CN119285713A - Senecavirus type A 3A protein antigen epitope peptide, monoclonal antibody and application thereof - Google Patents
Senecavirus type A 3A protein antigen epitope peptide, monoclonal antibody and application thereof Download PDFInfo
- Publication number
- CN119285713A CN119285713A CN202411408339.5A CN202411408339A CN119285713A CN 119285713 A CN119285713 A CN 119285713A CN 202411408339 A CN202411408339 A CN 202411408339A CN 119285713 A CN119285713 A CN 119285713A
- Authority
- CN
- China
- Prior art keywords
- type
- virus
- protein
- sai
- monoclonal antibody
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 41
- 241001632234 Senecavirus Species 0.000 title abstract description 9
- 102000036639 antigens Human genes 0.000 title description 9
- 108091007433 antigens Proteins 0.000 title description 9
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 144
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 144
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 43
- 210000004408 hybridoma Anatomy 0.000 claims abstract description 39
- 239000003814 drug Substances 0.000 claims abstract description 31
- 208000015181 infectious disease Diseases 0.000 claims abstract description 27
- 108020004707 nucleic acids Proteins 0.000 claims abstract description 22
- 102000039446 nucleic acids Human genes 0.000 claims abstract description 22
- 150000007523 nucleic acids Chemical class 0.000 claims abstract description 22
- 241000700605 Viruses Species 0.000 claims description 103
- 210000004027 cell Anatomy 0.000 claims description 33
- 230000009385 viral infection Effects 0.000 claims description 18
- 238000004321 preservation Methods 0.000 claims description 14
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 13
- 238000002360 preparation method Methods 0.000 claims description 11
- 239000012620 biological material Substances 0.000 claims description 7
- 241000486679 Antitype Species 0.000 claims description 5
- 241000780602 Senecio Species 0.000 claims 2
- 229940079593 drug Drugs 0.000 abstract description 7
- 150000001413 amino acids Chemical class 0.000 abstract description 6
- 230000000890 antigenic effect Effects 0.000 abstract 3
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 description 48
- 230000014509 gene expression Effects 0.000 description 24
- 239000007788 liquid Substances 0.000 description 17
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 16
- 230000001580 bacterial effect Effects 0.000 description 14
- 239000000243 solution Substances 0.000 description 13
- 239000003480 eluent Substances 0.000 description 11
- 239000006228 supernatant Substances 0.000 description 11
- 230000006698 induction Effects 0.000 description 10
- 238000002965 ELISA Methods 0.000 description 9
- 238000012216 screening Methods 0.000 description 9
- PXHVJJICTQNCMI-UHFFFAOYSA-N Nickel Chemical compound [Ni] PXHVJJICTQNCMI-UHFFFAOYSA-N 0.000 description 8
- 239000002671 adjuvant Substances 0.000 description 8
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 8
- 239000000427 antigen Substances 0.000 description 7
- 239000003795 chemical substances by application Substances 0.000 description 7
- 229960005486 vaccine Drugs 0.000 description 7
- 241000282898 Sus scrofa Species 0.000 description 6
- 238000004458 analytical method Methods 0.000 description 6
- 210000001124 body fluid Anatomy 0.000 description 6
- 239000010839 body fluid Substances 0.000 description 6
- 238000001514 detection method Methods 0.000 description 6
- 230000009257 reactivity Effects 0.000 description 6
- 239000000523 sample Substances 0.000 description 6
- 241000894006 Bacteria Species 0.000 description 5
- 101710172711 Structural protein Proteins 0.000 description 5
- 239000013642 negative control Substances 0.000 description 5
- 239000013641 positive control Substances 0.000 description 5
- 230000009465 prokaryotic expression Effects 0.000 description 5
- 238000011282 treatment Methods 0.000 description 5
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 4
- 238000011725 BALB/c mouse Methods 0.000 description 4
- 208000007212 Foot-and-Mouth Disease Diseases 0.000 description 4
- 241000699666 Mus <mouse, genus> Species 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 241000282887 Suidae Species 0.000 description 4
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 4
- 230000004913 activation Effects 0.000 description 4
- 239000004202 carbamide Substances 0.000 description 4
- 239000002552 dosage form Substances 0.000 description 4
- 239000003937 drug carrier Substances 0.000 description 4
- 210000003000 inclusion body Anatomy 0.000 description 4
- 239000000411 inducer Substances 0.000 description 4
- 238000000034 method Methods 0.000 description 4
- 229910052759 nickel Inorganic materials 0.000 description 4
- 230000000174 oncolytic effect Effects 0.000 description 4
- 230000002265 prevention Effects 0.000 description 4
- 238000005406 washing Methods 0.000 description 4
- 229920000936 Agarose Polymers 0.000 description 3
- 206010003445 Ascites Diseases 0.000 description 3
- 108020004414 DNA Proteins 0.000 description 3
- 241000710198 Foot-and-mouth disease virus Species 0.000 description 3
- 241001465754 Metazoa Species 0.000 description 3
- 241000699670 Mus sp. Species 0.000 description 3
- 241000709664 Picornaviridae Species 0.000 description 3
- 239000003085 diluting agent Substances 0.000 description 3
- 201000010099 disease Diseases 0.000 description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 239000013604 expression vector Substances 0.000 description 3
- 239000000945 filler Substances 0.000 description 3
- 230000003053 immunization Effects 0.000 description 3
- 230000002163 immunogen Effects 0.000 description 3
- 230000001939 inductive effect Effects 0.000 description 3
- 230000003834 intracellular effect Effects 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 238000003259 recombinant expression Methods 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 239000011347 resin Substances 0.000 description 3
- 229920005989 resin Polymers 0.000 description 3
- 239000012089 stop solution Substances 0.000 description 3
- 239000000758 substrate Substances 0.000 description 3
- 208000024891 symptom Diseases 0.000 description 3
- 239000013598 vector Substances 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- 238000001262 western blot Methods 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 238000011537 Coomassie blue staining Methods 0.000 description 2
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 2
- 241000588724 Escherichia coli Species 0.000 description 2
- 102100023727 Mitochondrial antiviral-signaling protein Human genes 0.000 description 2
- 101710142315 Mitochondrial antiviral-signaling protein Proteins 0.000 description 2
- 206010028980 Neoplasm Diseases 0.000 description 2
- 108091092724 Noncoding DNA Proteins 0.000 description 2
- 108700026244 Open Reading Frames Proteins 0.000 description 2
- 239000012648 POLY-ICLC Substances 0.000 description 2
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 description 2
- 101800001523 Protein 3AB Proteins 0.000 description 2
- 239000004480 active ingredient Substances 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- 230000007910 cell fusion Effects 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 238000011097 chromatography purification Methods 0.000 description 2
- 230000001419 dependent effect Effects 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 238000003745 diagnosis Methods 0.000 description 2
- 239000012153 distilled water Substances 0.000 description 2
- 239000012636 effector Substances 0.000 description 2
- 230000009368 gene silencing by RNA Effects 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 210000004754 hybrid cell Anatomy 0.000 description 2
- 230000028993 immune response Effects 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 229930027917 kanamycin Natural products 0.000 description 2
- 229960000318 kanamycin Drugs 0.000 description 2
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 2
- 229930182823 kanamycin A Natural products 0.000 description 2
- 108700021021 mRNA Vaccine Proteins 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 108020004999 messenger RNA Proteins 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 239000013612 plasmid Substances 0.000 description 2
- 239000013600 plasmid vector Substances 0.000 description 2
- 108700002563 poly ICLC Proteins 0.000 description 2
- 229940115270 poly iclc Drugs 0.000 description 2
- 229920001184 polypeptide Polymers 0.000 description 2
- 238000001556 precipitation Methods 0.000 description 2
- 102000004196 processed proteins & peptides Human genes 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 239000011535 reaction buffer Substances 0.000 description 2
