Phosphorylated polypeptide antigen vaccine, preparation method therefor and application thereof
12414987 ยท 2025-09-16
Assignee
Inventors
Cpc classification
Y02A50/30
GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
A61K2039/55561
HUMAN NECESSITIES
C07K14/4711
CHEMISTRY; METALLURGY
A61K2039/62
HUMAN NECESSITIES
A61P25/28
HUMAN NECESSITIES
C07K2317/34
CHEMISTRY; METALLURGY
A61K2039/6037
HUMAN NECESSITIES
C07K16/44
CHEMISTRY; METALLURGY
International classification
A61K39/00
HUMAN NECESSITIES
A61P25/28
HUMAN NECESSITIES
Abstract
The present invention discloses a phosphorylated polypeptide antigen vaccine, comprising at least two polypeptide fragments or conservatively modified variants thereof from human full-length Tau protein, wherein the polypeptide fragments or conservatively modified variants thereof contain phosphorylation sites. The present invention also discloses a complex vaccine formed by coupling a phosphorylated polypeptide antigen vaccine with a carrier. The polypeptide antigen vaccine and the complex vaccine can be used for preventing and/or treating tauopathy comprising Alzheimer's disease (AD).
Claims
1. A phosphorylated polypeptide antigen vaccine, which comprises at least two polypeptide fragments or conservatively modified variants thereof from human full-length Tau protein, wherein the polypeptide fragments or conservatively modified variants thereof contain phosphorylation sites, wherein the polypeptide fragments are connected directly by peptide bonds or connected by amino acid linkers.
2. The phosphorylated polypeptide antigen vaccine of claim 1, wherein the polypeptide fragments are connected directly by peptide bonds.
3. The phosphorylated polypeptide antigen vaccine of claim 1, wherein the polypeptide fragments are derived from phosphorylation modification site-rich regions of human full-length Tau protein.
4. The phosphorylated polypeptide antigen vaccine of claim 3, wherein the phosphorylation sites include two or more of phosphorylated amino acid sites corresponding to positions 18, 202, 205, 212, 214, 231, 235, 238, 262, 396 and 404 of the amino acid sequence of human full-length Tau protein, namely, 18(P-Tyr.sub.18), 202(P-Ser.sub.202), 205(P-Thr.sub.205), 212(P-Thr.sub.212), 214(P-Ser.sub.214), 231(P-Thr.sub.231), 235(P-Ser.sub.235), 238(P-Ser.sub.238), 262(P-Ser.sub.262), 396(P-Ser.sub.396) and 404(P-Ser.sub.404).
5. The phosphorylated polypeptide antigen vaccine of claim 1, wherein the conservatively modified variant of the polypeptide fragment is a variant obtained by conservatively substitution of one or more amino acids of the polypeptide fragment with functionally similar amino acids.
6. The phosphorylated polypeptide antigen vaccine of claim 1, which has an amino acid sequence as represented by any one of SEQ ID NOs: 1-1331 and the amino acid sequence contains two or more phosphorylation sites selected from 18(P-Tyr.sub.18), 202(P-Ser.sub.202), 205(P-Thr.sub.205), 212(P-Thr.sub.212), 214(P-Ser.sub.214), 231(P-Thr.sub.231), 235(P-Ser.sub.235), 238(P-Ser.sub.238), 262(P-Ser.sub.262), 396(P-Ser.sub.396) and 404(P-Ser.sub.404).
7. A complex vaccine formed by coupling the phosphorylated polypeptide antigen vaccine of claim 1 with a carrier.
8. The complex vaccine of claim 7, wherein the carrier is selected from the group consisting of human serum albumin, keyhole limpet hemocyanin, bacterium-like particles (BLP), immunoglobulin molecules, thyroglobulin, ovalbumin, bovine serum albumin component V, influenza hemagglutinin, PAN-DR binding peptide (PADRE polypeptide), malaria circumsporozoite (CS) protein, hepatitis B surface antigen (HB.sub.SAg.sub.19-28), Heat Shock Protein (HSP) 65, Bacille Calmette-Gurin (BCG), cholera toxin, attenuated cholera toxin variants, diphtheria toxin, norovirus capsid P protein, recombinant Streptococcus C5a peptidase, Streptococcus pyogenes ORF1224, Streptococcus pyogenes ORF1664, Streptococcus pyogenes ORF2452, pneumolysin, attenuated pneumolysin toxicity variants, Chlamydia pneumoniae ORFT367, Chlamydia pneumoniae ORFT858, Tetanus toxoid, HIV gp120T1, microbial surface components recognizing adhesive matrix molecules (MSCRAMMS), growth factor/hormone and/or chemokines.
9. A method for preparing the complex vaccine of claim 7, comprising the following steps: 1) artificially synthesizing the phosphorylated polypeptide antigen vaccine of claim 1; 2) preparing a carrier to be coupled to the phosphorylated polypeptide antigen vaccine; 3) mixing the phosphorylated polypeptide antigen vaccine with the carrier to perform coupling reaction; and 4) separating and purifying the conjugate obtained in 3), thereby obtaining a complex vaccine.
10. The method of claim 9, wherein the carrier in step 2) is a norovirus capsid P protein, preferably PP-3C or PP-3K, and step 2) specifically comprises: i) obtaining an expression vector comprising a nucleic acid encoding a PP-3C or PP-3K protein; ii) transferring the expression vector into a receptor cell; iii) expressing the PP-3C or PP-3K protein, and allowing it to self-assemble into a recombinant P particle in the receptor cell; preferably, step 2) also comprises isolation and purification steps; and more preferably, ion exchange chromatography and/or hydrophobic chromatography are used for purification.
11. The method of claim 9, wherein in step 3), PP-3C is used as a vaccine carrier for coupling, a preferred buffer system is an ammonium bicarbonate buffer system, and a preferred pH ranges from 7.5 to 8.8; preferably, the phosphorylated polypeptide antigen vaccine and the carrier are mixed in a ratio of 10:1 to 100:1, and a preferred reaction temperature ranges from 2 C. to 10 C.; or in step 3), PP-3K is used as a vaccine carrier for coupling, and a preferred buffer system is a phosphate buffer system, and a preferred pH ranges from 7.0 to 8.5; preferably, the phosphorylated polypeptide antigen vaccine and the carrier are mixed in a ratio of 10:1 to 100:1, and a preferred reaction temperature ranges from 2 C. to 25 C.
12. The method of claim 9, wherein the carrier in step 2) is bacterium-like particles (BLP), and step 3) specifically comprises: i) obtaining a purified protein adaptorC-PA protein with a sequence as represented by SEQ ID NO: 1364; and ii) connecting the carrierbacterium-like particles (BLP)obtained in step 2) with the C-PA protein to obtain C-PA-BLP; and iii) preforming coupling reaction of C-PA-BLP with the phosphorylated polypeptide antigen vaccine; wherein a Tris buffer system is used as a buffer system and a preferred pH ranges from 7.2 to 8.8; a preferred C-PA protein concentration is 0.1 mg/mL to 1.5 mg/mL, and a preferred reaction temperature ranges from 2 C. to 30 C.; or an ammonium bicarbonate buffer system is used as a buffer system, and a preferred pH ranges from 7.5 to 8.8; preferably, the phosphorylated polypeptide antigen vaccine and the C-PA-BLP are mixed in a ratio of 10:1 to 100:1, and a preferred reaction temperature ranges from 2 C. to 10 C.
13. The method of claim 9, wherein step 4) comprises removing coupling agent and polypeptide antigens which are not successfully connected by methods including desalting chromatography, dialysis and ultrafiltration.
14. A vaccine composition, wherein the vaccine composition comprises the phosphorylated polypeptide antigen vaccine of claim 1 or a complex vaccine; preferably, the vaccine composition further comprises a pharmaceutically acceptable adjuvant; more preferably, the pharmaceutically acceptable adjuvant is selected from one or more of CpG, MF59, AS02, AS03, Freund's complete adjuvant and Freund's incomplete adjuvant; wherein the complex vaccine is formed by coupling the phosphorylated polypeptide antigen vaccine of claim 1 with a carrier.
15. Use of the phosphorylated polypeptide antigen vaccine of claim 1 or a complex vaccine or a vaccine composition for preparing a medicament for prevention and/or treatment of neurodegenerative disorders; and/or for preparing a medicament for maintaining or improving, preferably recovering, and more preferably completely recovering the cognitive memory of mammals, especially human beings; wherein the complex vaccine is formed by coupling the phosphorylated polypeptide antigen vaccine of claim 1 with a carrier; wherein the vaccine composition comprises the phosphorylated polypeptide antigen vaccine of claim 1 or the complex vaccine; preferably, the vaccine composition further comprises a pharmaceutically acceptable adjuvant; more preferably, the pharmaceutically acceptable adjuvant is selected from one or more of CpG, MF59, AS02, AS03, Freund's complete adjuvant and Freund's incomplete adjuvant.
16. Use of claim 15, wherein the neurodegenerative disorders are selected from one or more of AD, Creutzfeldt-Jacob Syndrome, Dementia pugilistica, Down's Syndrome, Gerstmann-Strussler-Scheinker disease, inclusion-body myositis, prion protein cerebral amyloid angiopathy, traumatic brain injury, amyotrophic lateral sclerosis/parkinsonism syndrome-dementia syndrome, argyrophilic grain dementia, corticobasal degeneration, diffuse neurofibrillary tangles with calcification, frontotemporal dementia with parkinsonism syndrome linked to chromosome 17, Hallevorden-Spatz disease, multiple system atrophy, Niemann-Pick disease type C, Pick's disease, progressive subcortical gliosis and progressive supranuclear panencephalitis; preferably the neurodegenerative disorder is AD; preferably, the vaccine or vaccine composition is preferably immunized subcutaneously, intraperitoneally or intramuscularly, and more preferably immunized intramuscularly.
17. The phosphorylated polypeptide antigen vaccine of claim 1, wherein the phosphorylated polypeptide antigen vaccine contains an additional cysteine residue at its C-terminal.
18. The phosphorylated polypeptide antigen vaccine of claim 3, wherein the polypeptide fragments are derived from regions of human full-length Tau protein as follows: amino acids at positions 14 to 22 of human full-length Tau protein, amino acids at positions 194 to 266 of human full-length Tau protein, and/or amino acids at positions 392 to 408 of human full-length Tau protein.
19. The phosphorylated polypeptide antigen vaccine of claim 4, wherein the phosphorylation sites include all phosphorylated amino acid sites corresponding to positions 18, 202, 205, 212, 214, 231, 235, 238, 262, 396 and 404 of the amino acid sequence of human full-length Tau protein, namely, 18(P-Tyr.sub.18), 202(P-Ser.sub.202), 205(P-Thr.sub.205), 212(P-Thr.sub.212), 214(P-Ser.sub.214), 231(P-Thr.sub.231), 235(P-Ser.sub.235), 238(P-Ser.sub.238), 262(P-Ser.sub.262), 396(P-Ser.sub.396) and 404(P-Ser.sub.404).
20. The phosphorylated polypeptide antigen vaccine of claim 5, wherein the conservatively modified variant of the polypeptide fragment is a variant obtained by conservatively substitution of 1 to 10 amino acids of the polypeptide fragment with functionally similar amino acids.
21. The phosphorylated polypeptide antigen vaccine of claim 20, wherein the conservatively modified variant of the polypeptide fragment is a variant obtained by conservatively substitution of 1 to 6 amino acids of the polypeptide fragment with functionally similar amino acids.
