3 0.192 18L1DE.sub.121-124/59dE 114.7 0.133 18L1?CDE.sub.137-138/59dES 122.9 0.192 18L1?CDE.sub.137-138/59dE 121.4 0.188 18L1?Ch4.sub.432-433/59dE 107.5 0.179 18L1?Ch4.sub.434-435/59dE 105.2 0.196 18L1?CDE.sub.134-135/59dES 112.4 0.133 18L1?CDE.sub.134-135/59dE 110.5 0.167 18L1?CDE.sub.131-138/59dE 109.2 0.184 18L1?CDE.sub.121-124/59dE 121.7 0.165 18L1DE.sub.137-138/59dES-mut1 120.5 0.178 18L1DE.sub.137-138/59dES-mut2 118.4 0.165 18L1DE.sub.137-138/59dES-mut3 115.7 0.182 18L1DE.sub.137-138/59dES-mut4 119.2 0.133 18L1DE.sub.137-138/59dES-mut5 122.3 0.142 18L1DE.sub.137-138/59dES-mut6 125.6 0.191
Example 9: Transmission Electron Microscopy Observation of Chimeric VLPs
[0117] The chimeric proteins were purified separately according to the chromatographic purification method described in Example 8. The assembled chimeric proteins 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 chimeric proteins expressed by both E. coli and insect cells could be assembled into cVLPs with a diameter of about 50 nm. Among them, the electron microscopy images of 18L1?CDE.sub.134-135/59dE, 18L1?CDE.sub.134-135/59dES, 18L1?CDE.sub.137-138/59dE, 18L1?CDE.sub.137-138/59dES cVLPs were as shown in
Example 10: Immunization of Mice with Chimeric VLPs and Determination of Neutralizing Antibody Titers
[0118] 4-6 weeks old BALB/c mice were randomly divided into groups, 5 mice per group, and 10 ?g cVLP in combination with 50 ?g Al(OH); and 5 ?g MPL adjuvant were used to immunize mice by subcutaneous injection at Weeks 0, 4, 7, 10 for a total of 4 times. Tail vein blood was collected 2 weeks after the 4th immunization and serum was isolated.
[0119] 24 types of HPV pseudoviruses were used to detect the neutralizing antibody titers of the immune serum. The results showed that after immunizing mice with various cVLPs produced by E. coli and insect cell expression systems, the levels and neutralization range of the induced cross-neutralizing antibodies were different with each other. Among them, as shown in Table 5, the antiserum of 18L1?CDE.sub.134-135/59dES and 18L1?CDE.sub.137-138/59dES cVLPs expressed by insect cells could neutralize at least 23 types of pseudoviruses, and the immune serum of 18L1?CDE.sub.134-135/59dE and 18L1?CDE.sub.137-138/59dE cVLPs could neutralize at least 19 types of pseudoviruses. It was worth mentioning that the antiserum of 18L1?CDE.sub.134-135/59dES and 18L1?CDE.sub.137-138/59dES cVLP could neutralize all 6 ?7-HPVs used for detection, in particular for 18L1?CDE.sub.137-138/59dES cVLP, the antibody titers to cross-neutralize ?7-HPVs were all above 250, and it was the cVLP with the strongest cross-neutralization ability against ?7-HPVs reported so far.
[0120] In addition, after immunizing mice with the above strategy, the cVLPs constructed with 18L1 mutants with 32-amino acid truncation at C-terminus in the present invention could also induce high levels of neutralizing antibodies. Among them, the level of each type of neutralizing antibody induced by 18L1DE.sub.137-138/59dES-mut4 was comparable to those induced by 18L1?CDE.sub.137-138/59dES. It was worth noticing that the HPV39 and HPV59 neutralizing antibody titers of 18L1DE.sub.137-138/59dES-mut4 immune serum were both greater than 10.sup.3 (as shown in Table 5 and
[0121] Methods of pseudovirus preparation and pseudoviral neutralization experiments were all publicly available, for example, the patent CN 104418942A.
TABLE-US-00007 TABLE 5 Neutralizing antibody titers induced by different cVLPs in mice 18L1?CDE.sub.134-135/ 18L1?CDE.sub.134-135/ 18L1?CDE.sub.137-138/ 18L1?CDE.sub.137-138/ 59dE 59dES 59dE 59dES Average ?7 HPV 18 213333 230400 640000 204800 titer of subgenus HPV 39 75.3 171 19.3 327 neutralizing HPV 45 34 689 250.5 767 antibodies HPV 59 400.3 166 375 442 HPV 68 ND* 109 25.5 257 HPV 70 109 700 135 770 ?9 HPV 16 ND 346 ND 329 subgenus HPV 31 17.3 48 ND 69 HPV 33 9 97 31.8 105 HPV 35 134 60 31.8 34 HPV 52 9 60 7 60 HPV 58 ND 60 ND 75 ?5 HPV 26 10 34 7 125 subgenus HPV 51 ND ND ND 31 ?6 HPV 53 12 25 5 50 subgenus HPV 56 ND 13 ND ND HPV 66 7 19 4 30 ?11 HPV 73 41.7 182 31.5 137 subgenus ?10 HPV 6 25 31 13 37 subgenus HPV 11 41.7 120 37.5 70 ?4 HPV 2 33.3 130 38 150 subgenus HPV 27 ND 160 25.5 170 HPV 57 83.3 75 17 62 ?1 HPV 5 27 35 7 105 subgenus 18L1DE.sub.137-138/ 18L1?Ch4.sub.432-433/ 18L1?Ch4.sub.434-435/ 59dES- 59dE 59dES mut4 Average ?7 HPV 18 256000 51200 140000 titer of subgenus HPV 39 52 ND 1031 neutralizing HPV 45 77.5 ND 319 antibodies HPV 59 103 80 1425 HPV 68 ND ND 188 HPV 70 50 25 531 ?9 HPV 16 ND ND 319 subgenus HPV 31 ND ND 60 HPV 33 ND ND 125 HPV 35 20 ND 20 HPV 52 ND ND 9 HPV 58 ND ND 48 ?5 HPV 26 ND ND 105 subgenus HPV 51 ND 12 34 ?6 HPV 53 ND ND 50 subgenus HPV 56 ND ND ND HPV 66 ND ND 31.5 ?11 HPV 73 ND ND 150 subgenus ?10 HPV 6 10 ND 31 subgenus HPV 11 9 8 62 ?4 HPV 2 20 ND 170 subgenus HPV 27 ND ND 137 HPV 57 ND 21 60 ?1 HPV 5 13 ND 150 subgenus *ND indicates that no neutralizing antibody was detected at the lowest dilution