- 238000007789 sealing Methods 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 238000000654 solvent vapour annealing Methods 0.000 description 2
- 238000000527 sonication Methods 0.000 description 2
- 208000003265 stomatitis Diseases 0.000 description 2
- 208000006531 swine vesicular disease Diseases 0.000 description 2
- 208000005925 vesicular stomatitis Diseases 0.000 description 2
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 1
- 108020004705 Codon Proteins 0.000 description 1
- 108700010070 Codon Usage Proteins 0.000 description 1
- 208000035473 Communicable disease Diseases 0.000 description 1
- 108010041986 DNA Vaccines Proteins 0.000 description 1
- 229940021995 DNA vaccine Drugs 0.000 description 1
- 241000709661 Enterovirus Species 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 102000004457 Granulocyte-Macrophage Colony-Stimulating Factor Human genes 0.000 description 1
- 108010017213 Granulocyte-Macrophage Colony-Stimulating Factor Proteins 0.000 description 1
- 108700039932 Hepatitis A virus 3A Proteins 0.000 description 1
- 101800000120 Host translation inhibitor nsp1 Proteins 0.000 description 1
- 108010002350 Interleukin-2 Proteins 0.000 description 1
- 101800000517 Leader protein Proteins 0.000 description 1
- 101710107904 NADH-ubiquinone oxidoreductase subunit 9 Proteins 0.000 description 1
- 101800000512 Non-structural protein 1 Proteins 0.000 description 1
- 108700001237 Nucleic Acid-Based Vaccines Proteins 0.000 description 1
- 206010035226 Plasma cell myeloma Diseases 0.000 description 1
- 108091036407 Polyadenylation Proteins 0.000 description 1
- 101710132845 Protein P1 Proteins 0.000 description 1
- 229940022005 RNA vaccine Drugs 0.000 description 1
- 101100276209 Rhizobium meliloti (strain 1021) cya1 gene Proteins 0.000 description 1
- 241000837158 Senecavirus A Species 0.000 description 1
- 208000032140 Sleepiness Diseases 0.000 description 1
- 206010041349 Somnolence Diseases 0.000 description 1
- 210000001015 abdomen Anatomy 0.000 description 1
- 208000022531 anorexia Diseases 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 230000000840 anti-viral effect Effects 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 230000008827 biological function Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 239000002775 capsule Substances 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000005574 cross-species transmission Effects 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 101150084863 cya gene Proteins 0.000 description 1
- 101150101102 cyaA gene Proteins 0.000 description 1
- 125000004122 cyclic group Chemical group 0.000 description 1
- 206010061428 decreased appetite Diseases 0.000 description 1
- 230000005860 defense response to virus Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- 238000003113 dilution method Methods 0.000 description 1
- DGLRDKLJZLEJCY-UHFFFAOYSA-L disodium hydrogenphosphate dodecahydrate Chemical compound O.O.O.O.O.O.O.O.O.O.O.O.[Na+].[Na+].OP([O-])([O-])=O DGLRDKLJZLEJCY-UHFFFAOYSA-L 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 241001493065 dsRNA viruses Species 0.000 description 1
- 235000013601 eggs Nutrition 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 210000002472 endoplasmic reticulum Anatomy 0.000 description 1
- 210000003527 eukaryotic cell Anatomy 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 210000000003 hoof Anatomy 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 238000002649 immunization Methods 0.000 description 1
- 238000010166 immunofluorescence Methods 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 239000002955 immunomodulating agent Substances 0.000 description 1
- 230000002584 immunomodulator Effects 0.000 description 1
- 229940121354 immunomodulator Drugs 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000002458 infectious effect Effects 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 230000015788 innate immune response Effects 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 239000007928 intraperitoneal injection Substances 0.000 description 1
- 208000030175 lameness Diseases 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 229940057995 liquid paraffin Drugs 0.000 description 1
- 229940126582 mRNA vaccine Drugs 0.000 description 1
- 210000001700 mitochondrial membrane Anatomy 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 235000019796 monopotassium phosphate Nutrition 0.000 description 1
- 201000000050 myeloid neoplasm Diseases 0.000 description 1
- 239000002105 nanoparticle Substances 0.000 description 1
- 230000000955 neuroendocrine Effects 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 229940023146 nucleic acid vaccine Drugs 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 230000003950 pathogenic mechanism Effects 0.000 description 1
- 229940023041 peptide vaccine Drugs 0.000 description 1
- 230000003285 pharmacodynamic effect Effects 0.000 description 1
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 239000001103 potassium chloride Substances 0.000 description 1
- 235000011164 potassium chloride Nutrition 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 210000001236 prokaryotic cell Anatomy 0.000 description 1
- 230000000069 prophylactic effect Effects 0.000 description 1
- 229940021993 prophylactic vaccine Drugs 0.000 description 1
- 238000011321 prophylaxis Methods 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 229940126583 recombinant protein vaccine Drugs 0.000 description 1
- 230000010076 replication Effects 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- 239000006152 selective media Substances 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 230000000405 serological effect Effects 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 230000037321 sleepiness Effects 0.000 description 1
- 210000004989 spleen cell Anatomy 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 230000004936 stimulating effect Effects 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 229940126585 therapeutic drug Drugs 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 229940021747 therapeutic vaccine Drugs 0.000 description 1
- 238000009210 therapy by ultrasound Methods 0.000 description 1
- 238000011200 topical administration Methods 0.000 description 1
- 230000002463 transducing effect Effects 0.000 description 1
- 108091005703 transmembrane proteins Proteins 0.000 description 1
- 102000035160 transmembrane proteins Human genes 0.000 description 1
- 210000003956 transport vesicle Anatomy 0.000 description 1
- 239000012137 tryptone Substances 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 230000029812 viral genome replication Effects 0.000 description 1
- 239000013603 viral vector Substances 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
Landscapes
- Peptides Or Proteins (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
本发明属于生物医药技术领域，具体公开了一种A型塞内卡病毒3A蛋白抗原表位肽或其编码核酸，与A型塞内卡病毒3A蛋白抗原表位特异性结合的单克隆抗体，分泌该单克隆抗体的杂交瘤细胞株及其在制备预防和/或治疗A型塞内卡病毒感染的药物、或者检测和/或诊断A型塞内卡病毒感染的试剂中的应用。本发明提供的单克隆抗体由保藏编号为CCTCC NO：C202466的杂交瘤细胞株SVA‑3A‑5A7分泌产生，该单抗能够特异性结合A型塞内卡病毒3A蛋白（抗原表位位于3A蛋白胞外结构域第5‑36位氨基酸），可用于制备预防和/或治疗A型塞内卡病毒感染的药物，或者检测和/或诊断A型塞内卡病毒感染的试剂。
The present invention belongs to the field of biomedicine technology, and specifically discloses a type A Senecavirus 3A protein antigenic epitope peptide or its encoding nucleic acid, a monoclonal antibody that specifically binds to the type A Senecavirus 3A protein antigenic epitope, a hybridoma cell line that secretes the monoclonal antibody, and its use in preparing a drug for preventing and/or treating type A Senecavirus infection, or a reagent for detecting and/or diagnosing type A Senecavirus infection. The monoclonal antibody provided by the present invention is secreted and produced by the hybridoma cell line SVA-3A-5A7 with a deposit number of CCTCC NO: C202466. The monoclonal antibody can specifically bind to the type A Senecavirus 3A protein (the antigenic epitope is located at the 5-36 amino acids of the extracellular domain of the 3A protein), and can be used to prepare a drug for preventing and/or treating type A Senecavirus infection, or a reagent for detecting and/or diagnosing type A Senecavirus infection.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to a type A sai virus 3A protein epitope peptide or encoding nucleic acid thereof, a monoclonal antibody specifically combined with the type A sai virus 3A protein epitope, a hybridoma cell strain secreting the monoclonal antibody and application of the hybridoma cell strain in preparation of medicines for preventing and/or treating type A sai virus infection or reagents for detecting and/or diagnosing type A sai virus infection.