22. The phosphorylated polypeptide antigen vaccine of claim 21, wherein the conservatively modified variant of the polypeptide fragment is a variant obtained by conservatively substitution of 1 to 4 amino acids of the polypeptide fragment with functionally similar amino acids.
23. The phosphorylated polypeptide antigen vaccine of claim 22, wherein the conservatively modified variant of the polypeptide fragment is a variant obtained by conservatively substitution of 1 to 3 amino acids of the polypeptide fragment with functionally similar amino acids.
24. The phosphorylated polypeptide antigen vaccine of claim 23, wherein the conservatively modified variant of the polypeptide fragment is a variant obtained by conservatively substitution of 1 amino acid of the polypeptide fragment with functionally similar amino acids.
25. The phosphorylated polypeptide antigen vaccine of claim 6, wherein the phosphorylated polypeptide antigen vaccine has an amino acid sequence which has at least 80%, at least 85%, at least 90%, at least 95% sequence identity to any one of SEQ ID NOs: 1-1331.
26. The phosphorylated polypeptide antigen vaccine of claim 25, wherein the phosphorylated polypeptide antigen vaccine has an amino acid sequence which has at least 98% sequence identity to any one of SEQ ID NOs: 1-1331.
27. The phosphorylated polypeptide antigen vaccine of claim 26, wherein the phosphorylated polypeptide antigen vaccine has an amino acid sequence which has at least 99% sequence identity to any one of SEQ ID NOs: 1-1331.
28. The phosphorylated polypeptide antigen vaccine of claim 25, wherein the phosphorylated polypeptide antigen vaccine has an amino acid sequence as represented by any one of SEQ ID NO: 201, SEQ ID NO: 225, SEQ ID NO: 306, SEQ ID NO: 387, SEQ ID NO: 468, SEQ ID NO: 558, SEQ ID NO: 567, SEQ ID NO: 769, SEQ ID NO: 784, SEQ ID NO: 875, SEQ ID NO: 1020, SEQ ID NO: 1101, SEQ ID NO: 1182, SEQ ID NO: 1272, SEQ ID NO: 1313 and SEQ ID NO: 1330, and the amino acid sequence contains two or more phosphorylation sites selected from 18(P-Tyr.sub.18), 202(P-Ser.sub.202), 205(P-Thr.sub.205), 212(P-Thr.sub.212), 214(P-Ser.sub.214), 231(P-Thr.sub.231), 235(P-Ser.sub.235), 238(P-Ser.sub.238), 262(P-Ser.sub.262), 396(P-Ser.sub.396) and 404(P-Ser.sub.404).
29. The phosphorylated polypeptide antigen vaccine of claim 28, wherein the phosphorylated polypeptide antigen vaccine has an amino acid sequence as represented by any one of SEQ ID NO: 201, SEQ ID NO: 225, SEQ ID NO: 306, SEQ ID NO: 387, SEQ ID NO: 468, SEQ ID NO: 558, SEQ ID NO: 567, SEQ ID NO: 769, SEQ ID NO: 784, SEQ ID NO: 875, SEQ ID NO: 1020, SEQ ID NO: 1101, SEQ ID NO: 1182, SEQ ID NO: 1272, SEQ ID NO: 1313 and SEQ ID NO: 1330, and the amino acid sequence respectively contain phosphorylation sites as follows: 18(P-Tyr.sub.18), 202(P-Ser.sub.202), 205(P-Thr.sub.205); 18(P-Tyr.sub.18), 202(P-Ser.sub.202), 205(P-Thr.sub.205); 18(P-Tyr.sub.18), 212(P-Thr.sub.212), 214(P-Ser.sub.214); 18(P-Tyr.sub.18), 231(P-Thr.sub.231), 235(P-Ser.sub.235); 18(P-Tyr.sub.18), 238(P-Ser.sub.238), 262(P-Ser.sub.262); 18(P-Tyr.sub.18), 396(P-Ser.sub.396), 404(P-Ser.sub.404); 202(P-Ser.sub.202), 205(P-Thr.sub.205), 231(P-Thr.sub.231), 235(P-Ser.sub.235); 202(P-Ser.sub.202), 205(P-Thr.sub.205), 231(P-Thr.sub.231), 235(P-Ser.sub.235); 202(P-Ser.sub.202), 205(P-Thr.sub.205), 238(P-Ser.sub.238), 262(P-Ser.sub.262); 202(P-Ser.sub.202), 205(P-Thr.sub.205), 396(P-Ser.sub.396), 404(P-Ser.sub.404); 202(P-Ser.sub.202), 205(P-Thr.sub.205), 396(P-Ser.sub.396), 404(P-Ser.sub.404); 212(P-Thr.sub.212), 214(P-Ser.sub.214), 231(P-Thr.sub.231), 235(P-Ser.sub.235); 212(P-Thr.sub.212), 214(P-Ser.sub.214), 238(P-Ser.sub.238), 262(P-Ser.sub.262); 212(P-Thr.sub.212), 214(P-Ser.sub.214), 396(P-Ser.sub.396), 404(P-Ser.sub.404); 238(P-Ser.sub.238), 262(P-Ser.sub.262), 396(P-Ser.sub.396), 404(P-Ser.sub.404); 238(P-Ser.sub.238), 262(P-Ser.sub.262), 396(P-Ser.sub.396) and 404(P-Ser.sub.404).
30. The complex vaccine of claim 8, wherein the carrier is bacterium-like particles (BLP).
31. The complex vaccine of claim 30, wherein the BLP is coupled with a phosphorylated polypeptide antigen vaccine by means of a protein adaptor.
32. The complex vaccine of claim 31, wherein the protein adaptor has a sequence as represented by SEQ ID NO: 1364.
33. The complex vaccine of claim 8, wherein the carrier is norovirus capsid P protein.
34. The complex vaccine of claim 33, wherein the norovirus capsid P protein is PP-3C with a sequence as represented by SEQ ID NO: 1357 or PP-3K with a sequence as represented by SEQ ID NO: 1359.
Description
BRIEF DESCRIPTION OF DRAWINGS
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DETAILED DESCRIPTION OF THE INVENTION
(16) The content of the present invention is illustrated below through specific embodiments. It should be understood that the specific embodiments are for illustrative purposes only, and do not mean that the content of the present invention is limited to the specific embodiments.
Example 1: Preparation of Phosphorylated Polypeptide Antigen Vaccines
(17) In this example, based on human full-length Tau protein (SEQ ID NO: 1332), the inventor designed the following 15 phosphorylated polypeptide antigen vaccines comprising two polypeptide fragments from the human full-length Tau protein; the specific sequence information of the 15 phosphorylated polypeptide antigen vaccines is shown in Table 1 below:
(18) TABLE-US-00001 TABLE1 Sequenceinformationofthe15phosphorylatedpolypeptideantigenvaccines Thecorrespondingfull-length Sequence Vaccine Tauproteinfragmetsand No. No. Sequences phosphorylationsites SEQID A1 HAGTYGLGDSSPGSP Tau14-22[pY18],Tau198- NO:201 GTPGSRC 209[pS202,pT205] SEQID A2 HAGTYGLGDRSGYSS Tau14-22[pY18],Tau194- NO:225 PGSPGTPGSRSRTPC 213[pS202,pT205] SEQID A3 HAGTYGLGDSRSRTP Tau14-22[pY18],Tau208- NO:306 SLPTPC 218[pT212,pS214] SEQID A4 HAGTYGLGDAVVRTPP Tau14-22[pY18],Tau227- NO:387 KSPSSAC 239[pT231,pS235] SEQID A5 HAGTYGLGDKSPSSA Tau14-22[pY18],Tau234- NO:468 KSRSKIGSTENLC 242[pS238],Tau258- 266[pS262] SEQID A6 HAGTYGLGDIVYKSPV Tau14-22[pY18],Tau392- NO:558 VSGDTSPRHLC 408[pS396,pS404] SEQID A7 SSPGSPGTPGSRAVV Tau198-209[pS202,pT205], NO:567 RTPPKSPSSAC Tau227-239[pT231,pS235] SEQID A8 SGYSSPGSPGTPGSR Tau195-213[pS202,pT205], NO:769 SRTPVVRTPPKSPSSAC Tau228-239[pT231,pS235] SEQID A9 SSPGSPGTPGSRKSP Tau198-209[pS202,pT205], NO:784 SSAKSRSKIGSTENLC Tau234-242[pS238],Tau258- 266[pS262] SEQID A10 SSPGSPGTPGSRIVYK Tau198-209[pS202,pT205], NO:875 SPVVSGDTSPRHLC Tau395-406[pS396,pS404] SEQID A11 SGYSSPGSPGTPGSR Tau195-213[pS202,pT205], NO:1020 SRTPKSPVVSGDTSPRC Tau395-406[PS396,pS404] SEQID A12 SRSRTPSLPTPAVVRT Tau208-218[pT212,pS214], NO:1101 PPKSPSSAC Tau227-239[pT231,pS235] SEQID A13 SRSRTPSLPTPKSPSS Tau208-218[pT212,pS214], NO:1182 AKSRSKIGSTENLC Tau234-242[pS238],Tau258- 266[pS262] SEQID A14 SRSRTPSLPTPIVYKSP Tau208-218[pT212,pS214], NO:1272 VVSGDTSPRHLC Tau392-408[pS396,pS404] SEQID A15 SPSSAKSRSKIGSTEN Tau235-242[pS238],Tau258- NO:1313 VYKSPVVSGDTSPRH 265[pS262],Tau393- C 407[pS396,pS404] SEQID A16 KSPSSAKSRSKIGSTE Tau234-242[pS238],Tau258- NO:1330 NLKSPVVSGDTSPRC 266[pS262],Tau395- 406[pS396,pS404]
(19) The aforementioned 15 phosphorylated polypeptide antigen vaccines were synthesized and prepared by GL biochem (Shanghai) Ltd., and are presented in lyophilized form.
Example 2: Preparation of a Cysteine-Modified Norovirus P Protein (PP-3C)
(20) The preparation of norovirus P protein plasmid using pET26b(+) as a vector has been described in Chinese invention patent application 2015104155561. In this example, three point mutations were performed using this plasmid as a template, one amino acid in each of the three loop structures of the norovirus P particle was mutated to a cysteine, and the protein was expressed and purified.
(21) 2.1 Construction of PP-3C Plasmid
(22) Three rounds of site-directed mutagenesis were performed by site-directed mutation method using the pET26b-P protein plasmid which has already been constructed in Chinese invention patent application 2015104155561. Respectively, 5ATCGCTGGAACACAA3 in loop1 was mutated to 5ATCGCTTGCACACAA3. The specific method was as follows:
(23) TABLE-US-00002 SEQIDNO:1334(forward): CTGTGAACATCGCTACTTTCCGCGGCGACGTCACACACATCGCTTGCAC ACAAAACTAC, and SEQIDNO:1335(reverse): GTAGTTTTGTGTGCAAGCGATGTGTGTGACGTCGCCGCGGAAAGTAGCG ATGTTCACAG;
the entire plasmid was subjected to PCR reaction, wherein the PCR reaction system was a KOD-Plus DNA polymerase system (purchased from TOYOBO Corporation), and the total volume of the reaction system was 50 L (5 L buffer, 0.2 mM dNTP, 1 mM magnesium sulfate, 0.3 M upstream primer and 0.3 M downstream primer, 50 ng template DNA, 1 L KOD enzyme, and water which was added to a final volume of 50 L). PCR was carried out in accordance with the reaction system instructions to obtain 20 L of PCR products. 1 L of DpnI enzyme (purchased from NEB Corporation) was added to the PCR products. Digestion was carried out at 37 C. for 1 h. 10 L of the digested products were added to Tran1-Blue competent cells (purchased from Beijing TransGen Biotech, Ltd.), and placed on ice for 30 min. After that, the cells were subjected to heat shock at 42 C. for 45 s, placed on ice for 2 min and then were added to 600 L of fluid LB medium without resistance, and recovered at 200 rpm at 37 C. for 1 h. The culture broth was plated on a LB solid culture plate containing kanamycin (15 g/mL), and were placed upside down at 37 C. overnight. The mutant plasmid clones were obtained, and the sequence was verified by sequencing.
(24) The plasmid obtained by the first round of point mutation was subjected to a second round of point mutation. 5ACCTCAAACGAT3 in loop2 was mutated to 5ACCTGCAACGA3. The specific method was as follows:
(25) TABLE-US-00003 SEQIDNO:1336(forward): GCAATTCAGCACAGACACCTGCAACGATTTCGAGACTGGCC, and SEQIDNO:1337(reverse): GGCCAGTCTCGAAATCGTTGCAGGTGTCTGTGCTGAATTGC;
the entire plasmid was subjected to PCR reaction, wherein the PCR reaction system was a KOD-Plus DNA polymerase system (purchased from TOYOBO Corporation), and the total volume of the reaction system was 50 L (5 L buffer, 0.2 mM dNTP, 1 mM magnesium sulfate, 0.3 M upstream primer and 0.3 M downstream primer, 50 ng template DNA, 1 L KOD enzyme, and water which was added to a final volume of 50 L). PCR was carried out in accordance with the reaction system instructions to obtain 20 L of PCR products. 1 L of DpnI enzyme (purchased from NEB Corporation) was added to the PCR products. Digestion was carried out at 37 C. for 1 h. 10 L of the digested products were added to Tran1-Blue competent cells (purchased from Beijing TransGen Biotech, Ltd.), and placed on ice for 30 min. After that, the cells were subjected to heat shock at 42 C. for 45 s, placed on ice for 2 min. and then were added to 600 L of fluid LB medium without resistance, and recovered at 200 rpm at 37 C. for 1 h. The culture broth was plated on a LB solid culture plate containing kanamycin (15 g/mL), and were placed upside down at 37 C. overnight. The mutant plasmid clones were obtained, and the sequence was verified by sequencing.
(26) The plasmid obtained by the second round of point mutation was subjected to a third round of point mutation. 5GACGGCAGCACC3 in loop3 was mutated to 5GACTGCAGCACC3. The specific method was as follows:
(27) TABLE-US-00004 SEQIDNO:1338(forward): CCGTGGGTGTCGTTCAAGACTGCAGCACCACTCACCAGAACG, and SEQIDNO:1339(reverse): CGTTCTGGTGAGTGGTGCTGCAGTCTTGAACGACACCCACGG;
(28) the entire plasmid was subjected to PCR reaction, wherein the PCR reaction system was a KOD-Plus DNA polymerase system (purchased from TOYOBO Corporation), and the total volume of the reaction system was 50 L (5 L buffer, 0.2 mM dNTP, 1 mM magnesium sulfate, 0.3 UM upstream primer and 0.3 M downstream primer, 50 ng template DNA, 1 L KOD enzyme, and water which was added to a final volume of 50 L). PCR was carried out in accordance with the reaction system instructions to obtain 20 L of PCR products. 1 L of DpnI enzyme (purchased from NEB Corporation) was added to the PCR products. Digestion was carried out at 37 C. for 1 h. 10 L of the digested products were added to Tran1-Blue competent cells (purchased from Beijing TransGen Biotech, Ltd.), and placed on ice for 30 min. After that, the cells were subjected to heat shock at 42 C. for 45 s, placed on ice for 2 min. and then were added to 600 L of fluid LB medium without resistance, and recovered at 200 rpm at 37 C. for 1 h. The culture broth was plated on a LB solid culture plate containing kanamycin (15 g/mL), and were placed upside down at 37 C. overnight. The mutant plasmid clones were obtained, and the sequence was verified by sequencing. The resulting plasmid of pET26b-PP-3C is shown in
(29) 2.2 Construction of the P Protein that has been Successfully Mutated, Namely the PP-3C Gene, on a pCold IV Carrier
(30) An EcoR I restriction site was introduced at the 5 end of the PP-3C gene (whose sequence is represented by SEQ ID NO: 1358) by PCR method, and a Hind Ill restriction site was introduced at the 3 end. The specific method was as follows:
(31) TABLE-US-00005 SEQIDNO:1340(forward): GGGAATTCCATATGAAGCCCTTCTCGGTCCCTATCCTGACAG, and SEQIDNO:1341(reverse): CCCAAGCTTTTACAAGGCTCTGCGACGACCGGCTC;
the pET26b-PP-3C plasmid obtained above was subjected to PCR reaction, wherein the PCR reaction system was a KOD-Plus DNA polymerase system (purchased from TOYOBO Corporation), and the total volume of the reaction system was 50 L (5 L buffer, 0.2 mM dNTP, 1 mM magnesium sulfate, 0.3 UM upstream primer and 0.3 M downstream primer, 50 ng template DNA, 1 L KOD enzyme, and water which was added to a final volume of 50 L). PCR was carried out in accordance with the reaction system instructions to obtain 50 L of PCR products. The products were subjected to agarose gel electrophoresis, and the fragments of interest were recovered using gel recovery kit (purchased from Tiangen Biotech Co., Ltd.) to obtain a PP-3C gene fragment with an EcoRI restriction site at the 5 end and a Hind III restriction site at the 3 end.
(32) 1 L of Hind III enzyme and 1 L of EcoR I enzyme (purchased from Takara Corporation) and 5 L of enzyme digestion buffer (purchased from Takara Corporation) were added respectively to 2 g of the aforementioned recovered fragment, and finally sterile water was added to the system to reach a final volume of 50 L. Digestion was carried out at 37 C. for 2 hours. The products were purified using common DNA product purification kit (purchased from Tiangen Biotech Co., Ltd.) to obtain the fragment of interest having sticky ends.
(33) 1 L of Hind III enzyme and 1 L of EcoR I enzyme (purchased from Takara Corporation) and 5 L of enzyme digestion buffer (purchased from Takara Corporation) were added respectively to 2 g of pCold IV vector plasmid (purchased from Novagen Corporation), and finally sterile water was added to the system to reach a final volume of 50 L. Digestion was carried out at 37 C. for 2 hours. The products were subjected to agarose gel electrophoresis, and the vector fragments were recovered using gel recovery kit (purchased from Tiangen Biotech Co., Ltd.) to obtain a plasmid vector having double enzyme-digested sticky ends
(34) The above double enzyme-digested vector fragment and fragment of interest (with a molar ratio of 1:3, and a total volume of 15 L) were mixed, and 0.75 L of T4 ligase (purchased from Takara Corporation) and 1.5 L of ligase buffer (purchased from Takara Corporation) were added. The ligation was carried out at 16 C. overnight. 10 L of the ligated products were added to Tran1-Blue competent cells (purchased from Beijing TransGen Biotech, Ltd.), and placed on ice for 30 min. After that, the cells were subjected to heat shock at 42 C. for 45 s, placed on ice for 2 min, and then were added to 600 L of fluid LB medium without resistance, and recovered at 200 rpm at 37 C. for 1 h. The culture broth was plated on a LB solid culture plate containing ampicillin (15 g/mL), and were placed upside down at 37 C. overnight. The recombinant clones were obtained, and the sequence was verified by sequencing. A pColdIV-PP-3C plasmid that can express PP-3C protein was obtained, as shown in
(35) 2.3 Expression of the Cysteine-Modified Norovirus P Protein
(36) 1 L of the recombinant plasmids prepared in the above examples were respectively added to 100 L of E. coli BL21 competent cells (purchased from TransGen Corporation), ice-bathed for 30 min, heat shocked for 90 s in a water-bath at 42 C., and then ice-bathed for 2 min. 600 UL of LB medium was added to the mixture, and cultured at 180 rpm/min at 37 C. for 1 h. The mixture was coated evenly on a LB solid medium containing kanamycin (15 g/mL) resistance and cultured at 37 C. for 24 h to obtain strains that can stably express recombinant proteins. A growing colony was picked and inoculated into 20 mL of LB medium. The mixture was cultured at 220 rpm at 37 C. When the OD value of the culture mixture reached 1.0, induction by Isopropyl -D-Thiogalactoside (IPTG at a final concentration of 0.5 mmol/L) was carried out at 220 rpm at 16 C. overnight. After the induction, the culture broth was centrifuged at 4,000 rpm for 20 min. The supernatant was discarded, and the bacterial precipitates were resuspended with PBS. Centrifugation was conducted again at 4,000 rpm for 20 min and the supernatant was discarded to obtain the bacterial precipitates containing proteins of interest.
(37) 2.4 Purification of the Cysteine-Modified Norovirus P Protein
(38) The bacterial precipitates obtained in 2.3 were resuspended by adding 20 mL of protein buffer (pH8.0, containing 50 mM Tris and 300 mM KCl). The bacteria were lysed by sonication on ice for 30 min. The mixture was centrifuged at 12,000 rpm at 4 C. for 30 min. Subsequently, the supernatant was taken and allowed to pass through 0.45 m filter membrane to obtain crude extracts of proteins.
(39) The structure of the recombinant PP-3C particle protein is shown in
(40) The crude extracts of proteins were purified using an anion exchange column (purchased from GE Corporation). The specific scheme was as follows: First, the exchange column was rinsed with ultrapure water in a volume of about 100 mL, followed by equilibration with PB solution (pH 5.0) at a flow rate of 2 mL/min. Then 20 mL of the crude extracts of proteins were added to the exchange column at a flow rate of 1 ml/min. After the sample was completely loaded onto the column, the exchange column was rinsed with PB solution (pH 7.0) to remove hybrid protein, and then eluted with PB solution containing 0.5 mol/L NaCl. The proteins at peak value were collected to obtain the proteins of interest.
(41) The proteins were further purified by a hydrophobic chromatography column (purchased from GE Corporation). The specific scheme was as follows: First, the column was rinsed with ultrapure water, and then the hydrophobic column was rinsed with PB solution (pH 7.0) at a flow rate of 2 mL/min. After the column was equilibrated well, the protein sample was loaded onto it. After the sample was completely loaded onto the column, the column was eluted by gradient with PB (pH 7.0) and 1 mol/L NaCl solution for 2 hours. The concentration of NaCl decreases from 1 mol/L to 0.1 mol/L. The proteins were collected at peak value.
(42) The sizes of the resulting P particle protein monomers were identified by reductive SDS-PAGE.