Background
A-type Seikovia virus (Senecavirus A, SVA) is a single stranded RNA virus without a envelope, belonging to the family picornaviridae, the only member of the genus Seikovia. The major susceptible animal of the virus is swine. The clinical symptoms caused by SVA infection are very similar to swine infectious vesicular disease such as Foot and Mouth Disease (FMD), swine Vesicular Disease (SVD), vesicular Stomatitis (VS) and the like. Infected pigs mostly show anorexia, sleepiness, lameness, and clinical symptoms such as blisters in the mouth, nose and hoof crowns. Meanwhile, SVA has oncolytic property, can selectively infect tumor cells with neuroendocrine property, and shows the prospect of cancer treatment in early clinical experiments. Due to the widespread popularity of SVAs, and the characteristic natural oncolytic effects, it is increasingly well known.
SVA virus particles are in icosahedral structure, spherical and have diameters of 17-25nm. The full length of the SVA genome is approximately 7300nt, consisting of one 6543nt open reading frame, 668nt 5 'non-coding region, and 68nt 3' non-coding region in poly (A) tail. The open reading frame of SVA can encode approximately 2180 amino acids with the typical L-4-3-4 genomic layout of other picornaviruses, namely, leader protein L, intermediate protein P1 (split into four structural proteins VP1, VP2, VP3, and VP 4), P2 (split into three non-structural proteins 2A, 2B, and 2C), P3 (split into four non-structural proteins 3A, 3B, 3C, and 3D). It was found by prediction that the 3A protein is a transmembrane protein, and consists of an extracellular domain, a transmembrane domain and an intracellular domain. By sequence comparison, the extracellular structural sequence and the transmembrane domain sequence of the 3A protein are conserved, while the intracellular domain sequence is highly different, which may be related to the interaction between host proteins.
3A is one of the non-structural proteins of SVA, and related researches on 3A proteins are very few at present. However, it was found that the 3A protein of other viruses of the family Picornaviridae plays an important role. As in the 3A protein of foot-and-mouth disease, the differences in the intracellular domain sequences may be related to the cross-species transmission mechanism. The membrane-anchored region of hepatitis A Virus 3A protein can bind to the transducing protein MAVS on the outer mitochondrial membrane, resulting in degradation of MAVS by the 3C protein, inhibiting the host's natural immune response. The 3A protein of the 71 enterovirus can inhibit the expression of host antiviral response related proteins by preventing the formation of endoplasmic reticulum transport vesicles, and meanwhile, the 3A protein can bind and protect long-chain ds RNA from being cut by Dicer, so that RNAi (ribonucleic acid interference) channels can be effectively inhibited, and the replication of viruses is promoted. However, SVA 3A eggs have not been reported in the aspects of evading host cell antiviral natural immune response, so that screening of 3A monoclonal antibodies is helpful for the research of biological functions of 3A proteins.
Meanwhile, in the analysis of in vivo antibody kinetics of test pigs after challenge, the level of the antibodies generated against the nonstructural protein 3AB is obviously higher than that of other nonstructural proteins and structural proteins VP1. The 3A protein not only exists in a mature form, but also can be used as a component part of the precursor protein 3AB, and has better immunogenicity when expressed in early and late stages of virus infection. Therefore, the sensitivity of the SVA antibody detection method established based on the 3A protein is probably higher, and the SVA antibody detection method has potential as a diagnostic target.
Disclosure of Invention
The invention mainly solves the technical problem of providing an A-type sai virus 3A protein epitope peptide which is used for stimulating and inducing organisms to generate effector molecules (antibodies) and effector cells and playing an immune effect so as to kill the A-type sai virus.
Secondly, the invention provides application of an epitope peptide of the 3A protein of the A-type sai virus or encoding nucleic acid thereof.
Again, the invention provides a monoclonal antibody that specifically binds to an epitope of the type a sai virus 3A protein, and a hybridoma cell line that secretes the monoclonal antibody.
The invention further provides application of the anti-A-type sai virus 3A protein monoclonal antibody or hybridoma cell strain.
Finally, the present invention provides a medicament for preventing and/or treating an infection by a type a sai virus, or a reagent for detecting and/or diagnosing an infection by a type a sai virus.
In order to solve the technical problems, the invention provides the following technical scheme:
an epitope peptide of a type A sai virus 3A protein, which comprises an amino acid sequence shown as SEQ ID NO. 1.
The antigen epitope provided by the invention is positioned at the 5 th to 36 th amino acids of the extracellular domain of the A-type sai virus 3A protein (GenBank: QOJ79820.1, amino acid sequence region of 1397 th to 1486 th, 90aa, shown as SEQ ID NO: 2).
A coding nucleic acid of an epitope peptide of a 3A protein of an A-type sai virus.
Specifically, the encoding nucleic acid of the epitope peptide is DNA or RNA. Sequence optimization can be performed according to codon preference without changing the encoded amino acid sequence.
A biological material comprising an epitope peptide of the 3A protein of the type a sai virus or a nucleic acid encoding the same.
In particular, the biological material includes, but is not limited to, recombinant expression vectors (e.g., plasmid vectors, viral vectors), gene expression cassettes, recombinant bacteria, host cells (e.g., prokaryotic cells, eukaryotic cells), and the like.
An application of an A-type sai virus 3A protein epitope peptide or a coding nucleic acid and a biological material thereof.
As a preferred embodiment of the invention, the use includes any one or more of the following:
(1) The application in preparing the medicine for preventing and/or treating the A-type sai virus infection;
(2) Use in the preparation of a reagent for detecting and/or diagnosing an infection with a type a sai virus.
A medicament for the prevention and/or treatment of an infection by a type a sai virus, said medicament comprising one or more of the following active ingredients:
(1) An epitope peptide of a 3A protein of the A-type sai virus;
(2) Encoding nucleic acid of an epitope peptide of a 3A protein of the A-type sai virus;
(3) A biological material comprising an epitope peptide of the 3A protein of the type a sai virus or a nucleic acid encoding the same.
In particular, the medicament is a prophylactic and/or therapeutic vaccine. The vaccine can be a nucleic acid vaccine, wherein the nucleic acid comprises encoding nucleic acid of an epitope peptide of a 3A protein of the A-type sai virus, such as a DNA vaccine and an RNA vaccine. The vaccine can also be a polypeptide vaccine, a recombinant protein vaccine or a synthetic long peptide vaccine, wherein the vaccine comprises an A type sai virus 3A protein antigen epitope peptide.
As a preferred embodiment of the present invention, the vaccine further comprises an immunomodulator or adjuvant selected from one or more of poly-ICLC (poly-ICLC), 1018ISS, amplivax, MF, AS03, AS04, AS15, BCG, CP-870, CP-893, cpG7909, cyaA, cyclic dinucleotides (e.g., STING), dSLIM, GM-CSF, IL-2, IC30, IC31, montanide ISA TM (e.g., montanide ISA 51), etc.
An agent for detecting and/or diagnosing an infection with a type a sai virus, said agent comprising one or more of the following:
(1) An epitope peptide of a 3A protein of the A-type sai virus;
(2) And detecting a primer and/or a probe of the encoding nucleic acid of the A-type sai virus 3A protein epitope peptide.
Specifically, the reagent is an immunodetection and/or diagnosis reagent, and the reagent contains an antigen epitope peptide of a type A sai virus 3A protein, and can be used for detecting whether antibodies generated after infection of the type A sai virus exist in pig body fluid.
Specifically, the reagent is a molecular detection and/or diagnosis reagent, and the reagent comprises a primer, a probe and the like for detecting encoding nucleic acid of an antigen epitope peptide of the A-type sai virus 3A protein, and can be used for detecting whether the A-type sai virus (namely the A-type sai virus 3A protein) exists in pig body fluid.
As a preferred embodiment of the present invention, the agent further comprises a pharmaceutically acceptable carrier, adjuvant, etc.
Specifically, for an immunodetection and/or diagnostic reagent, the reagent may further comprise one or more of a sample diluent, an enzyme-labeled plate, a blocking solution, a wash solution, a substrate, a stop solution, a negative control, a positive control, and the like.
Specifically, for molecular detection and/or diagnostic reagents, the reagents further comprise one or more of a polymerase (RNA and/or DNA dependent polymerase), an amplification reaction buffer, a positive control, a negative control, and the like.
An anti-A type sai virus 3A protein monoclonal antibody is secreted by a Hybridoma cell strain SVA-3A-5A7 (hybrid mia CELL LINE SVA-3A-5A 7) which is preserved in China center for type culture collection and has a preservation number of CCTCC NO: C202466.
Specifically, the monoclonal antibody specifically recognizes and binds to amino acids 5-36 of the extracellular domain of the type A sai virus 3A protein, especially the 3A protein.
Specifically, the monoclonal antibody is of the IgG2b type, and the light chain type is of the Kappa type.
A hybridoma cell strain for preparing an anti-type A sai virus 3A protein monoclonal antibody. The hybridoma cell strain is preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of C202466.
Specifically, the hybridoma cell strain is prepared by immunizing a mouse by taking an A-type sai virus 3A protein as an immunogen.
An application of anti-A type sai virus 3A protein monoclonal antibody and hybridoma cell strain.
As a preferred embodiment of the invention, the use includes any one or more of the following:
(1) The application in preparing the medicine for preventing and/or treating the A-type sai virus infection;
(2) Use in the preparation of a reagent for detecting and/or diagnosing an infection with a type a sai virus.
A medicament for the prevention and/or treatment of an infection by a type a sai virus, said medicament comprising one or more of the following active ingredients:
(1) An anti-a sai virus 3A protein monoclonal antibody;
(2) A hybridoma cell strain for preparing the anti-type A sai virus 3A protein monoclonal antibody.
As a preferred embodiment of the present invention, the pharmaceutical dosage form is a pharmaceutically acceptable dosage form comprising a pharmaceutically acceptable carrier, adjuvant and/or adjuvant.
As a preferred embodiment of the present invention, the medicament may be administered by topical administration at a pharmaceutically acceptable dose.
An agent for detecting and/or diagnosing an infection with a type a sai virus, said agent comprising one or more of the following:
(1) An anti-a sai virus 3A protein monoclonal antibody;
(2) A hybridoma cell strain for preparing the anti-type A sai virus 3A protein monoclonal antibody.
In particular, the reagent can be used to detect the presence or absence of type a sai virus (i.e., sai virus type a 3A protein) in porcine body fluid.
As a preferred embodiment of the present invention, the agent further comprises a pharmaceutically acceptable carrier, adjuvant, etc., as described above.
The invention has the beneficial effects that:
According to the sequence of the A-type sai virus 3A protein (GenBank: QOJ79820.1, amino acid sequence region of 1397-1486, 90 aa) published by NCBI, a prokaryotic expression system is constructed to express and purify the 3A protein, and the 3A protein is used as an immunogen to immunize a BALB/c mouse to prepare a hybridoma cell strain.
The monoclonal antibody provided by the invention is secreted by a Hybridoma cell strain SVA-3A-5A7 (hybrid oma CELL LINE SVA-3A-5A 7) which is preserved in China center for type culture collection and has the preservation number of CCTCC NO: C202466, can be specifically combined with an A-type saint virus 3A protein, and can be used for preparing a medicament for preventing and/or treating A-type saint virus infection, or a reagent for detecting and/or diagnosing A-type saint virus infection.
According to the invention, through analysis of epitope regions of the 3A protein, most regions of the 3A protein have better antigenicity, and the epitope recognized by the monoclonal antibody 5A7 is positioned at amino acids 5-36 of the extracellular domain of the 3A protein. The synthesized epitope peptide can also be used for preparing medicines for preventing and/or treating A-type sai virus infection, or reagents for detecting and/or diagnosing A-type sai virus infection, and has good clinical application prospect and higher economic value.