(43) Then the tetracosamers of P protein particles were further isolated and purified using a Superdex 200 molecular sieve (purchased from GE Corporation). The procedures were as follows: The column was rinsed with ultrapure water at a flow rate of 1 mL/min for one volume of the column, and then again with about 120 mL of PB buffer (pH=5). After that, 2 mL of protein extraction solution was added to the column and washed with PB buffer at a flow rate of 1 mL/min. The proteins at peak value were collected to obtain the tetracosamers of P particle proteins. Three proteins were tested for their polymer structures by native polyacrylamide gel electrophoresis. As shown in
Example 3: Preparation of a Lysine-Modified Norovirus P Protein (PP-3K)
(44) In this example, three point mutations were performed using the norovirus P protein plasmid (which was obtained by constructing the PP gene in the pET26b-PP plasmid provided in the Chinese patent application for invention 2015104155561 into pET28a by homologous recombination method) as a template, a lysine is inserted into each of the three loops loop structure of the norovirus P particle, and the protein was expressed and purified.
(45) 3.1 Construction of pET28a-PP Plasmid
(46) The PP genes in pET 26b-PP plasmid were enriched by PCR method. The specific method was as follows:
(47) TABLE-US-00006 SEQIDNO:1361(forward): AACTTTAAGAAGGAGATATACCATGGGCAAGCCCTTCTCGGTCCCTA, and SEQIDNO:1362(reverse): CGGATCTCAGTGGTGGTGGTGGTGGTGCTCGAGTTACAAGGCTCTGCG ACGACCGGC;
the pET26b-P protein plasmid was subjected to PCR reaction, wherein the PCR reaction system was a KOD-Plus DNA polymerase system (purchased from TOYOBO Corporation), and the total volume of the reaction system was 50 L (5 L buffer, 0.2 mM dNTP, 1 mM magnesium sulfate, 0.3 M upstream primer and 0.3 M downstream primer, 50 ng template DNA, 1 L KOD enzyme, and water which was added to a final volume of 50 L). PCR was carried out in accordance with the reaction system instructions to obtain 50 L of PCR products. The products were subjected to agarose gel electrophoresis, and the fragments of interest were recovered using gel recovery kit (purchased from Tiangen Biotech Co., Ltd.).
(48) 1 L of BamHI enzyme and 1 L of XhoI enzyme (purchased from Takara Corporation) and 5 L of enzyme digestion buffer (purchased from Takara Corporation) were added respectively to 2 g of pET28a vector plasmid (purchased from Novagen Corporation), and finally sterile water was added to the system to reach a final volume of 50 L. Digestion was carried out at 37 C. for 2 hours. The products were subjected to agarose gel electrophoresis, and the vector fragments were recovered using gel recovery kit (purchased from Tiangen Biotech Co., Ltd.) to obtain a linear plasmid vector.
(49) The above double enzyme-digested vector fragment and fragment of interest (with a molar ratio of 1:3, and a total volume of 5 L) were mixed, and 5 L of homologous recombination mix (purchased from Taihe Corporation) was added. The ligation was carried out at 25 C. for 15 min. 10 L of the ligated products were added to DH10B competent cells (purchased from Taihe Corporation), and placed on ice for 30 min. After that, the cells were subjected to heat shock at 42 C. for 30 s, placed on ice for 2 min. and then were added to 600 L of fluid LB medium without resistance, and recovered at 200 rpm at 37 C. for 1 h. The culture broth was plated on a LB solid culture plate containing kanamycin (15 g/mL), and were placed upside down at 37 C. overnight. The recombinant clones were obtained, and the sequence was verified by sequencing. A pET28a-PP plasmid that can express PP protein was obtained, as shown in
(50) 3.2 Construction of PP-3K Plasmid
(51) Three rounds of site-directed mutagenesis were performed by site-directed mutation method using the pET28a-PP plasmid which has already been constructed. Respectively, 5 CGCTGGAACACAAA3 in loop1 was mutated to 5 CGCTGGAAAGACACAAA3. The specific method was as follows:
(52) TABLE-US-00007 SEQIDNO:1340(forward): GACGTCACACACATCGCTGGAAAGACACAAAACTACACCATGAAC, and SEQIDNO:1341(reverse): GTTCATGGTGTAGTTTTGTGTCTTTCCAGCGATGTGTGTGACGTC;
the entire plasmid was subjected to PCR reaction, wherein the PCR reaction system was a KOD-Plus DNA polymerase system (purchased from TOYOBO Corporation), and the total volume of the reaction system was 50 L (5 L buffer, 0.2 mM dNTP, 1 mM magnesium sulfate, 0.3 M upstream primer and 0.3 M downstream primer, 50 ng template DNA, 1 L KOD enzyme, and water which was added to a final volume of 50 L). PCR was carried out in accordance with the reaction system instructions to obtain 20 L of PCR products. 1 L of DpnI enzyme (purchased from NEB Corporation) was added to the PCR products. Digestion was carried out at 37 C. for 1 h. 10 L of the digested products were added to Tran1-Blue competent cells (purchased from Beijing TransGen Biotech, Ltd.), and placed on ice for 30 min. After that, the cells were subjected to heat shock at 42 C. for 45 s, placed on ice for 2 min, and then were added to 600 L of fluid LB medium without resistance, and recovered at 200 rpm at 37 C. for 1 h. The culture broth was plated on a LB solid culture plate containing kanamycin (15 g/mL), and were placed upside down at 37 C. overnight. The mutant plasmid clones were obtained, and the sequence was verified by sequencing.
(53) The PCR product obtained by the first round of site-directed mutation was subjected to a second round of site-directed mutation. 5 ACCTCAAACGAT 3 in loop2 was mutated to 5 ACCTCAAAGAACGAT3. The specific method was as follows:
(54) TABLE-US-00008 SEQIDNO:1342(forward): CAATTCAGCACAGACACCTCAAAGAACGATTTCGAGACTGGCCAG, and SEQIDNO:1343(reverse): CTGGCCAGTCTCGAAATCGTTCTTTGAGGTGTCTGTGCTGAATTG;
the entire plasmid was subjected to PCR reaction, wherein the PCR reaction system was a KOD-Plus DNA polymerase system (purchased from TOYOBO Corporation), and the total volume of the reaction system was 50 L (5 L buffer, 0.2 mM dNTP, 1 mM magnesium sulfate, 0.3 M upstream primer and 0.3 M downstream primer, 50 ng template DNA, 1 L KOD enzyme, and water which was added to a final volume of 50 L). PCR was carried out in accordance with the reaction system instructions to obtain 20 L of PCR products. 1 L of DpnI enzyme (purchased from NEB Corporation) was added to the PCR products. Digestion was carried out at 37 C. for 1 h. 10 L of the digested products were added to Tran1-Blue competent cells (purchased from Beijing TransGen Biotech, Ltd.), and placed on ice for 30 min. After that, the cells were subjected to heat shock at 42 C. for 45 s, placed on ice for 2 min, and then were added to 600 L of fluid LB medium without resistance, and recovered at 200 rpm at 37 C. for 1 h. The culture broth was plated on a LB solid culture plate containing kanamycin (15 g/mL), and were placed upside down at 37 C. overnight. The mutant plasmid clones were obtained, and the sequence was verified by sequencing.
(55) The PCR product obtained by the second round of site-directed mutation was subjected to a third round of site-directed mutation. 5 GACGGCAGCACC 3 in loop3 was mutated to 5 GACGGCAGCACC3. The specific method was as follows:
(56) TABLE-US-00009 SEQIDNO:1344(forward): GTGGGTGTCGTTCAAGACGGCAAGAGCACCACTCACCAGAACGAA, and SEQIDNO:1345(reverse): TTCGTTCTGGTGAGTGGTGCTCTTGCCGTCTTGAACGACACCCAC;
the entire plasmid was subjected to PCR reaction, wherein the PCR reaction system was a KOD-Plus DNA polymerase system (purchased from TOYOBO Corporation), and the total volume of the reaction system was 50 L (5 L buffer, 0.2 mM dNTP, 1 mM magnesium sulfate, 0.3 M upstream primer and 0.3 M downstream primer, 50 ng template DNA, 1 L KOD enzyme, and water which was added to a final volume of 50 L). PCR was carried out in accordance with the reaction system instructions to obtain 20 L of PCR products. 1 L of DpnI enzyme (purchased from NEB Corporation) was added to the PCR products. Digestion was carried out at 37 C. for 1 h. 10 L of the digested products were added to Tran1-Blue competent cells (purchased from Beijing TransGen Biotech, Ltd.), and placed on ice for 30 min. After that, the cells were subjected to heat shock at 42 C. for 45 s, placed on ice for 2 min. and then were added to 600 L of fluid LB medium without resistance, and recovered at 200 rpm at 37 C. for 1 h. The culture broth was plated on a LB solid culture plate containing kanamycin (15 g/mL), and were placed upside down at 37 C. overnight. The mutant plasmid clones were obtained, and the sequence was verified by sequencing (the sequence of the PP-3K gene is represented by SEQ ID NO: 1360). The pET28a-P protein-3K plasmid is shown as
3.3 Expression of the Lysine-Modified Norovirus P Protein
(57) 1 L of the recombinant plasmids prepared in the above examples were respectively added to 100 L of E. coli BL21 competent cells (purchased from TransGen Corporation), ice-bathed for 30 min, heat shocked for 90 s in a water-bath at 42 C., and then ice-bathed for 2 min. 600 UL of LB medium was added to the mixture, and cultured at 180 rpm/min at 37 C. for 1 h. The mixture was coated evenly on a LB solid medium containing kanamycin (15 g/mL) resistance and cultured at 37 C. for 24 h to obtain strains that can stably express recombinant proteins. A growing colony was picked and inoculated into 20 mL of LB medium. The mixture was cultured at 220 rpm at 37 C. When the OD value of the culture mixture reached 1.0, induction by Isopropyl -D-Thiogalactoside (IPTG at a final concentration of 0.5 mmol/L) was carried out at 220 rpm at 16 C. overnight. After the induction, the culture broth was centrifuged at 4,000 rpm for 20 min. The supernatant was discarded, and the bacterial precipitates were resuspended with PBS. Centrifugation was conducted again at 4,000 rpm for 20 min and the supernatant was discarded to obtain the bacterial precipitates containing proteins of interest.
(58) 3.4 Purification of the Lysine-Modified Norovirus P Protein
(59) The bacterial precipitates obtained in 3.3 were resuspended by adding 20 ml of protein buffer (pH8.0, containing 50 mM Tris and 300 mM KCl). The bacteria were lysed by sonication on ice for 30 min. The mixture was centrifuged at 12,000 rpm at 4 C. for 30 min. Subsequently, the supernatant was taken and allowed to pass through 0.45 m filter membrane to obtain crude extracts of proteins.
(60) The structure of the recombinant PP-3K particle is shown as
(61) The crude extracts of proteins were purified using an anion exchange column (purchased from GE Corporation). The specific scheme was as follows: First, the exchange column was rinsed with ultrapure water in a volume of about 100 mL, followed by equilibration with PB solution (pH 5.0) at a flow rate of 2 mL/min. Then 20 mL of the crude extracts of proteins were added to the exchange column at a flow rate of 1 ml/min. After the sample was completely loaded onto the column, the exchange column was rinsed with PB solution (pH 7.0) to remove hybrid protein, and then eluted with PB solution containing 0.5 mol/L NaCl. The proteins at peak value were collected to obtain the proteins of interest.
(62) The proteins were further purified by a hydrophobic chromatography column (purchased from GE Corporation). The specific scheme was as follows: First, the column was rinsed with ultrapure water, and then the hydrophobic column was rinsed with PB solution (pH 7.0) at a flow rate of 2 mL/min. After the column was equilibrated well, the protein sample was loaded onto it. After the sample was completely loaded onto the column, the column was eluted by gradient with PB (pH 7.0) and 1 mol/L NaCl solution for 2 hours. The concentration of NaCl decreases from 1 mol/L to 0.1 mol/L. The proteins were collected at peak value.