Drawings
FIG. 1 shows SDS-PAGE in experimental examples to verify optimal induction of 3A protein IPTG;
FIG. 2 shows the optimal expression pattern of the 3A protein by SDS-PAGE in experimental examples;
FIG. 3 shows the expression level of 3A protein when SDS-PAGE is performed to verify that the induction is performed at 37℃for 6 hours in experimental examples;
FIG. 4 shows the expression level of 3A protein when SDS-PAGE is performed to verify that induction is performed at 25℃for 12 hours in experimental examples;
FIG. 5 shows the expression level of 3A protein when SDS-PAGE is performed to verify that the induction is performed at 16℃for 20 hours in experimental examples;
FIG. 6 shows the difference in protein concentration after elution of 3A protein imidazole by SDS-PAGE in experimental examples;
In the figure, 1:3A protein passes through a nickel column, 2:3A protein passes through liquid, 3:20mM imidazole eluent, 4:40mM imidazole eluent, first milliliter eluent after 5:250mM imidazole is eluted, third milliliter eluent after 6:250mM imidazole is eluted, fifth milliliter eluent after 7:250mM imidazole is eluted, seventh milliliter eluent after 8:250mM imidazole is eluted, ninth milliliter eluent after 9:250mM imidazole is eluted, eleventh milliliter eluent after 10:250mM imidazole is eluted, thirteenth milliliter eluent after 11:250mM imidazole is eluted, and fifteenth milliliter eluent after 12:250mM imidazole is eluted;
FIG. 7 shows the reactivity of Western blot to determine the reactivity of the monoclonal antibody SVA-3A-5A7 to 3A protein in experimental examples;
FIG. 8 shows the reactivity of immunofluorescence-identified monoclonal antibodies with SVA in experimental examples;
FIG. 9 shows the identification of the subtype 5A7 of monoclonal antibody in experimental examples;
FIG. 10 shows the epitope prediction of 3A protein in experimental examples;
FIG. 11 shows the reaction of his antibodies with amino acid regions at positions 5-36 and 67-87 of 3A in experimental examples;
FIG. 12 shows the reaction of the 5A7 antibody with the amino acid regions at positions 5-36 and 67-87 of 3A in experimental example;
The drawings in the examples or experimental examples are briefly described above in order to more clearly illustrate the technical solutions to be protected by the present invention. It should be understood that the above-described drawings are not to be construed as limiting the scope of the present invention in any way. Other relevant drawings may be made by those of ordinary skill in the art without undue burden from these drawings.
Detailed Description
The technical scheme of the present invention will be clearly and completely described in the following in connection with specific examples and experimental examples.
It should be understood by those skilled in the art that the following examples and experimental examples are only for illustrating the technical scheme and effect of the present invention, and should not be construed as limiting the scope of the present invention in any way. Based on the following examples, other technical solutions, such as modified, deformed or simply replaced, obtained by a person skilled in the art without any inventive effort, are all within the scope of the present invention.
The methods used in the examples and experimental examples are conventional methods unless otherwise specified.
The raw materials, reagents, equipment, etc. used in examples and experimental examples were commercially available products unless otherwise specified.
The preservation information related to the invention is as follows:
Preservation information
The preservation name is Hybridoma cell strain SVA-3A-5A7 (hybrid CELL LINE SVA-3A-5A 7).
The preservation number is CCTCC NO, C202466.
The preservation unit is China center for type culture Collection, and the address is China, wuhan and university of Wuhan.
The preservation date is 2024, 8 and 17.
Example 1
The embodiment provides an antigen epitope peptide of a 3A protein of an A-type sai virus and a coding nucleic acid thereof, wherein the antigen epitope peptide comprises an amino acid sequence shown as SEQ ID NO. 1.
The embodiment also provides a recombinant bacterium or host cell containing the A-type sai virus 3A protein epitope peptide.
The embodiment also provides a recombinant expression vector (such as a plasmid vector and a virus vector) or a gene expression cassette containing the encoding nucleic acid of the A-type sai virus 3A protein epitope peptide.
The embodiment also provides application of the A-type sai virus 3A protein epitope peptide, recombinant bacteria or host cells in preparing medicaments for preventing and/or treating infection of the A-type sai virus.
The embodiment also provides an application of the encoding nucleic acid of the A-type sai virus 3A protein epitope peptide, the recombinant expression vector or the gene expression cassette in preparing a reagent for detecting and/or diagnosing the A-type sai virus infection.
Example 2
The present example provides a medicament for preventing infection by a type a sai virus, which is an mRNA vaccine comprising a pharmaceutically effective amount of mRNA delivered by Lipid Nanoparticles (LNPs), and an appropriate amount of an adjuvant. The mRNA is codon optimized and can code an antigen epitope peptide of an A-type sai-kava virus 3A protein (the amino acid sequence is shown as SEQ ID NO: 1).
The embodiment also provides a medicine for treating the A-type sai virus infection, wherein the medicine is a synthetic polypeptide vaccine, and the vaccine comprises a pharmacodynamic amount of an epitope peptide (the same as the above) of the A-type sai virus 3A protein and a proper amount of an adjuvant.
The embodiment also provides a reagent for detecting and/or diagnosing the infection of the A-type sai virus, wherein the reagent is an enzyme-linked immunosorbent assay reagent, and the reagent comprises an antigen epitope peptide of the 3A protein of the A-type sai virus, a sample diluent, an ELISA plate, a sealing liquid, a washing liquid, a substrate, a stop solution, a negative control and a positive control. The reagent is mainly used for detecting whether antibodies generated after infection of the type A sai virus exist in pig body fluid.
The embodiment also provides a reagent for detecting and/or diagnosing the infection of the type A sai virus, wherein the reagent is a molecular diagnostic reagent, and the reagent comprises a primer and a probe for detecting encoding nucleic acid of the epitope peptide of the 3A protein of the type A sai virus, polymerase (polymerase dependent on RNA and/or DNA), an amplification reaction buffer solution, positive control and negative control. The reagent is mainly used for detecting whether the A-type saint virus (namely the A-type saint virus 3A protein) exists in the body fluid of pigs.
Example 3
The embodiment provides an anti-A-type sai virus 3A protein monoclonal antibody which is secreted and generated by a Hybridoma cell strain SVA-3A-5A7 (hybrid om CELL LINE SVA-3A-5A 7) which is preserved in China center for type culture collection and has the preservation number of CCTCC NO: C202466. The monoclonal antibody is of an IgG2b type, the light chain type is of a Kappa type, and the monoclonal antibody can specifically identify and bind with the 3A protein of the A-type sai virus, particularly the 5 th-36 th amino acid of the extracellular domain of the 3A protein.
The embodiment also provides a hybridoma cell strain for preparing the anti-A-type sai virus 3A protein monoclonal antibody, wherein the hybridoma cell strain is preserved in China center for type culture collection (China center for type culture collection), and the preservation number is CCTCC NO: C202466. The hybridoma cell strain is prepared by immunizing mice by taking an A-type sai virus 3A protein as an immunogen.
The embodiment also provides an application of the anti-A type sai virus 3A protein monoclonal antibody or hybridoma cell strain in preparing a medicine for preventing and/or treating A type sai virus infection.
The embodiment also provides an application of the anti-A type sai virus 3A protein monoclonal antibody or hybridoma cell strain in preparing a reagent for detecting and/or diagnosing the A type sai virus infection.
Example 4
This example provides a medicament for the prophylaxis and/or treatment of infection by type a sai virus comprising a pharmaceutically effective amount of an anti-sai virus type a 3A protein monoclonal antibody (as in example 3) and an appropriate amount of an adjuvant.
The embodiment also provides a reagent for detecting and/or diagnosing the infection of the A-type sai virus, wherein the reagent is an enzyme-linked immunosorbent assay reagent, and the reagent comprises an anti-A-type sai virus 3A protein monoclonal antibody, a sample diluent, an ELISA plate, a sealing liquid, a washing liquid, a substrate, a stop solution, a negative control and a positive control. The reagent is mainly used for detecting whether the A-type saint virus (namely the A-type saint virus 3A protein) exists in the body fluid of pigs.
In other embodiments of the invention, the medicament further comprises other pharmaceutically effective components for use in the combination or auxiliary prevention and/or treatment of an infection by a type a sai virus.
In other embodiments of the invention, the dosage form of the medicament is other pharmaceutically acceptable dosage forms, such as capsules, tablets and the like, which contain other pharmaceutically acceptable carriers and auxiliary materials.
In other embodiments of the present invention, the dosage of the drug is a pharmaceutically acceptable dosage, and the dosage of the drug is determined according to the disease condition and is in compliance with the medical prescription.
Experimental example
1. Experimental materials
The expression strain containing the recombinant plasmid PET-28a-SVA-3A is preserved by Henan agricultural university laboratory.
BALB/c mice were purchased from Henan laboratory animal center.
0.01M PBS 8g sodium chloride, 0.2g potassium chloride, 3.63g disodium hydrogen phosphate dodecahydrate and 0.24g potassium dihydrogen phosphate are weighed and dissolved in deionized water, the volume is fixed to 1L, and the mixture is sterilized under high pressure for standby.
8M Urea 480g of urea was weighed and dissolved in 0.01M PBS to a volume of 1L.
Weighing 2g of Tryptone, 1g of Yeast Extract and 2g of sodium chloride, placing into a 500mL conical flask, adding 200mL of single distilled water, completely dissolving, adjusting pH to 7.0, sterilizing under high pressure, and storing at 4 ℃ for later use.
2. Experimental method
1. Preparation of SAV 3A protein
1.1 Screening of inducer IPTG concentration
A frozen pET-28a-3A expression strain is taken and inoculated into 5mL of liquid LB medium containing k + (50 mg/mL) and cultured on a 37 ℃ air shaking table. When the OD600nm value of the bacterial liquid is measured to be between 0.6 and 0.8, the bacterial liquid is added into 5 groups of 5mL of liquid LB culture medium containing k + (50 mg/mL) according to the volume ratio of 1:100, and the parent bacteria are placed at 4 ℃ for short-term storage for later experiments. And then placing 5 groups of bacterial solutions on an air shaking table for culture, and when the OD600nm value of the bacterial solutions is between 0.6 and 0.8, respectively adding IPTG with the final concentration of 0, 0.3, 0.5, 0.8 and 1mM into the 5 groups of bacterial solutions, and continuing to induce the expression for 6 hours. After induction, the 5 groups of bacterial solutions were centrifuged at 12000rpm for 2min to enrich the bacterial cells, the supernatant was discarded, 250. Mu.L of PBS was added for resuspension, and 10. Mu.L of SDS-PAGE samples were obtained after resuspension for SDS-PAGE analysis. The results are shown in FIG. 1.
1.2 Determination of protein supernatant expression and precipitation expression
The preserved bacterial liquid is added into 5mL of antibiotic-free LB culture medium according to the volume ratio of 1:100 for activation for 1h. After activation kanamycin was added at a final concentration of 50g/mL and subsequently incubated on an air shaker until OD600nm was between 0.6 and 0.8. The inducer IPTG was added at a final concentration of 0.5mM and the induction of expression was continued for 6h. After induction expression, the bacterial liquid is taken out and centrifuged at 12000rpm for 2min to enrich bacterial cells, the supernatant is removed, 250 mu L of PBS is added for resuspension, and the working parameters of an ultrasonic crusher are set to be that under the condition of 0 ℃, the ultrasonic operation is started for 1s and stopped for 1s, and the total ultrasonic operation is carried out for 15min. After completion of the sonication, the inclusion bodies were dissolved by using 250. Mu.L of 8M urea, and 10. Mu.L of each of the supernatant and the inclusion body solution was taken out to prepare SDS-PAGE samples, and SDS-PAGE analysis was performed. The results are shown in FIG. 2.
1.3 Screening for optimal temperature and time of inducible expression
The preserved bacterial liquid is added into 3 groups of 5mL LB medium without antibiotics according to the volume ratio of 1:100 for activation for 1h. After activation kanamycin was added at a final concentration of 50. Mu.g/mL and subsequently incubated on an air shaker until OD600nm was between 0.6 and 0.8. The inducer IPTG was added to each group at a final concentration of 0.5mM, and induction was performed at 37℃for 6 hours, 25℃for 12 hours, and 16℃for 20 hours, respectively. After induction expression, taking bacterial liquid 12000rpm for centrifugation for 2min, discarding supernatant, adding 250 mu L PBS into the enriched bacterial body for resuspension, and setting the working parameters of an ultrasonic crusher to be 1s at 0 ℃, stopping ultrasonic treatment for 15min. After completion of the sonication, the inclusion bodies were dissolved by using 250. Mu.L of 8M urea, and 10. Mu.L of each of the supernatant and the inclusion body solution was taken out to prepare SDS-PAGE samples, and SDS-PAGE analysis was performed. The results are shown in FIGS. 3-5.
1.4Ni-Agarose Resin chromatographic purification
The Ni-Agarose Resin filler was added to the column by mixing and washing 3 times with PBS before use. The nickel column was equilibrated with TBS containing 10mM imidazole. And then, the crushed bacterial supernatant is blown and evenly mixed with the filler, and the mixture is transferred into a centrifuge tube to be inversely mixed for 3 hours at the temperature of 4 ℃ so that the protein and the filler are fully combined. Adding the well-mixed protein into a chromatographic column, respectively adding 10mL of 20mM and 40mM of imidazole to wash out the hybrid protein, eluting the target protein with 15mL of 250mM of imidazole, and collecting the eluent according to each 1 mL. The nickel column is washed by distilled water and then preserved by 20% absolute ethanol. The eluate was placed in a dialysis bag with a pore size of 8000D, dialyzed overnight at 4℃in 0.01MPBS, the protein concentration was determined, sub-packaged and stored at-80 ℃. SDS-PAGE samples were prepared from 10mM, 20mM and 250mM imidazole eluates and analyzed by SDS-PAGE. The results are shown in FIG. 6.