(63) The sizes of P particle protein monomers were identified by reductive SDS-PAGE. Then the tetracosamers of P protein particles were further isolated and purified using a Superdex 200 molecular sieve (purchased from GE Corporation). The procedures were as follows: The column was rinsed with ultrapure water at a flow rate of 1 mL/min for one volume of the column, and then again with about 120 mL of PB buffer (pH=5). After that, 2 mL of protein extraction solution was added to the column and washed with PB buffer at a flow rate of 1 mL/min. The proteins at peak value were collected to obtain the tetracosamers of P particle proteins. Three proteins were tested for their polymer structures by native polyacrylamide gel electrophoresis, as shown in
Example 4: Coupling Reaction of PP-3C with a Phosphorylated Polypeptide Antigen Vaccine
(64) Example 4-1: The purified PP-3C was subjected to a buffer-exchange treatment with a desalting column from GE Corporation to change the buffer system of the protein to a 0.1 M ammonium bicarbonate solution (pH=7.5). The PP-3C after the buffer-exchange treatment was quantified, and was diluted to a concentration of 0.3 mg/mL. In the ratio of the molar amount of protein to the molar amount of polypeptide of 1:30, each of the phosphorylated polypeptide antigen vaccines in lyophilized form prepared in Example 1 was added and mixed slowly. The mixed system was incubated at 2 C. to 8 C. with slow shaking for 48 hours. The resulting product was concentrated by ultrafiltration to remove unbound polypeptides, and the buffer system was changed to a PBS buffer system.
(65) Example 4-2: The purified PP-3C was subjected to a buffer-exchange treatment with a desalting column from GE Corporation to change the buffer system of the protein to a 1 M ammonium bicarbonate solution (pH=8.0). The PP-3C after the buffer-exchange treatment was quantified, and was diluted to a concentration of 0.5 mg/mL. In the ratio of the molar amount of protein to the molar amount of polypeptide of 1:50, each of the phosphorylated polypeptide antigen vaccines in lyophilized form prepared in Example 1 was added and mixed slowly. The mixed system was incubated at 2 C. to 8 C. with slow shaking for 48 hours. The resulting product was concentrated by ultrafiltration to remove unbound polypeptides, and the buffer system was changed to a PBS buffer system.
(66) Example 4-3: The purified PP-3C was subjected to a buffer-exchange treatment with a desalting column from GE Corporation to change the buffer system of the protein to a 0.1 M ammonium bicarbonate solution (pH=7.8). The PP-3C after the buffer-exchange treatment was quantified, and was diluted to a concentration of 0.3 mg/mL. In the ratio of the molar amount of protein to the molar amount of polypeptide of 1:100, each of the phosphorylated polypeptide antigen vaccines in lyophilized form prepared in Example 1 was added and mixed slowly. The mixed system was incubated at 2 C. to 8 C. with slow shaking for 48 hours. The resulting product was concentrated by ultrafiltration to remove unbound polypeptides, and the buffer system was changed to a PBS buffer system.
(67) Example 4-4: The purified PP-3C was subjected to a buffer-exchange treatment with a desalting column from GE Corporation to change the buffer system of the protein to a 0.1 M ammonium bicarbonate solution (pH=8.8). The PP-3C after the buffer-exchange treatment was quantified, and was diluted to a concentration of 0.3 mg/mL. In the ratio of the molar amount of protein to the molar amount of polypeptide of 1:30, each of the phosphorylated polypeptide antigen vaccines in lyophilized form prepared in Example 1 was added and mixed slowly. The mixed system was incubated at 2 C. to 8 C. with slow shaking for 72 hours. The resulting product was concentrated by ultrafiltration to remove unbound polypeptides, and the buffer system was changed to a PBS buffer system.
Example 5: Coupling Reaction of PP-3K with a Phosphorylated Polypeptide Antigen Vaccine
(68) Example 5-1: The purified PP-3K solution was quantified by BCA and the pH was adjusted to 7.2. This protein solution was mixed with a 1 mg/mL sulfo-maleimide (sulfo-SMCC) solution in the ratio of the molar amount of SMCC to the molar amount of PP-3K of 5:1, and the mixture was reacted in a water bath at 25 C. for 30 min. The mixture was subjected to a desalting treatment with a desalting column from GE Corporation, the system was changed to a PBS solution (pH=7.2-7.4), and unbound sulfo-SMCC was removed. The reaction product was taken and each of the lyophilized products of the phosphorylated polypeptide antigen vaccines prepared in Example 1 was added to the system in the ratio of the molar amount of PP-3K to the molar amount of phosphorylated polypeptide of 1:5, and mixed slowly. Then, the reaction system was reacted in a water bath at 25 C. for 30 min. The reaction product was collected, and the mixture was subjected to a desalting treatment with a desalting column from GE Corporation, the system was changed to a PBS solution (pH=7.2-7.4), and unbound phosphorylated polypeptides were removed.
(69) Example 5-2: The purified PP-3K solution was quantified by BCA. This protein solution was mixed with a 1 mg/mL sulfo-maleimide (sulfo-SMCC) solution in the ratio of the molar amount of SMCC to the molar amount of PP-3K of 20:1, and the mixture was reacted in a water bath at 25 C. for 30 min. The mixture was subjected to a desalting treatment with a desalting column from GE Corporation, the system was changed to a PB solution (pH=8.5), and unbound sulfo-SMCC was removed. The reaction product was taken and each of the lyophilized products of the phosphorylated polypeptide antigen vaccines prepared in Example 1 was added to the system in the ratio of the molar amount of PP-3K to the molar amount of phosphorylated polypeptides of 1:30, and mixed slowly. Then, the reaction system was reacted in a water bath at 25 C. for 30 min. The reaction product was collected, and the mixture was subjected to a desalting treatment with a desalting column from GE Corporation, the system was changed to a PB solution (pH=8.5), and unbound phosphorylated polypeptides were removed.
(70) Example 5-3: The purified PP-3K solution was quantified by BCA. This protein solution was mixed with a 1 mg/mL sulfo-maleimide (sulfo-SMCC) solution in the ratio of the molar amount of SMCC to the molar amount of PP-3K of 20:1, and the mixture was reacted in a water bath at 25 C. for 30 min. The mixture was subjected to a desalting treatment with a dialysis bag having a molecular weight cut-off of 20 kd (the dialysis volume ratio is 1,000:1), and was dialyzed overnight at 2 C. to 8 C., the system was changed to a PB solution (pH=7.0), and unbound sulfo-SMCC was removed. The reaction product was taken and each of the lyophilized products of the phosphorylated polypeptide antigen vaccines prepared in Example 1 was added to the system in the ratio of the molar amount of PP-3K to the molar amount of phosphorylated polypeptides of 1:50, and mixed slowly. Then, the reaction system was reacted in a water bath at 25 C. for 30 min. The mixture was subjected to a desalting treatment with a dialysis bag having a molecular weight cut-off of 20 kDa (the dialysis volume ratio is 1,000:1), and was dialyzed overnight at 2 C. to 8 C., the system was changed to a PB solution (pH=7.0), and unbound phosphorylated polypeptides were removed.
(71) Example 5-4: The purified PP-3K solution was quantified by BCA. This protein solution was mixed with a 1 mg/mL sulfo-maleimide (sulfo-SMCC) solution in the ratio of the molar amount of SMCC to the molar amount of PP-3K of 20:1, and the mixture was reacted with slow shaking overnight at 2 C. to 8 C. The mixture was subjected to a desalting treatment with a dialysis bag having a molecular weight cut-off of 20 kd (the dialysis volume ratio is 1,000:1), and was dialyzed overnight at 2 C. to 8 C., the system was changed to a PBS solution (pH=7.2-7.4), and unbound sulfo-SMCC was removed. The reaction product was taken and each of the lyophilized products of the phosphorylated polypeptide antigen vaccines prepared in Example 1 was added to the system in the ratio of the molar amount of PP-3K to the molar amount of phosphorylated polypeptides of 1:50, and mixed slowly. Then, the reaction system was reacted with slow shaking overnight at 2 C. to 8 C. The mixture was subjected to a desalting treatment with a dialysis bag having a molecular weight cut-off of 20 kd (the dialysis volume ratio is 1,000:1), and was dialyzed overnight at 2 C. to 8 C., the system was changed to a PBS solution (pH=7.2-7.4), and unbound phosphorylated polypeptides were removed.
(72) Example 5-5: The purified PP-3K solution was quantified by BCA. This protein solution was mixed with a 20 mg/ml dimethylformamide (DMF) solution of maleimide (SMCC) in the ratio of the molar amount of SMCC to the molar amount of PP-3K of 10:1, and the mixture was reacted in a water bath at 25 C. for 30 min. The mixture was subjected to a desalting treatment with a desalting column from GE Corporation, the system was changed to a PBS solution (pH=7.2-7.4), and unbound SMCC was removed. The reaction product was taken and each of the lyophilized products of the phosphorylated polypeptide antigen vaccines prepared in Example 1 was added to the system in a ratio of the molar amount of PP-3K to the molar amount of phosphorylated polypeptides of 1:30, and mixed slowly. Then, the reaction system was reacted in a water bath at 25 C. for 30 min. The reaction product was collected, and the mixture was subjected to a desalting treatment with a desalting column from GE Corporation, the system was changed to a PBS solution (pH=7.2), and unbound phosphorylated polypeptides were removed.
(73) Example 5-6: The purified PP-3K solution was quantified by BCA. This protein solution was mixed with a 20 mg/ml dimethylformamide (DMF) solution of maleimide (SMCC) in the ratio of the molar amount of SMCC to the molar amount of PP-3K of 30:1, and the mixture was reacted with slow shaking overnight at 2 C. to 8 C. The mixture was subjected to a desalting treatment with a desalting column from GE Corporation, the system was changed to a PBS solution (pH=7.2-7.4), and unbound SMCC was removed. The reaction product was taken and each of the lyophilized products of the phosphorylated polypeptide antigen vaccines prepared in Example 1 was added to the system in the ratio of the molar amount of PP-3K to the molar amount of phosphorylated polypeptides of 1:50, and mixed slowly. The mixture was reacted with slow shaking overnight at 2 C. to 8 C. The reaction product was collected, and the mixture was subjected to a desalting treatment with a desalting column from GE Corporation, the system was changed to a PBS solution (pH=7.2), and unbound phosphorylated polypeptides were removed.
(74) Example 5-7: The purified PP-3K solution was quantified by BCA. This protein solution was mixed with a 20 mg/ml dimethylformamide (DMF) solution of maleimide (SMCC) in the ratio of the molar amount of SMCC to the molar amount of PP-3K of 5:1, and the mixture was reacted in a water bath at 25 C. for 30 min. The mixture was subjected to a desalting treatment with a dialysis bag having a molecular weight cut-off of 20 kDa (the dialysis volume ratio is 1,000:1), and was dialyzed overnight at 2 C. to 8 C., the system was changed to a PBS solution (pH=7.2-7.4), and unbound SMCC was removed. The reaction product was taken and each of the lyophilized products of the phosphorylated polypeptide antigen vaccines prepared in Example 1 was added to the system in the ratio of the molar amount of PP-3K to the molar amount of phosphorylated polypeptides of 1:100, and mixed slowly. The mixture was reacted with slow shaking overnight at 2 C. to 8 C. The reaction product was collected, and the mixture was subjected to a desalting treatment with a dialysis bag having a molecular weight cut-off of 20 kDa (the dialysis volume ratio is 1,000:1), and was dialyzed overnight at 2 C. to 8 C., the system was changed to a PBS solution (pH=7.2), and unbound phosphorylated polypeptides were removed.