2. Preparation of monoclonal antibodies
2.1 Screening of positive hybridoma cell lines
Purified SVA 3A protein was inoculated into BALB/c mice by subcutaneous multipoint injection at a dose of 50. Mu.g/mouse. After multiple immunizations, serum titers of immunized mice were detected by a 3A indirect ELISA method, and when the titers reached cell fusion conditions, mouse spleen cells and mouse myeloma cells (SP 2/0) were taken for cell fusion. Screening and removing unsuccessfully fused cells by using HAT selective medium, changing the liquid after 5-7 days of cell mass grows to a proper size, extracting the cell culture liquid after 2-3 days, performing indirect ELISA detection by using a 3A protein coated ELISA plate, and preliminarily determining that the cells in the wells of 2 cell culture plates can generate antibodies capable of reacting with the 3A prokaryotic expression protein. And then screening out specific cell strains by using ELISA plates coated by the 3A protein, SVA virus liquid and a vector pET-28a irrelevant protein expressed by an homologous escherichia coli expression system. Cell counting is carried out by a limiting dilution method, 200 cells are taken for subcloning, and whether target cells exist in the cell holes is determined by an indirect ELISA method. And after repeated times, amplifying and timely freezing the screened cell strains. Cell supernatants were taken to verify the effect with 3A protein and SVA virus. The results are shown in FIGS. 7-8.
2.2 Culture of hybridoma cells and preparation of monoclonal antibody ascites
SVA-5A7 hybridoma cells were removed from the liquid nitrogen tank and thawed rapidly in a 37℃water bath, the cells were washed with serum-free hybridoma medium, cultured in a T75-based flask with 1% HT hybridoma culture, and maintained in a good state until the cells grew to a full and clear state (i.e., optimal state).
Healthy BALB/c mice were selected and 500. Mu.L of liquid paraffin was injected by intraperitoneal injection one week before the injection of cells. After washing the hybridoma cells with serum-free DMEM, 1×10 6 hybridoma cells were resuspended with 500 μl of serum-free DMEM, and when the abdomen of the mice began to swell, ascites were extracted. The ascites was centrifuged at 12000rpm for 5min, the uppermost layer of the fat and the bottom precipitate were discarded, and then filtered through a 0.22 μm filter for use. Subtype identification was performed using the antibody subtype identification kit, and the results are shown in fig. 9.
Prediction and identification of 2.3va 3a protein epitope
The 3A protein is analyzed by using an epitope prediction website, and the cutting position of the 3A protein is selected according to the distribution condition of the epitope region, as shown in figure 10. The truncated protein was obtained by ligating the base sequence of the truncated protein to pET-28a plasmid and expressing E.coli BL21 (DE 3) expressing bacteria. The reactivity between the monoclonal anti-SVA-3A-5A 7 and 3A truncated proteins was then identified by Western blot. The results are shown in FIGS. 11-12.
3. Experimental results
1. 3A protein expression and purification
1.1 Determination of optimal concentration of inducer IPTG
As shown in FIG. 1, when the IPTG concentration was in the range of 0.3-0.8mM, the expression level of the 3A protein supernatant was high, and the optimum concentration of IPTG was finally determined to be 0.5mM.
1.2 Determination of expression of protein supernatant and expression pattern of precipitation
As shown in FIG. 2, the 3A protein supernatant was expressed in a high amount by SDS-PAGE Coomassie blue staining.
1.3 Screening for optimal temperature and time of inducible expression
As shown in FIGS. 3-5, the expression level of the 3A protein was highest when the induction was performed at 37℃for 6 hours.
1.4Ni-Agarose Resin chromatographic purification
As shown in FIG. 6, proteins bound to the nickel column were found to be competitively eluted by SDS-PAGE Coomassie blue staining with 250mM imidazole, and higher concentrations of the protein of interest were found to be contained in the first to fifteenth milliliters of eluate.
2. Preparation of 3A monoclonal antibodies
2.1 Screening of positive hybridoma cell lines
As shown in FIG. 7, the antibodies produced by the cell lines screened by Western blot verification reacted specifically with the expressed and purified 3A protein, a distinct band appeared, whereas no band appeared when incubated with the pET-28a blank vector prokaryotic expression protein. The monoclonal antibody generated by the screened cell strain has good specificity, can recognize the 3A protein and has good reactivity with the 3A protein.
As shown in FIG. 8, the antibodies produced by the screened cell lines were confirmed by Immunofluorescence (IFA) to react with IBRS-2 cells after SVA infection, and fluorescence was observed in the cells, whereas no IBRS-2 cells not infected with SVA. The monoclonal antibodies produced by the selected cell lines are shown to have good reactivity with SVA virus.
The finally selected cell strain is named SVA-3A-5A7, a part of the cell strain is taken and sent to China center for type culture collection of university of Wuhan for patent program preservation, the preservation name is Hybridoma cell strain SVA-3A-5A7 (hybrid CELL LINE SVA-3A-5A 7), and the preservation number is CCTCC NO: C202466.
2.2 Monoclonal antibodies produced by hybridoma cell line SVA-3A-5A7
As shown in FIG. 9, the results of the subtype identification of the monoclonal antibody indicate that monoclonal antibody 5A7 is of the IgG2b type and the light chain types are all Kappa type.
2.3 3A protein epitope prediction and identification
The 3A protein consists of an extracellular domain, a transmembrane domain and an intracytoplasmic domain, and as shown in FIG. 10, the analysis of the monoclonal antibody 5A7 by an epitope prediction website can mainly identify the 5 th to 36 th amino acid regions and 67 th to 87 th amino acid regions of the 3A protein.
As shown in FIG. 11, the amino acid regions at positions 5-36 and 67-87 of the 3A protein are normally expressed by a prokaryotic expression system.
As shown in FIG. 12, the identification result shows that the 5A7 monoclonal antibody reacts with the 5-36 amino acid region of the 3A protein.
4. Conclusion(s)
SVAs have gradually assumed a global popularity since their discovery in 2002. The clinical symptoms of the disease are very similar to animal A virulent infectious diseases-foot-and-mouth disease released by world animal health organization, and influence the global live pig trade. And is well known in tumor therapy for its oncolytic properties. However, the pathogenic mechanisms of SVA are still poorly understood, which severely restricts the progress of development of efficient prevention and control products and oncolytic agents for SVA. The SVA 3A protein plays an important role in the innate immunity against the host and in the viral replication process. The development of a study of the 3A protein will help to further understand the biological properties of SVA.
The invention obtains a large amount of purified A-type sai virus 3A protein by means of prokaryotic expression, and prepares monoclonal antibodies aiming at the A-type sai virus 3A protein. Provides a material basis for the mechanism research of the 3A protein in the future, the establishment of the clinical serological detection method of the Seneca virus and the screening of clinical therapeutic drugs.