Example 6: Determination of the Ligation Efficiency of the Phosphorylated Polypeptide Antigen Product Coupled with PP-3C
(75) A10 mM dithiothreitol solution was added to the phosphorylated polypeptide antigen product coupled with PP-3C at a volume ratio of 1:1, and mixed homogeneously, and the mixture was left to stand at room temperature and react for 16 hours. The reaction product was centrifuged at 16,000 g at 4 C. for 15 min. The supernatants were taken for HPLC determination. Using 0.5 mg/mL, 0.25 mg/mL, 0.125 mg/mL, 0.0625 mg/mL, 0.03125 mg/mL, 0.03125 mg/mL, and 0.015625 mg/mL phosphorylated polypeptide dissolved in 10 mM of a dithiothreitol solution as standard, a standard curve of the concentration of the standard versus the peak area of the phosphorylated polypeptide was plotted for quantitatively analyzing the concentration of the test sample (the results are shown in
Example 7: Determination of the Ligation Efficiency of the Phosphorylated Polypeptide Antigen Product Coupled with PP-3K
(76) The phosphorylated polypeptide antigen product coupled with PP-3K was subjected to a mass spectrometric analysis, and the concentration of the test sample was quantitatively analyzed using the phosphorylated polypeptide dissolved in PBS as standard (the results are not shown).
Example 8: Analysis of the Immunogenicity of Phosphorylated Polypeptide Antigen Vaccines in Wild Mice
(77) The phosphorylated polypeptide antigens with sequences as represented by SEQ ID NO: 201, SEQ ID NO: 225, SEQ ID NO: 306, SEQ ID NO: 387, SEQ ID NO: 468, SEQ ID NO: 558, SEQ ID NO: 567, SEQ ID NO: 769, SEQ ID NO: 784, SEQ ID NO: 875, SEQ ID NO: 1020, SEQ ID NO: 1101, SEQ ID NO: 1182, SEQ ID NO: 1272, SEQ ID NO: 1313 and SEQ ID NO: 1330 were dissolved to 1 mg/mL, and mixed with a complete Freund's adjuvant or an incomplete Freund's adjuvant at a volume ratio of 1:1 to form a water-in-oil emulsion so as to prepare vaccines A1 to A16. Each wild mouse was injected intramuscularly with 50 L. Each wild mouse was vaccinated four times at two-week intervals using complete Freund's adjuvant for the first immunization and incomplete Freund's adjuvant for the second, third and fourth immunizations. Two weeks after the fourth injection, the mice were subjected to blood sampling to obtain mouse serum for ELISA analysis.
(78) The polypeptide (whose sequence is represented by SEQ ID NO: 1346) which was coupled with BSA and phosphorylated at a position corresponding to amino acid 18 of full-length Tau protein was used as the coating antigen to detect the antiserum titer against a phosphorylation Y18 site; the phosphorylatd polypeptide (whose sequence is represented by SEQ ID NO: 1347) which was coupled with BSA and phosphorylated at positions corresponding to amino acids 202 and 205 of full-length Tau protein was used as the coating antigen to detect the antiserum titer against phosphorylation S202 and T205 sites; the phosphorylatd polypeptide (whose sequence is represented by SEQ ID NO: 1348) which was coupled with BSA and phosphorylated at a position corresponding to amino acid 212/214 of full-length Tau protein was used as the coating antigen to detect the antiserum titer against phosphorylation T212 and S214 sites; the phosphorylatd polypeptide (whose sequence is represented by SEQ ID NO: 1349) which was coupled with BSA and phosphorylated at a position corresponding to amino acid 231/235 of full-length Tau protein was used as the coating antigen to detect the antiserum titer against phosphorylation T231 and S235 sites; the phosphorylatd polypeptide (whose sequence is represented by SEQ ID NO: 1350) which was coupled with BSA and phosphorylated at a position corresponded to amino acid 238 of full-length Tau protein was used as the coating antigen to detect the antiserum titer against a phosphorylation S238 site; the phosphorylatd polypeptide (whose sequence is represented by SEQ ID NO: 1351) which was coupled with BSA and phosphorylated at a position corresponding to amino acid 262 of full-length Tau protein was used as the coating antigen to detect the antiserum titer against a phosphorylation S262 site; the phosphorylatd polypeptide (whose sequence is represented by SEQ ID NO: 1352) which was coupled with BSA and phosphorylated at a position corresponding to amino acid 396 of full-length Tau protein was used as the coating antigen to detect the antiserum titer against a phosphorylation S396 site; the phosphorylatd polypeptide (whose sequence is represented by SEQ ID NO: 1353) which was coupled with BSA and phosphorylated at a position corresponding to amino acid 404 of full-length Tau protein was used as the coating antigen to detect the antiserum titer against a phosphorylated S404 site.
(79) The above antigens were respectively dissolved in carbonate buffer with pH=9.5, diluted to 1 g/mL, and added to an ELISA plate (100 L/well) at 4 C. overnight. The liquid in the plate was discarded, and after the 96-well plate was washed three times with PBST (300 L/well), the liquid in the plate was discarded. A3% BSA (PBS) solution was added (200 L/well), and incubation was carried out at 37 C. for 1 hour. The liquid in the plate was discarded, and after the 96-well plate was washed three times with PBST (300 L/well), the liquid in the plate was discarded. Mouse serum diluted by gradient with a 1% BSA (PBS) solution was added (100 L/well), and incubation was carried out at 37 C. for 2 hour. The liquid in the plate was discarded, and after the 96-well plate was washed three times with PBST (300 L/well), the liquid in the plate was discarded. A goat-anti-mouse HRP labled antibody solution diluted with a 1% BSA (PBS) solution was added (100 L/well), and incubation was carried out at 37 C. for 1 hour. The liquid in the plate was discarded, and after the 96-well plate was washed three times with PBST (300 L/well), the liquid in the plate was discarded. TMB was added (300 L/well) and incubation was carried out in the dark at room temperature for 25 min. 1M sulfuric acid aqueous solution was added to terminate the reaction. Absorbance was detected at 450 nm using a microplate reader
(80) The levels of the antibodies against different antigens in the serum after the fourth immunization by vaccines A1 to A16 are shown in
Example 9: Evaluation of the Immunogenicity of Phosphorylated Polypeptide Antigens Coupled with the Norovirus P Protein PP-3C in Wild Mice Immunized with the Same by Intramuscular Injection
(81) The PP-3C protein was coupled with A1, A5, A9, A10, A11, A12 and A15 respectively to prepare complex vaccines, and wild mice were immunized with the complex vaccines by intramuscular injection. Each group contained 6 mice. The immune dosage of the protein was 25 g/animal. The immunization was carried out at week 0, week 2, week 4 and week 6. Blood was taken two weeks after every immunization to obtain serum for ELISA assessment. The assessment was performed by the same method as in Example 8. The results are shown in
Example 10: Evaluation of the Immunogenicity of Phosphorylated Polypeptide Antigens Coupled with the Norovirus P Protein PP-3C in Wild Mice Immunized with the Same by Intraperitoneal Injection
(82) The PP-3C protein was coupled with A2, A3, A7, A11 and A14 respectively to prepare complex vaccines, and wild mice were immunized with the complex vaccines by intraperitoneal injection. Each group contained 6 mice. The immune dosage of the protein was 25 g/animal. The immunization was carried out at week 0, week 2, week 4 and week 6. Blood was taken two weeks after every immunization to obtain serum for ELISA assessment. The assessment was performed by the same method as in Example 10. The results were shown in
Example 11: Evaluation of the Immunogenicity of Phosphorylated Polypeptide Antigens Coupled with the Norovirus P Protein PP-3C in Wild Mice Immunized with the Same by Subcutaneous Injection
(83) The PP-3C protein was coupled with A4, A6, A8, A11, A13 and A16 respectively to prepare complex vaccines, and wild mice were immunized with the complex vaccines by subcutaneous injection. Each group contained 6 mice. The immune dosage of the protein was 25 g/animal. The immunization was carried out at week 0, week 2, week 4 and week 6. Blood was taken two weeks after every immunization to obtain serum for ELISA assessment. The assessment was performed by the same method as in Example 10. The results were shown in
Example 12: Evaluation of the Immunogenicity of Phosphorylated Polypeptide Antigens Coupled with a Norovirus P Protein in Wild Mice
(84) Take vaccine A11 as an example. Wild mice were intramuscularly immunized with A11 coupled with PP-3C or PP-3K and combined with CpG, AS02, AS03, MF59 and AS02+CpG adjuvants respectively. Each group contained 6 mice. The immune dosage of the protein was 25 g/animal. The immunization was carried out at week 0, week 2, week 4, week 6 and week 12. Blood was taken two weeks after every immunization to obtain serum for ELISA assessment. In the ELISA experiments, the phosphorylated polypeptide (whose sequence is SEQ ID NO: 1354) coupled with BSA was used as the coating antigen, and was phosphorylated at sites corresponding to amino acids 202, 205, 396 and 404 of full-length Tau protein. A standard curve was plotted with the concentration of the AT8 antibody (Thermo scientific MN1020) diluted by gradient versus the O.D. value to calibrate the concentration of the antibodies in the mouse serum. The change in antibody concentration in each group is shown in
Example 13: Evaluation of the Immunogenicity of Phosphorylated Polypeptide Antigen A5 Coupled with a Norovirus P Protein in Wild Mice
(85) Wild mice were intramuscularly immunized with the vaccine prepared by combining phosphorylated polypeptide antigen A5 coupled with PP-3K with AS02+CpG adjuvant. Each group contained 6 mice. The immune dosage of the protein was 25 g/animal. The immunization was carried out at week 0, week 2, week 4, week 6 and week 12. Blood was taken two weeks after every immunization to obtain serum for ELISA assessment. In the ELISA experiments, the phosphorylated polypeptide (whose sequence is SEQ ID NO: 1355) coupled with BSA was used as the coating antigen, and was phosphorylated at sites corresponding to amino acids 18, 238 and 262 of full-length Tau protein. The change in the concentration of antibodies in the mouse serum is shown in
Example 14: Evaluation of the Immunogenicity of Phosphorylated Polypeptide Antigen A5 Coupled with a Norovirus P Protein in P301S Transgenic Mice
(86) P301S transgenic mice were intramuscularly immunized with the vaccine prepared by combining phosphorylated polypeptide antigen A5 coupled with PP-3K with AS02+CpG adjuvant. Each group contained 8 mice. The immune dosage of the protein was 25 g/animal. The immunization was carried out at week 0, week 2, week 4, week 6 and week 12. Blood was taken two weeks after every immunization to obtain serum for ELISA assessment. In the ELISA experiments, the phosphorylated polypeptide (whose sequence is SEQ ID NO: 1355) coupled with BSA was used as the coating antigen, and was phosphorylated at sites corresponding to amino acids 18, 238 and 262 of full-length Tau protein. The change in the concentration of antibodies in the mouse serum is shown in
Example 15: Evaluation of the Immunogenicity of Phosphorylated Polypeptide Antigen A11 Coupled with a Norovirus P Protein in P301S Transgenic Mice
(87) P301S transgenic mice were intramuscularly immunized with the vaccine prepared by combining phosphorylated polypeptide antigen A11 coupled with PP-3C with AS02+CpG adjuvant. Each group contained 12 mice. The immune dosage of the protein was 25 g/animal. The immunization was carried out at week 0, week 2, week 4, week 6 and week 12. Blood was taken two weeks after every immunization to obtain serum for ELISA assessment. In the ELISA experiments, the phosphorylated polypeptide (whose sequence is SEQ ID NO: 1354) coupled with BSA was used as the coating antigen, and was phosphorylated at sites corresponding to amino acids 202, 205, 396 and 404 of full-length Tau protein. The change in the concentration of antibodies in the mouse serum is shown in
Example 16: Evaluation of the Immunogenicity of Phosphorylated Polypeptide Antigen A11 Coupled with a Norovirus P Protein in P301S Transgenic Mice
(88) P301S transgenic mice were intramuscularly immunized with the vaccine prepared by combining phosphorylated polypeptide antigen A11 coupled with PP-3K with AS02+CpG adjuvant. Each group contained 12 mice. The immune dosage of the protein was 25 g/animal. The immunization was carried out at week 0, week 2, week 4, week 6 and week 12. Blood was taken two weeks after every immunization to obtain serum for ELISA assessment. In the ELISA experiments, the phosphorylated polypeptide (whose sequence is SEQ ID NO: 1354) coupled with BSA was used as the coating antigen, and was phosphorylated at sites corresponding to amino acids 202, 205, 396 and 404 of full-length Tau protein. The change in the concentration of antibodies in the mouse serum is shown in
Example 17: Evaluation of the Immunogenicity of Phosphorylated Polypeptide Antigen A12 Coupled with a Norovirus P Protein in Wild Mice
(89) Wild mice were intramuscularly immunized with the vaccine prepared by combining phosphorylated polypeptide antigen A12 coupled with PP-3K with AS02+CpG adjuvant. Each group contained 6 mice. The immune dosage of the protein was 25 g/animal. The immunization was carried out at week 0, week 2, week 4, week 6 and week 12. Blood was taken two weeks after every immunization to obtain serum for ELISA assessment. In the ELISA experiments, the phosphorylated polypeptide (whose sequence is SEQ ID NO: 1356) coupled with BSA was used as the coating antigen, and was phosphorylated at sites corresponding to amino acids 212, 214, 231 and 235 of full-length Tau protein. The change in the concentration of antibodies in the mouse serum is shown in
Example 18: Evaluation of the Immunogenicity of Phosphorylated Polypeptide Antigen A12 Coupled with a Norovirus P Protein in P301S Transgenic Mice
(90) P301S transgenic mice were intramuscularly immunized with the vaccine prepared by combining phosphorylated polypeptide antigen A12 coupled with PP-3K with AS02+CpG adjuvant. Each group contained 12 mice. The immune dosage of the protein was 25 g/animal. The immunization was carried out at week 0, week 2, week 4, week 6 and week 12. Blood was taken two weeks after every immunization to obtain serum for ELISA assessment. In the ELISA experiments, the phosphorylated polypeptide (whose sequence is SEQ ID NO: 1356) coupled with BSA was used as the coating antigen, and was phosphorylated at sites corresponding to amino acids 212, 214, 231 and 235 of full-length Tau protein. The change in the concentration of antibodies in the mouse serum is shown in
Example 19: Evaluation of the Cellular Immunity of Phosphorylated Polypeptide Antigen A5 Coupled with a Norovirus P Protein in P301S Transgenic MiceELISPOT Detection
(91) A96-well plate was coated with the monoclonal antibody against cytokine interferon (from elispot kit purchased from BD Corporation) in a concentration of 5 g/mL (50 L/well), and covered at 4 C. overnight. After discarding the coating antibody and washing once with a complete medium containing 10% fetal bovine serum, 200 L of this complete medium was added to each well. Blocking was carried out at 37 C. for 1 hour, and then the medium was discarded. Mice used in the experiment were sacrificed by neck-pulling. Spleen cells of the mice were taken out and formulated into a cell suspension in a cell concentration of 107/mL. The cell suspension was added to the coated 96-well plate (100 L/well). 100 L of 1 g/mL prokaryotically expressed full-length Tau protein and nonphosphorylated A5 polypeptide antigen were added to each well. The plate was then cultured at 37 C. in an incubator containing 5% CO.sub.2 for 24 h to stimulate and activate the cells. 24 h later, the plate was washed two times with sterile water, and six times with sterile PBST (pH7.4, 0.01 mol/L PBS, containing 0.05% Tween-20) buffer to wash the cells away. 50 L of 2 g/mL antibody against interferon (from elispot kit purchased from BD Corporation) was added to each well and incubated at room temperature for 2 hours. The 96-well plate was washed, and horse radish peroxidase labeled biotin secondary antibody (from elispot kit purchased from BD Corporation) was added (50 L/well). The plate was cultured at room temperature for 2 h, and washed four times with PBST, and two times with PBS. 50 L of Elispot color developing solution (AEC substrate) was added to each well and reacted in the dark at room temperature for 5-60 min. The staining solution was discarded, and the plate was washed with distilled water. After being dried overnight, the sample was calculated for the number of activated cells using a microscope.
(92) The experimental results are shown in
Example 20: Evaluation of the Cellular Immunity of Phosphorylated Polypeptide Antigen A11 Coupled with a Norovirus P Protein in P301S Transgenic MiceELISPOT Detection
(93) The experiment was performed by the same method as in Example 19. The stimulus was 100 L of 1 g/mL prokaryotically expressed full-length Tau protein and nonphosphorylated A11 polypeptide antigen.
(94) The experimental results are shown in
Example 21: Evaluation of the Cellular Immunity of Phosphorylated Polypeptide Antigen A11 Coupled with a Norovirus P Protein in P301S Transgenic MiceELISPOT Detection
(95) The experiment was performed by the same method as in Example 19. The stimulus was 100 L of 1 g/mL prokaryotically expressed full-length Tau protein and nonphosphorylated A11 polypeptide antigen.
(96) The experimental results are shown in
Example 22: Evaluation of the Cellular Immunity of Phosphorylated Polypeptide Antigen A12 Coupled with a Norovirus P Protein in P301S Transgenic MiceELISPOT Detection
(97) The experiment was performed by the same method as in Example 19. The stimulus was 100 L of 1 g/mL prokaryotically expressed full-length Tau protein and nonphosphorylated A12 polypeptide antigen.
(98) The experimental results are shown in
Example 23: Evaluation of the Behavior of P301S Transgenic Mice Immunized with Phosphorylated Polypeptide Antigen A5 Coupled with a Norovirus P Protein
(99) P301S transgenic mice were immunized with phosphorylated polypeptide antigen A5 which was coupled with PP-3K and combined with AS02+CpG adjuvant. Eight weeks after the start of the experiment, mice in the immune group were subjected to a rota-rod endurance test every two weeks. The mice were placed on the mouse rota-rod apparatus, and after the rota-rod apparatus was turned on, its rotation speed rose from 5 rpm to 40 rpm within 1.5 minutes and remained constant. The time until the mice fell from the rota-rod within 300 s was recorded. The experiment showed that the immunized mice had stronger endurance in the rota-rod experiment than the PBS group (the results are shown in
Example 24: Evaluation of the Behavior of P301S Transgenic Mice Immunized with Phosphorylated Polypeptide Antigen A11 Coupled with a Norovirus P Protein
(100) P301S transgenic mice were immunized with phosphorylated polypeptide antigen A11 which was coupled with PP-3C and combined with AS02+CpG adjuvant. Eight weeks after the start of the experiment, mice in the immune group were subjected to a rota-rod endurance test every two weeks. The mice were placed on the mouse rota-rod apparatus, and after the rota-rod apparatus was turned on, its rotation speed rose from 5 rpm to 40 rpm within 1.5 minutes and remained constant. The time until the mice fell from the rota-rod within 300 s was recorded. The experiment showed that the immunized mice had stronger endurance in the rota-rod experiment than the PBS group (the results are shown in
Example 25: Evaluation of the Behavior of P301S Transgenic Mice Immunized with Phosphorylated Polypeptide Antigen A11 Coupled with a Norovirus P Protein
(101) P301S transgenic mice were immunized with phosphorylated polypeptide antigen A11 which was coupled with PP-3K and combined with AS02+CpG adjuvant. Eight weeks after the start of the experiment, mice in the immune group were subjected to a rota-rod endurance test every two weeks. The mice were placed on the mouse rota-rod apparatus, and after the rota-rod apparatus was turned on, its rotation speed rose from 5 rpm to 40 rpm within 1.5 minutes and remained constant. The time until the mice fell from the rota-rod within 300 s was recorded. The experiment showed that the immunized mice had stronger endurance in the rota-rod experiment than the PBS group (the results are shown in
Example 26: Evaluation of the Behavior of P301S Transgenic Mice Immunized with Phosphorylated Polypeptide Antigen A12 Coupled with a Norovirus P Protein
(102) P301S transgenic mice were immunized with phosphorylated polypeptide antigen A12 which was coupled with PP-3K and combined with AS02+CpG adjuvant. Eight weeks after the start of the experiment, mice in the immune group were subjected to a rota-rod endurance test every two weeks. The mice were placed on the mouse rota-rod apparatus, and after the rota-rod apparatus was turned on, its rotation speed rose from 5 rpm to 40 rpm within 1.5 minutes and remained constant. The time until the mice fell from the rota-rod within 300 s was recorded. The experiment showed that the immunized mice had stronger endurance in the rota-rod experiment than the PBS group (the results are shown in
Example 27: Evaluation of the Behavior of P301S Transgenic Mice Immunized with Phosphorylated Polypeptide Antigen A5 Coupled with a Norovirus P Protein
(103) P301S transgenic mice were immunized with phosphorylated polypeptide antigen A5 couplded to PP-3K and combined with AS02+CpG adjuvant. Ten weeks after the start of the experiment, a nesting test was performed. All mice were transferred to new sterile mouse boxes. New sterile bedding was spread evenly to a thickness of 0.8 cm/box. Two 5 cm5 cm cotton pieces were placed in the center of the right side of each mouse box, the total weight of the cotton being 1.2 g to 1.3 g. The mice were transferred to the mouse boxes at 16:00 and the indoor lighting was turned off. The lighting was turned on at 7:00 the next day and the nesting effect of mice was scored at 8:00. The labels with mouse numbers were hidden and three trained operators performed the scoring independently of each other. The average of the three scores represents the score of one mouse in the nesting test.
(104) The scoring criteria were as follows: 1: the cotton was not completely torn; 2: the cotton was completely torn for nest building, the nest being shallow and flat; 3: the cotton was completely torn for nest building, but the nest had other notches besides those for exposing drinking water and the like, or the upper edge of the nest entrance was lower than the mouse's head; 4: the cotton was completely torn for nest building, and the nest entrance was higher than the mouse's head, or the nest was three-dimensional and had no notch.
(105) The nesting test can reflect the integrity of the consciousness of mice. The experiment showed that the immunized mice performed significantly better than the PBS group in the nesting test (the results are shown in
Example 28: Evaluation of the Behavior of P301S Transgenic Mice Immunized with Phosphorylated Polypeptide Antigen A11 Coupled with a Norovirus P Protein
(106) P301S transgenic mice were immunized with phosphorylated polypeptide antigen A11 coupled with PP-3C and combined with AS02+CpG adjuvant. Ten weeks after the start of the experiment, a nesting test was performed by the same method as in Example 27. The experiment showed that the immunized mice performed significantly better than the PBS group in the nesting test (the results are shown in
Example 29: Evaluation of the Behavior of P301S Transgenic Mice Immunized with Phosphorylated Polypeptide Antigen A11 Coupled with a Norovirus P Protein
(107) P301S transgenic mice were immunized with phosphorylated polypeptide antigen A11 coupled with PP-3K and combined with AS02+CpG adjuvant. Ten weeks after the start of the experiment, a nesting test was performed by the same method as in Example 27. The experiment showed that the immunized mice performed significantly better than the PBS group in the nesting test (the results are shown in
Example 30: Evaluation of the Behavior of P301S Transgenic Mice Immunized with Phosphorylated Polypeptide Antigen A12 Coupled with a Norovirus P Protein
(108) P301S transgenic mice were immunized with phosphorylated polypeptide antigen A12 coupled with PP-3K and combined with AS02+CpG adjuvant. Ten weeks after the start of the experiment, a nesting test was performed by the same method as in Example 27. The experiment showed that the immunized mice performed significantly better than the PBS group in the nesting test (the results are shown in
Example 31: Construction of pET28a-C-PA Plasmid
(109) The plasmid containing C-PA was obtained through sequence optimization and gene synthesis (GL Bio).
(110) The C-PA genes were enriched by PCR method and the sequence of the genes was represented by SEQ ID NO: 1363. The specific method was as follows:
(111) TABLE-US-00010 SEQIDNO:1365(forward): TTTAACTTTAAGAAGGAGATATACATATGGGTGGTGGTGGTTCTTGCGG CGGCG, and SEQIDNO:1366(reverse): GTGGTGGTGGTGGTGGTGCTCGAGTTATTTAATACGCAGATACTGGCCA ATCA;
(112) The plasmid containing C-PA was subjected to PCR reaction, wherein the PCR reaction system was a KOD-Plus DNA polymerase system (purchased from TOYOBO Corporation), and the total volume of the reaction system was 50 L (5 L buffer, 0.2 mM dNTP, 1 mM magnesium sulfate, 0.3 M upstream primer and 0.3 M downstream primer, 50 ng template DNA, 1 L KOD enzyme, and water which was added to a final volume of 50 L). PCR was carried out in accordance with the reaction system instructions to obtain 50 L of PCR products. The products were subjected to agarose gel electrophoresis, and the fragments of interest were recovered using gel recovery kit (purchased from Tiangen Biotech Co., Ltd.)
(113) 1 L of BamHI enzyme and 1 L of XhoI enzyme (purchased from Takara Corporation) and 5 L of enzyme digestion buffer (purchased from Takara Corporation) were added respectively to 2 g of pET28a vector plasmid (purchased from Novagen Corporation), and finally sterile water was added to the system to reach a final volume of 50 L. Digestion was carried out at 37 C. for 2 hours. The products were subjected to agarose gel electrophoresis, and the vector fragments were recovered using gel recovery kit (purchased from Tiangen Biotech Co., Ltd.) to obtain a linear plasmid vector.
(114) The above double enzyme-digested vector fragment and fragment of interest (with a molar ratio of 1:3, and a total volume of 5 L) were mixed, and 5 L of homologous recombination mix (purchased from Taihe Corporation) was added. The ligation was carried out at 25 C. for 15 min. 10 L of the ligated products were added to DH10B competent cells (purchased from Taihe Corporation), and placed on ice for 30 min. After that, the cells were subjected to heat shock at 42 C. for 30 s, placed on ice for 2 min, and then were added to 600 L of fluid LB medium without resistance, and recovered at 200 rpm at 37 C. for 1 h. The culture broth was plated on a LB solid culture plate containing kanamycin (15 g/mL), and were placed upside down at 37 C. overnight. The recombinant clones were obtained, and the sequence was verified by sequencing, which was represented by SEQ ID NO: 1363. A pET28a-C-PA plasmid that can express C-PA protein was obtained, as shown in
Example 32: Expression and Purification of a C-PA Protein
(115) 32.1 Expression of a C-PA Protein
(116) 1 L of the recombinant plasmids prepared in the above examples were respectively added to 100 L of E. coli BL21 competent cells (purchased from TransGen Corporation), ice-bathed for 30 min, heat shocked for 90 s in a water-bath at 42 C., and then ice-bathed for 2 min. 600 L of LB medium was added to the mixture, and cultured at 180 rpm/min at 37 C. for 1 h. The mixture was coated evenly on a LB solid medium containing kanamycin (15 g/mL) resistance and cultured at 37 C. for 24 h to obtain strains that can stably express recombinant proteins. A growing colony was picked and inoculated into 20 mL of LB medium. The mixture was cultured at 220 rpm at 37 C. When the OD value of the culture mixture reached 2.0, induction by Isopropyl -D-Thiogalactoside (IPTG at a final concentration of 1.0 mmol/L) was carried out at 220 rpm at 37 C. for six hours. After the induction, the culture broth was centrifuged at 4,000 rpm for 20 min. The supernatant was discarded, and the bacterial precipitates were resuspended with PBS. Centrifugation was conducted again at 4,000 rpm for 20 min and the supernatant was discarded to obtain the bacterial precipitates containing proteins of interest.
(117) 32.2 Purification of a C-PA Protein
(118) The bacterial precipitates obtained in 32.1 were resuspended by adding 20 mL of protein buffer (pH=7.0, containing 50 mM Tris and 300 mM KCl). The bacteria were lysed by sonication on ice for 30 min. The mixture was centrifuged at 12,000 rpm at 4 C. for 30 min and the supernatant was discarded. The collected precipitates were resuspended in 8M urea buffer (pH=7.0, containing 50 mM Tris, 300 mM KCl and 8M urea) and stirred overnight at 4 C. The collected solution was centrifuged at 16,000 rpm at 4 C. for 30 min. The supernatant was collected and transferred to a dialysis bag with a cut-off size of 10 kd. Dialysis was performed in the ratio of the volume of glycine dialysate to the volume of protein solution of 10:1, and the fluid was changed every 4 hours. The dialysis was performed 6 times to slowly renature the protein. The collected protein solution was concentrated by ultrafiltration to a protein concentration of 1.0 mg/mL to 2.0 mg/mL. The results of SDS-PAGE of the protein are shown in
Example 33: Coupling Reaction of Phosphorylated Polypeptide Antigen Vaccines with BLP
Example 33.1: Ligation Reaction of C-PA with BLP
(119) The method for preparing BLP was described in the Chinese Patent Application CN 105968213. 1 mL of 15 mg/mL BLP solution was centrifuged at 5,000 g at 4 C. for 30 min, and the supernatant was discarded. BLP was resuspended with 5 mL of C-PA solution and slowly shaked at 25 C. for 30 min to prepare C-PA-BLP. Centrifuge was performed at 5,000 g for 30 min at 4 C., the supernatant was discarded, and resuspension was performed with 10 mL sterile PBS (pH=7.2). This operation was repeated 4 times. The washed precipitates were resuspended with 1 mL PBS, OD600 was detected, and the concentration of C-PA-BLP was calibrated with a BLP standard.
Example 33.2: Ligation Reaction of C-PA with BLP
(120) The method for preparing BLP was described in the Chinese Patent Application CN 105968213. 1 mL of 15 mg/mL BLP solution was centrifuged at 5,000 g at 4 C. for 30 min, and the supernatant was discarded. BLP was resuspended with 3 mL of C-PA solution and slowly shaked at 4 C. for 6 h to prepare C-PA-BLP. Centrifuge was performed at 5,000 g for 30 min at 4 C., the supernatant was discarded, and resuspension was performed with 10 mL sterile PBS (pH=7.2). This operation was repeated 4 times. The washed precipitates were resuspended with 1 mL PBS, OD600 was detected, and the concentration of C-PA-BLP was calibrated with a BLP standard.
Example 33.3: Coupling Reaction of the Phosphorylated Polypeptide Antigen Vaccines with C-PA-BLP
(121) The phosphorylated polypeptide in each of the phosphorylated polypeptide antigen vaccines in lyophilized form prepared in Example 1 was diluted with a 0.1 M ammonium bicarbonate solution (pH=7.5) to a concentration of 1 mg/mL and was quantified. In the ratio of the molar amount of the C-PA protein to the molar amount of the polypeptide of 1:30, the polypeptide solution was slowly added to the precipitates obtained after centrifugation of C-PA-BLP, and mixed slowly. The mixed system was incubated at 2 C. to 8 C. with slow shaking for 48 hours. The obtained product was centrifuged and resuspended with PBS to remove unbound polypeptide.
Example 33.4: Coupling Reaction of the Phosphorylated Polypeptide Antigen Vaccines with C-PA-BLP
(122) The phosphorylated polypeptide in each of the phosphorylated polypeptide antigen vaccines in lyophilized form prepared in Example 1 was diluted with a 0.1 M ammonium bicarbonate solution (pH=8.0) to a concentration of 2 mg/mL and was quantified. In the ratio of the molar amount of the C-PA protein to the molar amount of the polypeptide of 1:100, the polypeptide solution was slowly added to the precipitates obtained after centrifugation of C-PA-BLP, and mixed slowly. The mixed system was incubated at 2 C. to 8 C. with slow shaking for 48 hours. The obtained product was centrifuged and resuspended with PBS to remove unbound polypeptide.
Example 33.5: Coupling Reaction of the Phosphorylated Polypeptide Antigen Vaccines with C-PA-BLP
(123) The phosphorylated polypeptide in each of the phosphorylated polypeptide antigen vaccines in lyophilized form prepared in Example 1 was diluted with a 0.1 M ammonium bicarbonate solution (pH=8.0) to a concentration of 2 mg/mL and was quantified. In the ratio of the molar amount of the C-PA protein to the molar amount of the polypeptide of 1:100, the polypeptide solution was slowly added to the precipitates obtained after centrifugation of C-PA-BLP, and mixed slowly. The mixed system was incubated at 2 C. to 8 C. with slow shaking for 48 hours. The obtained product was centrifuged and resuspended with PBS to remove unbound polypeptide.
Example 34: Determination of the Ligation Efficiency of the Phosphorylated Polypeptide Antigen Product Coupled with BLP
(124) A10 mM dithiothreitol solution was added to the phosphorylated polypeptide antigen product coupled with C-PA-BLP at a volume ratio of 1:1, and mixed homogeneously, and the mixture was left to stand at room temperature to react for 16 hours. The reaction product was centrifuged at 16,000 g at 4 C. for 15 min. The supernatants were taken for HPLC determination. Using 0.5 mg/mL, 0.25 mg/ml, 0.125 mg/ml, 0.0625 mg/ml, 0.03125 mg/mL, 0.03125 mg/mL, and 0.015625 mg/mL phosphorylated polypeptide dissolved in 10 mM of a dithiothreitol solution as standard, a standard curve was plotted with the concentration of the standard versus the peak area of the phosphorylated peptide to quantitatively analyze the concentration of the test sample (the results are shown in
(125) It should be understood that specific embodiments described herein are only used for explaining the present invention, instead of limiting the present invention. The protection scope of the present invention is subject to the protection scope defined in claims. For those of ordinary skill in the art, a variety of changes and modifications can be made without departing from the spirit and scope of the present invention, and these changes and modifications should be considered to belong to the protection scope of the present invention.