Although the technical solutions of the present invention have been described in detail in the foregoing general description, the specific embodiments and the experimental examples, it should be noted that the examples and the experimental examples are only for illustrating the technical solutions and the technical effects of the present invention, and should not be construed as limiting the scope of the present invention. Simple variations, modifications or improvements made on the basis of the technical idea of the invention fall within the scope of the invention as claimed.
Claims (10)
1. The A-type sai virus 3A protein epitope peptide is characterized by comprising an amino acid sequence shown as SEQ ID NO. 1.
2. A nucleic acid encoding the epitope peptide of the type a sai virus 3A protein of claim 1.
3. A biological material comprising the saint-type a saint-virus 3A protein epitope peptide of claim 1, or the encoding nucleic acid of claim 2 thereof.
4. Use of an epitope peptide of the 3A protein of the type a saint virus according to claim 1, or of a nucleic acid encoding said protein of claim 2, or of a biological material according to claim 3, characterized in that said use comprises any one or more of the following:
(1) The application in preparing the medicine for preventing and/or treating the A-type sai virus infection;
(2) Use in the preparation of a reagent for detecting and/or diagnosing an infection with a type a sai virus.
5. A medicament for preventing and/or treating type A Senecio cinerea virus infection, or a reagent for detecting and/or diagnosing type A Senecio cinerea virus infection, characterized in that the medicament or reagent comprises one or more of the following components:
(1) The sai-a virus 3A protein epitope peptide of claim 1;
(2) A nucleic acid encoding an epitope peptide of the 3A protein of the saint-a virus of claim 2;
(3) A biological material comprising an epitope peptide of the 3A protein of the type a saint virus as claimed in claim 3 or a nucleic acid encoding the same.
6. An anti-A type sai virus 3A protein monoclonal antibody is secreted by a Hybridoma cell strain SVA-3A-5A7 (hybrid om a CELL LINE SVA-3A-5A 7) which is preserved in China center for type culture collection and has the preservation number of CCTCC NO: C202466, and the monoclonal antibody specifically recognizes and binds to the A type sai virus 3A protein epitope peptide of claim 1.
7. A hybridoma cell strain for preparing the anti-A-type sai virus 3A protein monoclonal antibody according to claim 6, wherein the hybridoma cell strain is preserved with a CCTCC NO. C202466 and is preserved in China center for type culture collection.
8. Use of the anti-sai virus type a 3A protein monoclonal antibody according to claim 6 or the hybridoma cell line according to claim 7, wherein the use comprises any one or more of the following:
(1) The application in preparing the medicine for preventing and/or treating the A-type sai virus infection;
(2) Use in the preparation of a reagent for detecting and/or diagnosing an infection with a type a sai virus.
9. A medicine for preventing and/or treating A-type sai virus infection is characterized in that the medicine comprises one or more of the following medicinal components:
(1) The anti-type a sai virus 3A protein monoclonal antibody of claim 6;
(2) A hybridoma cell line for the preparation of monoclonal antibodies against the sai-type a virus 3A protein according to claim 7.
10. A reagent for detecting and/or diagnosing an infection with a type A Session virus, characterized in that the reagent comprises one or more of the following components:
(1) The anti-type a sai virus 3A protein monoclonal antibody of claim 6;
(2) A hybridoma cell line for the preparation of monoclonal antibodies against the sai-type a virus 3A protein according to claim 7.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202411408339.5A CN119285713A (en) | 2024-10-10 | 2024-10-10 | Senecavirus type A 3A protein antigen epitope peptide, monoclonal antibody and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202411408339.5A CN119285713A (en) | 2024-10-10 | 2024-10-10 | Senecavirus type A 3A protein antigen epitope peptide, monoclonal antibody and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN119285713A true CN119285713A (en) | 2025-01-10 |
Family
ID=94150210
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202411408339.5A Pending CN119285713A (en) | 2024-10-10 | 2024-10-10 | Senecavirus type A 3A protein antigen epitope peptide, monoclonal antibody and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN119285713A (en) |
-
2024
- 2024-10-10 CN CN202411408339.5A patent/CN119285713A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| WO2021168875A1 (en) | Helicobacter pylori ferritin-based novel coronavirus s protein double-region subunit nanovaccine | |
| CN111560074A (en) | A novel coronavirus S protein single-domain subunit nanovaccine based on Helicobacter pylori ferritin | |
| CN113512096B (en) | Weever rhabdovirus recombinant G2 protein and application thereof | |
| CN105566497A (en) | Ferritin fusion proteins for use in vaccines and other applications | |
| CN101955545B (en) | Multi-target recombination gene and application of protein thereof in preventing and treating infection of helicobacter pylori | |
| Zhao et al. | Immersion vaccination of Mandarin fish Siniperca chuatsi against infectious spleen and kidney necrosis virus with a SWCNTs-based subunit vaccine | |
| EP4410843A1 (en) | Recombinant fusion protein derived from hr region of s2 protein of sars-cov-2 and application of recombinant fusion protein | |
| JP2013538195A (en) | Drugs and methods for prevention of HIV contamination, diseases caused by HIV including AIDS and diseases associated with HIV | |
| WO2023051701A1 (en) | Mrna, protein and vaccine against sars-cov-2 infection | |
| CN116693632A (en) | African swine fever virus epitope peptide, monoclonal antibody and application thereof | |
| WO2015200254A1 (en) | Compositions, methods and therapies for administering antigen peptide | |
| CN114989312B (en) | A Mycobacterium tuberculosis fusion protein DR2 and its application | |
| CN114874336A (en) | A kind of Echinococcus granulosus recombinant protein EgG1Y162-2 (4) and its application | |
| CN102406929B (en) | Co-expressed molecular adjuvant enhanced divalent foot and mouth disease protein engineering vaccine | |
| WO2025040122A1 (en) | Beta-coronavirus recombinant chimeric antigen, preparation method therefor and use thereof | |
| CN119285713A (en) | Senecavirus type A 3A protein antigen epitope peptide, monoclonal antibody and application thereof | |
| CN113527516B (en) | A-type seneca virus genetic engineering composite epitope protein, vaccine and application thereof | |
| CN117106102A (en) | Enterovirus multi-antigen epitope fusion protein, gene, vaccine and preparation method thereof | |
| CN105777909A (en) | A-type foot-and-mouth disease targeting composite epitope protein mediated by pig chemotactic factors and vaccine | |
| CN116462743A (en) | Acinetobacter baumannii translation elongation factor recombinant protein, preparation method and application | |
| CN111748042B (en) | African swine fever fusion protein containing endotoxin and preparation method and application thereof | |
| CN102321180B (en) | Polypeptide fragment composition and application in preparation of porcine reproductive and respiratory syndrome vaccine thereof | |
| Rezaei et al. | Antibodies produced toward recombinant RBD and nucleocapsid neutralize SARS-COV-2 | |
| CN118978575B (en) | Recombinant protein antigen of Acinetobacter baumannii A1S_1027, preparation method and application thereof | |
| CN114053400B (en) | Vaccine for preventing novel coronavirus pneumonia COVID-19 and preparation method thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |