CN116731195A - 表达pedv s蛋白优势抗原区域的重组prrsv疫苗株的构建及应用 - Google Patents
表达pedv s蛋白优势抗原区域的重组prrsv疫苗株的构建及应用 Download PDFInfo
- Publication number
- CN116731195A CN116731195A CN202210793115.5A CN202210793115A CN116731195A CN 116731195 A CN116731195 A CN 116731195A CN 202210793115 A CN202210793115 A CN 202210793115A CN 116731195 A CN116731195 A CN 116731195A
- Authority
- CN
- China
- Prior art keywords
- sne
- protein
- recombinant
- pedv
- rhun4
- 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
- 241001135989 Porcine reproductive and respiratory syndrome virus Species 0.000 title claims abstract description 55
- 102100031673 Corneodesmosin Human genes 0.000 title claims abstract description 52
- 101710139375 Corneodesmosin Proteins 0.000 title claims abstract description 52
- 241001135549 Porcine epidemic diarrhea virus Species 0.000 title claims abstract description 41
- 229960005486 vaccine Drugs 0.000 title claims abstract description 41
- 108091007433 antigens Proteins 0.000 title claims abstract description 25
- 239000000427 antigen Substances 0.000 title claims abstract description 24
- 102000036639 antigens Human genes 0.000 title claims abstract description 24
- 238000010276 construction Methods 0.000 title abstract description 5
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 28
- 208000005342 Porcine Reproductive and Respiratory Syndrome Diseases 0.000 claims abstract description 20
- 238000001514 detection method Methods 0.000 claims abstract description 13
- 239000002773 nucleotide Substances 0.000 claims abstract description 3
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 3
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 23
- 208000015181 infectious disease Diseases 0.000 claims description 22
- 208000033380 Paroxysmal exertion-induced dyskinesia Diseases 0.000 claims description 18
- 201000003320 childhood onset GLUT1 deficiency syndrome 2 Diseases 0.000 claims description 18
- 239000000499 gel Substances 0.000 claims description 12
- 230000012743 protein tagging Effects 0.000 claims description 11
- 230000000694 effects Effects 0.000 claims description 7
- 101150010882 S gene Proteins 0.000 claims description 6
- 108010076504 Protein Sorting Signals Proteins 0.000 claims description 5
- 239000003153 chemical reaction reagent Substances 0.000 claims description 5
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 claims description 4
- 239000003814 drug Substances 0.000 claims description 4
- 239000008194 pharmaceutical composition Substances 0.000 claims description 4
- 229920001184 polypeptide Polymers 0.000 claims description 4
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 4
- 230000015572 biosynthetic process Effects 0.000 claims description 3
- 238000003786 synthesis reaction Methods 0.000 claims description 3
- 239000004793 Polystyrene Substances 0.000 claims description 2
- 239000002671 adjuvant Substances 0.000 claims description 2
- 239000011324 bead Substances 0.000 claims description 2
- 229960002685 biotin Drugs 0.000 claims description 2
- 235000020958 biotin Nutrition 0.000 claims description 2
- 239000011616 biotin Substances 0.000 claims description 2
- 239000002552 dosage form Substances 0.000 claims description 2
- 229940079593 drug Drugs 0.000 claims description 2
- 239000007850 fluorescent dye Substances 0.000 claims description 2
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 claims description 2
- 238000004519 manufacturing process Methods 0.000 claims description 2
- 239000004005 microsphere Substances 0.000 claims description 2
- 239000007923 nasal drop Substances 0.000 claims description 2
- 229940100662 nasal drops Drugs 0.000 claims description 2
- 229940100652 nasal gel Drugs 0.000 claims description 2
- 229940052404 nasal powder Drugs 0.000 claims description 2
- 238000006386 neutralization reaction Methods 0.000 claims description 2
- 229920002223 polystyrene Polymers 0.000 claims description 2
- 239000000843 powder Substances 0.000 claims description 2
- 238000002360 preparation method Methods 0.000 claims description 2
- 230000002265 prevention Effects 0.000 claims description 2
- 238000012216 screening Methods 0.000 claims description 2
- 239000007921 spray Substances 0.000 claims description 2
- 241000700605 Viruses Species 0.000 abstract description 76
- 101150054963 SNE gene Proteins 0.000 abstract description 15
- 230000014509 gene expression Effects 0.000 abstract description 8
- 238000010353 genetic engineering Methods 0.000 abstract description 8
- 238000011160 research Methods 0.000 abstract description 8
- 102000004169 proteins and genes Human genes 0.000 abstract description 7
- 238000011161 development Methods 0.000 abstract description 6
- 238000001262 western blot Methods 0.000 abstract description 6
- 230000002068 genetic effect Effects 0.000 abstract description 4
- 125000003275 alpha amino acid group Chemical group 0.000 abstract description 2
- 229940125575 vaccine candidate Drugs 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 29
- 230000003472 neutralizing effect Effects 0.000 description 27
- 239000013612 plasmid Substances 0.000 description 21
- 239000000047 product Substances 0.000 description 16
- 239000002299 complementary DNA Substances 0.000 description 13
- 239000012634 fragment Substances 0.000 description 11
- 101710141454 Nucleoprotein Proteins 0.000 description 10
- 241000282887 Suidae Species 0.000 description 10
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 9
- 238000004113 cell culture Methods 0.000 description 9
- 238000006243 chemical reaction Methods 0.000 description 9
- 108091008146 restriction endonucleases Proteins 0.000 description 9
- 238000000338 in vitro Methods 0.000 description 8
- 238000000034 method Methods 0.000 description 8
- 238000011084 recovery Methods 0.000 description 8
- 238000012163 sequencing technique Methods 0.000 description 8
- 238000013518 transcription Methods 0.000 description 8
- 230000035897 transcription Effects 0.000 description 8
- 238000001976 enzyme digestion Methods 0.000 description 7
- 238000003776 cleavage reaction Methods 0.000 description 6
- 230000002829 reductive effect Effects 0.000 description 6
- 230000007017 scission Effects 0.000 description 6
- 239000000243 solution Substances 0.000 description 6
- 239000013598 vector Substances 0.000 description 6
- 241000711573 Coronaviridae Species 0.000 description 5
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 5
- 230000003321 amplification Effects 0.000 description 5
- 238000001962 electrophoresis Methods 0.000 description 5
- 238000003199 nucleic acid amplification method Methods 0.000 description 5
- 244000052769 pathogen Species 0.000 description 5
- 238000003757 reverse transcription PCR Methods 0.000 description 5
- 230000003612 virological effect Effects 0.000 description 5
- 229920000936 Agarose Polymers 0.000 description 4
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 4
- 108020004705 Codon Proteins 0.000 description 4
- 108020004414 DNA Proteins 0.000 description 4
- 108700026244 Open Reading Frames Proteins 0.000 description 4
- 229940031567 attenuated vaccine Drugs 0.000 description 4
- 230000012010 growth Effects 0.000 description 4
- 230000002458 infectious effect Effects 0.000 description 4
- 238000002844 melting Methods 0.000 description 4
- 238000001890 transfection Methods 0.000 description 4
- 206010012735 Diarrhoea Diseases 0.000 description 3
- 238000012408 PCR amplification Methods 0.000 description 3
- 101000702488 Rattus norvegicus High affinity cationic amino acid transporter 1 Proteins 0.000 description 3
- 239000011543 agarose gel Substances 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 238000000137 annealing Methods 0.000 description 3
- 230000009286 beneficial effect Effects 0.000 description 3
- 239000012228 culture supernatant Substances 0.000 description 3
- 238000012258 culturing Methods 0.000 description 3
- 230000000120 cytopathologic effect Effects 0.000 description 3
- 238000004925 denaturation Methods 0.000 description 3
- 230000036425 denaturation Effects 0.000 description 3
- 238000013461 design Methods 0.000 description 3
- 239000012154 double-distilled water Substances 0.000 description 3
- 238000002649 immunization Methods 0.000 description 3
- 230000003053 immunization Effects 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 230000034217 membrane fusion Effects 0.000 description 3
- 238000002156 mixing Methods 0.000 description 3
- 239000002245 particle Substances 0.000 description 3
- 238000012257 pre-denaturation Methods 0.000 description 3
- 102000005962 receptors Human genes 0.000 description 3
- 108020003175 receptors Proteins 0.000 description 3
- 230000002441 reversible effect Effects 0.000 description 3
- 239000002356 single layer Substances 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 238000002255 vaccination Methods 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- 241000283707 Capra Species 0.000 description 2
- 208000035473 Communicable disease Diseases 0.000 description 2
- 238000007400 DNA extraction Methods 0.000 description 2
- 102100038132 Endogenous retrovirus group K member 6 Pro protein Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 102000012750 Membrane Glycoproteins Human genes 0.000 description 2
- 108010090054 Membrane Glycoproteins Proteins 0.000 description 2
- 101150001779 ORF1a gene Proteins 0.000 description 2
- 101001028244 Onchocerca volvulus Fatty-acid and retinol-binding protein 1 Proteins 0.000 description 2
- 231100000645 Reed–Muench method Toxicity 0.000 description 2
- 239000006180 TBST buffer Substances 0.000 description 2
- 238000000246 agarose gel electrophoresis Methods 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 238000010367 cloning Methods 0.000 description 2
- 230000029087 digestion Effects 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 241001493065 dsRNA viruses Species 0.000 description 2
- 239000012091 fetal bovine serum Substances 0.000 description 2
- 239000012894 fetal calf serum Substances 0.000 description 2
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 2
- 239000003292 glue Substances 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 230000028993 immune response Effects 0.000 description 2
- 230000001965 increasing effect Effects 0.000 description 2
- 230000000670 limiting effect Effects 0.000 description 2
- 229940023832 live vector-vaccine Drugs 0.000 description 2
- 238000012423 maintenance Methods 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000001717 pathogenic effect Effects 0.000 description 2
- 238000004321 preservation Methods 0.000 description 2
- 230000035755 proliferation Effects 0.000 description 2
- 239000013639 protein trimer Substances 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 238000004064 recycling Methods 0.000 description 2
- 239000012096 transfection reagent Substances 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- NOIIUHRQUVNIDD-UHFFFAOYSA-N 3-[[oxo(pyridin-4-yl)methyl]hydrazo]-N-(phenylmethyl)propanamide Chemical compound C=1C=CC=CC=1CNC(=O)CCNNC(=O)C1=CC=NC=C1 NOIIUHRQUVNIDD-UHFFFAOYSA-N 0.000 description 1
- 101000977065 Acidithiobacillus ferridurans Uncharacterized 11.6 kDa protein in mobS 3'region Proteins 0.000 description 1
- 101000787133 Acidithiobacillus ferridurans Uncharacterized 12.3 kDa protein in mobL 3'region Proteins 0.000 description 1
- 101000787132 Acidithiobacillus ferridurans Uncharacterized 8.2 kDa protein in mobL 3'region Proteins 0.000 description 1
- 101000827262 Acidithiobacillus ferrooxidans Uncharacterized 18.9 kDa protein in mobE 3'region Proteins 0.000 description 1
- 241000004176 Alphacoronavirus Species 0.000 description 1
- 101000811747 Antithamnion sp. UPF0051 protein in atpA 3'region Proteins 0.000 description 1
- 241001292006 Arteriviridae Species 0.000 description 1
- 101000827603 Bacillus phage SPP1 Uncharacterized 10.2 kDa protein in GP2-GP6 intergenic region Proteins 0.000 description 1
- 101000827607 Bacillus phage SPP1 Uncharacterized 8.5 kDa protein in GP2-GP6 intergenic region Proteins 0.000 description 1
- 101000765604 Bacillus subtilis (strain 168) FlaA locus 22.9 kDa protein Proteins 0.000 description 1
- 101000961975 Bacillus thuringiensis Uncharacterized 13.4 kDa protein Proteins 0.000 description 1
- 206010006895 Cachexia Diseases 0.000 description 1
- 101000964407 Caldicellulosiruptor saccharolyticus Uncharacterized 10.7 kDa protein in xynB 3'region Proteins 0.000 description 1
- 101000964402 Caldicellulosiruptor saccharolyticus Uncharacterized protein in xynC 3'region Proteins 0.000 description 1
- 208000003322 Coinfection Diseases 0.000 description 1
- 101000861180 Cupriavidus necator (strain ATCC 17699 / DSM 428 / KCTC 22496 / NCIMB 10442 / H16 / Stanier 337) Uncharacterized protein H16_B0147 Proteins 0.000 description 1
- 208000005156 Dehydration Diseases 0.000 description 1
- 101710091045 Envelope protein Proteins 0.000 description 1
- 101710088172 HTH-type transcriptional regulator RipA Proteins 0.000 description 1
- 101000768777 Haloferax lucentense (strain DSM 14919 / JCM 9276 / NCIMB 13854 / Aa 2.2) Uncharacterized 50.6 kDa protein in the 5'region of gyrA and gyrB Proteins 0.000 description 1
- 101710126256 Hydrolase in agr operon Proteins 0.000 description 1
- 101000607404 Infectious laryngotracheitis virus (strain Thorne V882) Protein UL24 homolog Proteins 0.000 description 1
- 101000735632 Klebsiella pneumoniae Uncharacterized 8.8 kDa protein in aacA4 3'region Proteins 0.000 description 1
- 108091026898 Leader sequence (mRNA) Proteins 0.000 description 1
- 101000977779 Lymantria dispar multicapsid nuclear polyhedrosis virus Uncharacterized 33.9 kDa protein in PE 3'region Proteins 0.000 description 1
- 101000977786 Lymantria dispar multicapsid nuclear polyhedrosis virus Uncharacterized 9.7 kDa protein in PE 3'region Proteins 0.000 description 1
- 108010052285 Membrane Proteins Proteins 0.000 description 1
- 102000018697 Membrane Proteins Human genes 0.000 description 1
- 101000827630 Narcissus mosaic virus Uncharacterized 10 kDa protein Proteins 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 108090001074 Nucleocapsid Proteins Proteins 0.000 description 1
- -1 ORF1b Proteins 0.000 description 1
- 101150009852 ORF2 gene Proteins 0.000 description 1
- 239000012124 Opti-MEM Substances 0.000 description 1
- 229930040373 Paraformaldehyde Natural products 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 229940124158 Protease/peptidase inhibitor Drugs 0.000 description 1
- 101710137397 Protein ORF3 Proteins 0.000 description 1
- 229940096437 Protein S Drugs 0.000 description 1
- 101710188315 Protein X Proteins 0.000 description 1
- 241001112091 Pseudoviridae Species 0.000 description 1
- 238000010802 RNA extraction kit Methods 0.000 description 1
- 101001113905 Rice tungro bacilliform virus (isolate Philippines) Protein P4 Proteins 0.000 description 1
- 101710198474 Spike protein Proteins 0.000 description 1
- 101000818100 Spirochaeta aurantia Uncharacterized 12.7 kDa protein in trpE 5'region Proteins 0.000 description 1
- 101001037658 Streptomyces coelicolor (strain ATCC BAA-471 / A3(2) / M145) Glucokinase Proteins 0.000 description 1
- 108091036066 Three prime untranslated region Proteins 0.000 description 1
- 108091023045 Untranslated Region Proteins 0.000 description 1
- 208000036142 Viral infection Diseases 0.000 description 1
- 206010047700 Vomiting Diseases 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 230000000890 antigenic effect Effects 0.000 description 1
- 230000002238 attenuated effect Effects 0.000 description 1
- 210000003719 b-lymphocyte Anatomy 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 210000004899 c-terminal region Anatomy 0.000 description 1
- 238000010805 cDNA synthesis kit Methods 0.000 description 1
- 239000013592 cell lysate Substances 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 239000013553 cell monolayer Substances 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000012411 cloning technique Methods 0.000 description 1
- 238000011217 control strategy Methods 0.000 description 1
- 210000004748 cultured cell Anatomy 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 230000018044 dehydration Effects 0.000 description 1
- 238000006297 dehydration reaction Methods 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 230000004064 dysfunction Effects 0.000 description 1
- 208000026500 emaciation Diseases 0.000 description 1
- 230000003203 everyday effect Effects 0.000 description 1
- 239000000834 fixative Substances 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 238000012215 gene cloning Methods 0.000 description 1
- 230000006801 homologous recombination Effects 0.000 description 1
- 238000002744 homologous recombination Methods 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 230000003902 lesion Effects 0.000 description 1
- 239000002502 liposome Substances 0.000 description 1
- 239000012160 loading buffer Substances 0.000 description 1
- 230000008018 melting Effects 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 229940031348 multivalent vaccine Drugs 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 238000011022 operating instruction Methods 0.000 description 1
- 229920002866 paraformaldehyde Polymers 0.000 description 1
- 230000007918 pathogenicity Effects 0.000 description 1
- 239000000137 peptide hydrolase inhibitor Substances 0.000 description 1
- 239000000546 pharmaceutical excipient Substances 0.000 description 1
- 230000008488 polyadenylation Effects 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 238000010814 radioimmunoprecipitation assay Methods 0.000 description 1
- 230000010076 replication Effects 0.000 description 1
- 230000001850 reproductive effect Effects 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 208000023504 respiratory system disease Diseases 0.000 description 1
- 238000010839 reverse transcription Methods 0.000 description 1
- 239000000523 sample Substances 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000003892 spreading Methods 0.000 description 1
- 230000007480 spreading Effects 0.000 description 1
- 239000013595 supernatant sample Substances 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- 238000010257 thawing Methods 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 238000013519 translation Methods 0.000 description 1
- 108700026220 vif Genes Proteins 0.000 description 1
- 230000009385 viral infection Effects 0.000 description 1
- 229960004854 viral vaccine Drugs 0.000 description 1
- 230000008673 vomiting Effects 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/12—Viral antigens
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/525—Virus
- A61K2039/5256—Virus expressing foreign proteins
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
- A61K2039/552—Veterinary vaccine
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/70—Multivalent vaccine
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
- C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
- C12N2770/00011—Details
- C12N2770/10011—Arteriviridae
- C12N2770/10021—Viruses as such, e.g. new isolates, mutants or their genomic sequences
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
- C12N2770/00011—Details
- C12N2770/10011—Arteriviridae
- C12N2770/10034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
- C12N2770/00011—Details
- C12N2770/20011—Coronaviridae
- C12N2770/20022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
- C12N2770/00011—Details
- C12N2770/20011—Coronaviridae
- C12N2770/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
- G01N2333/08—RNA viruses
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
- G01N2333/08—RNA viruses
- G01N2333/165—Coronaviridae, e.g. avian infectious bronchitis virus
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Virology (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Organic Chemistry (AREA)
- Biomedical Technology (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Genetics & Genomics (AREA)
- Public Health (AREA)
- Pharmacology & Pharmacy (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Animal Behavior & Ethology (AREA)
- Veterinary Medicine (AREA)
- Cell Biology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Food Science & Technology (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Mycology (AREA)
- Epidemiology (AREA)
- General Engineering & Computer Science (AREA)
- Biophysics (AREA)
- Gastroenterology & Hepatology (AREA)
- Communicable Diseases (AREA)
- Oncology (AREA)
- Chemical Kinetics & Catalysis (AREA)
Abstract
本发明提供一种表达PEDV S蛋白优势抗原区域的重组PRRSV疫苗株的构建及应用,结果表明拯救出的重组病毒rHuN4‑F112‑SNE能够表达PEDV S蛋白优势抗原区域,且重组病毒的生物学特性与亲本毒相似。将重组病毒rHuN4‑F112‑SNE在Marc‑145细胞中连续传至30代,可见不同代次重组病毒中引入的SNE基因核苷酸序列及其编码的氨基酸序列均未发生缺失或突变,IFA和Western Blot检测结果显示,不同代次的重组病毒rHuN4‑F112‑SNE均能稳定表达SNE基因,且不影响亲本毒自身蛋白的表达,表明重组病毒rHuN4‑F112‑SNE株具有遗传稳定性。本研究成功获得了能够表达PEDV S蛋白优势抗原区域的重组PRRSV基因工程疫苗候选毒株(rHuN4‑F112‑SNE),可用于预防PRRS和PED新型基因工程疫苗的研制和开发。
Description
技术领域
本发明涉及生物领域,涉及一种表达PEDV S蛋白优势抗原区域的重组PRRSVrHuN4-F112-SNE疫苗株的构建及应用。
背景技术
猪流行性腹泻(Porcine epidemic diarrhea,PED)是造成世界养猪业巨大经济损失的主要传染病之一。猪流行性腹泻病毒(porcine epidemic diarrhea virus,PEDV)是引起该病的主要病原体。PEDV可以感染所有年龄的猪,主要表现为水样腹泻、呕吐、脱水、消瘦和死亡等,而死亡率随着猪的日龄不同而变化,其中10日龄以内的新生哺乳仔猪死亡率80-100%。尽管在成年猪中由于PEDV感染而导致的死亡率较低,但感染仍可导致猪的生长性能下降。PEDV是有囊膜的单股正链RNA病毒,属于冠状病毒科,冠状病毒属,α-冠状病毒亚群的成员。PEDV基因组长约28kb,包含5'和3'非翻译区(UTR)和至少7个开放阅读框(ORF),依次为ORF1a、ORF1b、刺突蛋白(S)、辅助蛋白ORF3、包膜蛋白(E)、膜蛋白(M)和核衣壳蛋白(N)。与其他冠状病毒一样,PEDV的S蛋白位于病毒颗粒的表面,是一种同源三聚体膜糖蛋白,由S1和S2两个亚基组成,S1亚基负责受体结合,S2亚基负责膜融合。其中S1亚基由N末端结构域(NTD)和负责受体结合的C端结构域(CTD)组成。S2亚基由胞外结构域、跨膜结构域和胞内结构域三个结构域组成;而在S2亚基的胞外域包含蛋白酶切割位点、融合肽(FP)和两个七肽重复区(HR1和HR2),它们在病毒和宿主细胞膜融合中起重要作用。S蛋白作为囊膜病毒粒子上的主要表面糖蛋白,也是中和抗体的目标,现已发现S蛋白中部分具有中和作用的抗原表位,如已经证实COE结构域(aa 499–638)含有被中和抗体识别的B细胞表位以及SS2、SS6和2C10等线性中和抗原表位,因此,这些中和性抗原表位也常常被作为疫苗开发的主要候选靶标。
猪繁殖与呼吸综合征病毒(Porcine Reproductive and Respiratory Syndromevirus,PRRSV)感染可引起各年龄段猪发生呼吸系统疾病以及母猪发生繁殖障碍等临床表现。PRRSV是一种有囊膜的,单股正链的RNA病毒,属于动脉炎病毒科,它与冠状病毒科一起都属于套式病毒目。PRRSV基因组的大小约为15kb,包括5'帽子结构和3'聚腺苷酸尾巴。基因组至少包含9个开放阅读框(ORF),即:ORF 1a,ORF 1b,ORF 2a,ORF 2b,ORF 3、ORF 4、ORF5、ORF 6和ORF 7。PRRSV主要有两种基因型:PRRSV-1(欧洲型)和PRRSV-2(北美洲型)。PRRSV自发现以来一直是全球养猪业面临的主要挑战之一,2011年仅在美国,给养猪业造成的损失就达6.64亿美元。为了控制PRRS,各个国家已经采用了多种策略,包括生物安全管理,检测和清除以及疫苗接种等,在不同的PRRS控制策略中,疫苗接种依然是最常用的方法。弱毒疫苗是通过将PRRSV在培养细胞中传代致弱而产生的,能够有效保护同源病毒的感染,对高度抗原变异的异源病毒产生部分保护。因此,研制新型安全高效的疫苗已成为当前PRRSV研究的重要目标。尽管一直以来人们在PRRSV致病性和免疫学等各方面做了大量研究工作,但PRRSV有效疫苗的设计仍然面临巨大挑战。近年来随着反向遗传操作技术的发展,PRRSV感染性克隆技术使得开发针对PRRSV基因组修饰或改造的减毒活疫苗成为可能,通过PRRSV感染性克隆可以设计提高抗原性并诱导强大保护性免疫反应的新型基因工程疫苗,例如,利用反向遗传操作技术构建了不同PRRSV的嵌合疫苗。而且,现在已有研究证实PRRSV可以作为病毒疫苗载体,尤其是针对猪的一些重要病原体,它们与PRRSV共同感染会导致更严重的临床后果,因此,以PRRS疫苗毒株作为载体,递送来自其他病原体的外源基因,能够保护猪免受PRRSV和其他病原的侵害,将有利于提高PRRSV疫苗的使用效率,适用于新型基因工程疫苗的研发。
针对PRRS和PED目前尚没有可提供双重保护的商品化疫苗,而依赖单一疫苗的频繁免疫接种,不仅增加了疫苗接种成本,而且会对猪群造成过度的免疫应激,影响猪的生产性能,因此,设计一种多价疫苗以用于同时预防PRRSV和PEDV两种不同病原的感染,可为养猪业对PRRS和PED两种重要传染病的防控提供新的助力。PEDV的S蛋白位于病毒颗粒的表面,主要介导识别受体和膜融合,能够诱导产生中和抗体,是研发冠状病毒候选疫苗的理想抗原。已有研究报道证实S蛋白中存在包括COE、SS2、SS6和和2C10等部分中和性抗原表位,但是并非S蛋白中所有的抗原表位都能诱导产生中和抗体,在完整的S蛋白或S1蛋白中还有大量诱导非中和抗体的抗原表位,而非中和性抗体中也包含与抗体依赖性增强(ADE)相关的抗体,而ADE作用可能会增强病毒的感染,增加疫苗的安全隐患。只有有针对性的选择S蛋白的中和性抗原表位,最大限度的减少S蛋白中的冗余表位,这样才会提高疫苗的免疫效率,并进一步降低了疫苗的安全隐患。PRRSV弱毒疫苗HuN4-F112株在临床上已被广泛用于预防高致病性PRRSV的感染,且已证实PRRSV基因组适合作为表达外源基因的载体,因此选用PRRSV弱毒疫苗株作为重组病毒的表达载体,可以进一步提高了PRRSV疫苗的使用效率,降低疫苗的免疫接种频次,节约疫苗免疫的成本,简化疫苗的接种程序,达到一针防两病的效果。本研究,我们使用PRRSV的HuN4-F112疫苗毒株为载体,在PRRSV基因组cDNA克隆的非结构和结构基因之间区域插入含有PEDV S蛋白的中和性抗原表位区域的SNE基因,生成用于外源基因表达的重组PRRSV全长基因cDNA克隆载体,获得的重组病毒rHuN4-F112-SNE株在细胞培养中可稳定表达SNE,研究表明rHuN4-F112-SNE株可能具有作为开发针对PRRS和PED的二价基因工程活疫苗的潜力。
发明内容
为了解决上述技术问题,本发明的目的是:
提供一种针对PEDV的S蛋白融合抗原表位肽,所述S蛋白融合抗原表位肽是选择S蛋白中的具有中和活性的抗原表位以及优势抗原区域进行串联组合和优化合成,并在前端引入S基因的信号肽序列,将合成后的基因命名为SNE,基因序列为1788bp,如SEQ ID NO.1所示。
进一步的,所述抗原表位肽N端或C端还可以偶联有多肽标记;优选地,所述多肽标记为生物素标记或荧光标记。
进一步提供了上述的抗原表位肽在制备开发针对PRRS和PED的二价基因工程活疫苗中的应用。
进一步的,本发明提供一种表达PEDV S蛋白优势抗原区域的重组PRRSV rHuN4-F112-SNE疫苗株,其特征在于所述PEDV S蛋白优势抗原区域为上述S蛋白融合抗原表位肽。
进一步的,本发明提供重组PRRSV rHuN4-F112-SNE疫苗株的应用,所述应用为以PRRS疫苗毒株作为载体,递送来自PED的外源基因,能够保护猪免受PRRSV和PED的侵害,将有利于提高PRRSV疫苗的使用效率,适用于新型基因工程疫苗的研发。
本发明还提供了上述的抗原表位肽在制备用于预防或治疗PRRS和PED的药物中的应用。
本发明进一步提供了上述的抗原表位肽作为抗PRRS和PED感染药物筛选靶标的应用。
进一步的,本发明的一个实施方案中,还提供了一种用于预防或治疗PRRS和PED感染的药物组合物,其包含上述的抗原表位肽和药学上可接受的辅料。
在根据本发明的一个实施方案中,还包含佐剂,所述佐剂可选AddaVax。
进一步的,在根据本发明的一个实施方案中,所述药物组合物为经鼻施用的剂型;优选为喷雾剂、滴鼻剂、粉末剂、凝胶制剂或微球制剂。
本发明进一步提供了一种用于PRRS和PED感染的诊断试剂,其包含上述的抗原表位肽,所述抗原表位肽包被于检测载体上,所述检测载体选自聚苯乙烯微量反应板、胶体金试剂条、磁珠和微流控芯片中的任一种。
有益效果
本发明中提供的重组抗原主要包含PEDV S蛋白的已知的5个中和性抗原表位区域,减少了完整S蛋白中大量非中和性冗余抗原表位,降低了抗体依赖性增强(ADE)效应的风险因素,提高了疫苗的安全性。
所构建的重组抗原SNE包含5个已知中和性抗原表位区域经过相互无缝串联,同时对SNE基因密码子优化为哺乳动物猪偏嗜的密码子,更利于重组抗原的表达。
所构建的重组抗原SNE基因大小是PRRSV基因组可容纳的基因大小,获得的重组病毒不容易发生突变和缺失,可在Marc-145细胞稳定传代,获得的重组病毒具有遗传稳定性。
所构建的重组病毒选择的抗原能够刺激机体集中产生抑制流行变异PEDV感染的中和性抗体,提高了重组抗原诱导抗体的靶向性。
所构建的重组病毒可同时诱导产生针对PRRSV和PEDV的特异性免疫反应,产生的中和抗体可有效降低PEDV的感染能力。
进一步的,本发明中提供的重组病毒rHuN4-F112-SNE株在细胞培养中可稳定表达PEDV S蛋白优势抗原,可以作为今后开发用于预防PRRS和PED的新型基因工程活载体疫苗的候选毒株。
附图说明
图1,其中,A:选择串联组合的SNE基因抗原性分析;B:SNE包含抗原表位在S蛋白三聚体表面分布模式图;C、D为SNE基因片段的扩增电泳鉴定图,其中,C为利用SNE-F1、SNE-R1为引物进行RT-PCR扩增,结果显示,扩增出含有信号肽的SNE基因大小约1800bp左右;D为利用SNE-F2、SNE-R2、SNE-F3、SNE-R3为两对引物进行PCR扩增,结果显示,扩增出融合有AscⅠ和EcoRⅤ限制性酶切位点的SNE基因片段大小约为2000bp左右;
图2 pHuN4-F112-SNE感染性克隆质粒的鉴定;
图3拯救病毒的细胞病变结果,其中A为拯救的重组病毒rHuN4-F112-SNE感染细胞48h病变,B为正常细胞对照;
图4重组病毒rHuN4-F112-SNE的RT-PCR检测结果;
图5重组病毒rHuN4-F112-SNE的IFA鉴定;
图6重组病毒rHuN4-F112-SNE表达蛋白的Western-blot鉴定
图7重组病毒rHuN4-F112-SNE噬斑形态鉴定
图8重组病毒rHuN4-F112-SNE与亲本病毒HuN4-F112株的多步生长曲线
图9不同代次重组病毒rHuN4-F112-SNE的IFA鉴定结果
图10不同代次重组病毒rHuN4-F112-SNE的Western-blot鉴定结果
具体实施方式
在本发明中所使用的术语,除非有另外说明,一般具有本领域普通技术人员通常理解的含义。下面结合具体的实施例,并参照数据进一步详细地描述本发明。应理解,这些实施例只是对本发明进行进一步说明,不能理解为对本发明保护范围的限定,该领域的技术工程师可根据上述发明的内容对本发明作出一些非本质的改进和调整。
毒株、抗体与主要试剂
PEDV FJzz1分离株(chen et al.,2019)、PRRSV HuN4-F112弱毒疫苗株以及含HuN4-F112全长cDNA感染性克隆质粒均由本实验室分离并保存(周艳君等,2011;zhang etal.,2011),抗PRRSV N蛋白单克隆抗体和抗PEDV S蛋白单克隆抗体由本实验室制备并保存。
主要试剂:RNA提取试剂盒、PCR产物胶回收试剂盒和质粒DNA提取试剂盒购自Omega公司;2×LA Taq Premix、DL2000 DNA Marker、DL5000 DNA Marke、pMD18-T载体和低熔点琼脂糖均购自Takara公司;AscⅠ、EcoRⅤ和SwaⅠ限制性内切酶购于NEB公司;SP6启动子体外转录试剂盒mMESSAGE mMACHINER SP6操作说明书购于Ambion公司;RevertAid FirstStrand cDNA Synthesis Kit、DMRI-C转染试剂盒以及FITC标记的羊抗鼠IgG荧光二抗均购自Thermo Scientific公司;
1.2 PEDV S基因优势抗原区域的选择
由于现有疫苗中主要选择完整的S蛋白或者S1蛋白为靶蛋白,但并非S蛋白中所有的抗原表位都能诱导产生中和性抗体,其中绝大部分属于非中和性抗原表位,这些非中和性抗原表位会诱导产生大量非中和性抗体,而这些非中和性抗体中也包含与ADE作用相关的抗体,由于ADE作用可能会增强病毒的感染,如果引入大量非中和性抗原表位可能会增加疫苗的安全隐患,降低疫苗的免疫效果。为了最大限度的去除这些不利因素,本研究根据Gene Bank中公布的PEDV流行变异毒株FJzz1株S基因序列(登录号:MK288006),利用DNASTAR软件分析S蛋白氨基酸序列,与已知的中和性抗原表位进行比对分析,选择PEDV流行变异毒株FJzz1株S蛋白中包含5个已知的具有中和活性的抗原表位区域,即:26-120aa;499-638aa;744-759aa;756-771aa;1273-1283aa,进一步将将这些优势中和抗原表位区域进行串联组合,抗原性分析结果显示不同表位区域串联组合后其抗原性较强(图1A),而且本研究选择的5个中和性抗原表位均分布于S蛋白三聚体的表面(图1B)。同时,为了提高引入外源基因的表达效率,我们在基因的前端引入S基因的信号肽序列,并将目标基因密码子优化为哺乳动物猪所偏嗜的密码子,合成后将其命名为SNE,基因序列为1788bp,如SEQ IDNO.1所示。
1.3引物设计
根据PEDV流行变异毒株FJzz1株S基因序列(登录号:MK288006),以及HuN4株Nsp12基因和ORF2基因序列(登录号:EF635006),利用Primer5.0软件设计3对引物用于扩增SNE基因,其中用SNE-F1/SNE-R1扩增片段为1869bp,用SNE-F2、SNE-R2、SNE-F3、SNE-R3两对引物融合扩增的片段为2056bp,三对引物序列(SEQ ID NO.2-7)中,在SNE-F3引物中引入AscⅠ限制性酶切位点,在SNE-R3引物中引入EcoRⅤ限制性酶切位点,
同时,根据PRRSV HuN4株基因组序列(登录号:EF635006),设计用于对重组质粒或重组病毒进行插入片段鉴定的特异性引物JD1和JD2,根据片段的大小可以区分出PRRSV亲本毒株(762bp)以及插入目标基因的重组毒株(2746bp),引物序列见SEQ ID NO.8、9。上述引物均由上海派森诺生物科技有限公司合成。
1.4目的片段扩增
利用Omega公司的Total RNA Kit I提取PEDV流行变异毒株FJzz1株基因组RNA,再用反转录试剂盒将RNA反转录为cDNA,然后以cDNA为模板,用SNE-F1/SNE-R1引物,进行第一轮PCR扩增,PCR反应体系为50μl:包括2×LA Taq Premix 25μl,引物SNE-F1、SNE-R1各2.0μL(10pmol/L),cDNA模板1.0μL和ddH2O 20.0μL。PCR反应程序为:98℃预变性30s,98℃变性10s,58℃退火30s,72℃延伸2min,进行35个循环,72℃延伸10min,4℃保存扩增产物。结果显示,扩增出含有信号肽的SNE基因大小约1800bp左右(图1C),将PCR产物用0.8%琼脂糖凝胶电泳后切胶,并利用胶回收试剂盒进行回收纯化。然后以胶回收的PCR产物为模板,以SNE-F2、SNE-R2、SNE-F3、SNE-R3为引物进行第二轮PCR扩增,PCR反应体系为50μl:包括2×LA Taq Premix 25μl,引物SNE-F2、SNE-R2、SNE-F3、SNE-R3各1.0μL(10pmol/L),cDNA模板1.0μL和ddH2O 20.0μL。PCR反应程序为:98℃预变性30s,98℃变性10s,58℃退火30s,72℃延伸2min,进行35个循环,72℃延伸10min,4℃保存扩增产物。将PCR产物用0.8%琼脂糖凝胶电泳后,切胶,利用胶回收试剂盒进行回收纯化,获得的SNE基因在5‘端引入了Asc I限制性酶切位点,在3‘末端引入了EcoR V限制性酶切位点,结果显示,扩增出融合有AscⅠ和EcoRⅤ限制性酶切位点的SNE基因片段大小约为2000bp左右(图1D),将其作为目标基因用于下一步基因克隆。
1.5 SNE重组PRRSV HuN4-F112全长cDNA感染性克隆的构建
将含HuN4-F112的全长cDNA克隆质粒pHuN4-F112利用限制性内切酶AscⅠ和EcoRⅤ进行双酶切处理,酶切体系为50μl,主要包括:限制性内切酶AscⅠ和EcoRⅤ各3μl,10×Buffer 5μl,pHuN4-F112质粒3μg,ddH2O补充至50μl。酶切反应条件为:37℃水浴3h,将酶切产物用0.8%琼脂糖凝胶进行电泳,分别切胶,利用胶回收试剂盒回收pHuN4-F112双酶切产物。
将回收的PCR产物SNE与AscⅠ/EcoRⅤ双酶切产物pHuN4-F112利用同源重组酶进行连接,同源重组连接体系为10μl(5×In-Fusion HD Enzyme premix 1μl,SNE胶回收产物6μl,pHuN4-F112质粒双酶切胶回收产物2μl,ddH2O 1μl)。然后,将其置于50℃连接60min,4℃静置5min。然后,将连接产物转入至TOP10感受态细胞中进行转化,转化产物涂布于含有Amp抗性的LB固体培养基平板上,37℃过夜培养,然后从中挑取单个菌落,于含有Amp抗性的LB液体培养基中继续在37℃摇床中扩大培养,并用质粒DNA提取试剂盒提取质粒,然后,利用JD1和JD2引物进行PCR鉴定,PCR反应体系为20μl:包括2×LA Taq Premix 10μl,引物JD1和JD2各1.0μL(10pmol/L),质粒模板1.0μL和ddH2O 7.0μL。PCR反应程序为:98℃预变性30s,95℃变性10s,58℃退火30s,72℃延伸2min,进行35个循环,72℃延伸10min,获得的PCR扩增产物通过0.8%琼脂糖凝胶电泳观察结果。pHuN4-F112-SNE阳性重组质粒可扩增出大小约2800bp左右的特异性条带,而亲本质粒pHuN4-F112则扩增出大小约760bp左右的目标条带(图2)。筛选出阳性质粒,继续进行测序鉴定,测序由上海派森诺生物科技有限公司完成。最后,将经过PCR和测序鉴定正确阳性质粒命名为pHuN4-F112-SNE,其全长核苷酸序列为SEQID NO.10所示。
1.6重组病毒的拯救
1.6.1 pHuN4-F112-SNE全长cDNA的线性化
将鉴定正确的重组PRRSV全长cDNA克隆质粒pHuN4-F112-SNE利用限制性内切酶SwaⅠ进行质粒的线性化处理,线性化体系为50μl:10×NEB buffer 5μl,限制性内切酶SwaⅠ5μl,pHuN4-F112-SNE质粒30μg,ddH2O补充至50μl。在25℃恒温条件下酶切过夜,待酶切反应结束后,利用胶回收试剂盒对酶切产物进行回收纯化。
1.6.2体外转录
将线性化的pHuN4-F112-SNE重组质粒DNA,根据SP6启动子体外转录试剂盒mMESSAGE mMACHINER SP6操作说明书进行体外转录,合成含有SNE基因的PRRSV基因组全长RNA,配置体外转录体系20μL:2×NTP/CAP 10μL,Enzyme Mix 2μL,10×Reaction Buffer 2μL,线性化pHuN4-F112-SNE重组质粒DNA 2μg,ddH2O补充至20μL。将体外转录体系置于37℃水浴条件下作用105min,收集pHuN4-F112-SNE重组质粒的体外转录RNA。
1.6.3体外转录RNA的转染
将经体外转录获得的RNA利用DMRI-C脂质体进行转染,预先将已经培养好的Marc-145细胞转接至6孔细胞培养板中,于37℃5%CO2条件下培养,当细胞形成80%以上的单层时,按照DMRI-C转染试剂说明进行转染,即:取12ul转染试剂DMRI-C加入至1ml opti-MEM培养基中,混匀后,将体外转录获得的RNA加入其中,并轻轻混匀,然后将其加入至预先用DMEM培养基洗涤处理后的6孔板中的Marc-145细胞内,置于37℃5%CO2培养箱中孵育6小时,弃去转染液,加入2ml含有2%胎牛血清的DMEM培养基,于37℃5%CO2培养箱中继续培养,每天观察细胞病变,待出现的细胞病变约80%时(图3),收集细胞培养上清液,于-80℃冻存备用,获得的重组病毒命名为rHuN4-F112-SNE。
1.6.4重组病毒传代培养
将重组病毒rHuN4-F112-SNE冻融后,经12000rpm,4℃离心10min,取上清液取200μL与500μL含有2%胎牛血清DMEM细胞维持液混合,并接种至预先用PBS洗涤后的Marc-145细胞中,于37℃5%CO2培养箱中孵育1-2h,弃去感作液,用PBS洗涤细胞,再加入含有2%胎牛血清DMEM细胞维持液继续培养,并观察细胞病变情况,待细胞病变达80%以上时,收集细胞培养上清液保存于-80℃,此后按照上述方法继续进行传代。
1.7重组病毒的鉴定
1.7.1重组病毒的RT-PCR鉴定
取200μL第五代重组病毒培养液,按照RNeasy Plus Mini Kit试剂盒说明提取病毒全基因组RNA,同时以亲本毒株HuN4-F112作为阳性对照。以反转录后的cDNA为模板,用特异性鉴定引物JD1、JD2进行PCR扩增,对重组病毒rHuN4-F112-SNE中插入的SNE基因进行鉴定。核酸电泳结果显示,第5代的重组病毒rHuN4-F112-SNE扩增出大小约为2800bp左右的单一目标条带(图4),对PCR产物进行克隆测序,结果显示,插入的SNE基因与原始序列完全一致。
1.7.1重组病毒测序鉴定
以第五代重组病毒的cDNA为模板,用特异性鉴定引物JD1/JD2进行插入片段的扩增,经0.8%琼脂糖凝胶电泳后,用胶回收试剂盒将扩增片段回收纯化,并克隆至pMD-18T载体,选取阳性质粒pMD-18T-SNE进行基因测序鉴定。
1.7.2重组病毒IFA鉴定
将亲本病毒HuN4-F112和第五代重组病毒rHuN4-F112-SNE分别感染Marc-145细胞,感染24小时后弃上清,用80%冷乙醇固定,然后分别并加入抗PRRSV N蛋白单克隆抗体和抗PEDV S蛋白单克隆抗体,37℃孵育45min,用PBS洗涤3次,加入1:1000稀释的山羊抗小鼠FITC标记的荧光二抗,于37℃避光孵育45min,经PBS洗涤3次,于倒置荧光显微镜下观察结果。结果显示,重组毒株rHuN4-F112-SNE与亲本病毒HuN4-F112株一致,都能被PRRSV N蛋白单克隆抗体特异性识别,产生特异性荧光。而利用抗PEDV S蛋白单克隆抗体检测时,仅重组病毒rHuN4-F112-SNE能观察到特异性亮绿色荧光,而亲本病毒HuN4-F112未见荧光(图5),表明插入的PEDV S蛋白优势抗原基因SNE获得了表达。
1.7.3重组病毒表达蛋白的Western-blot鉴定
将亲本病毒HuN4-F112和第五代重组病毒rHuN4-F112-SNE分别感染Marc-145细胞,感染至48-72小时后,待细胞病变明显且达到80%时,用无菌PBS洗涤细胞3次,加含有蛋白酶抑制剂的细胞裂解液RIPA裂解细胞,并收获细胞蛋白。经12000rpm 4℃离心10min,取上清,加入5×SDS上样缓冲液,煮沸10分钟。取10μL蛋白样品进行SDS-PAGE电泳,并将其转印到NC膜上,然后分别用经适当稀释的抗PRRSV N蛋白单克隆抗体和抗PEDV S蛋白单克隆抗体作为一抗,室温条件下孵育45min,TBST缓冲液洗涤3次,每次5min,然后用1:10000稀释的HRP标记的抗羊鼠IgG作为二抗,室温孵育45min,经TBST缓冲液洗涤3次后,用ECL显影液显影,在显影仪中观察显影结果。结果显示,利用抗PRRSV N蛋白单抗检测,亲本毒株和重组毒株均能够检测到大小约17KDa左右PRRSV N蛋白的表达。而利用抗PEDV S蛋白单抗检测,仅重组病毒rHuN4-F112-SNE能检测到大小约100KDa左右的特异性目标条带(图6)。表明本研究获得重组病毒rHuN4-F112-SNE不影响PRRSV自身蛋白的翻译,而且能够表达和修饰插入的目标蛋白SNE。
1.7.4重组病毒TCID50的测定
将重组毒株rHuN4-F112-SNE用含2%胎牛血清的DMEM进行10-1-10-10梯度稀释,然后分别将不同稀释的病毒液接种于预先在96孔细胞培养板中制备成单层的Marc-145细胞中,100μL/孔,每个稀释度重复8个孔,同时设2列未接毒的细胞做为阴性对照。按上述操作,重复接种2块96孔板,并置于5%CO2、37℃的细胞培养箱培养4-5天,观察记录细胞病变,并按照Reed-Muench法计算病毒TCID50。对重组毒株rHuN4-F112-SNE病毒滴度测定结果显示,其效价为6.12×106~8.16×106TCID50/ml。
1.7.5重组病毒噬斑形态观察
预先将Marc-145细胞接种于6孔细胞培养板中,使其形成细胞单层,然后接种0.01MOI的重组毒株rHuN4-F112-SNE,在5%CO2、37℃的细胞培养箱中感作2h,弃感作液,用无菌PBS清洗细胞2次,然后,加入1%的低熔点琼脂糖,室温静置10min,待低熔点琼脂胶完全凝固后,倒置于5%CO2、37℃的细胞培养箱中继续培养,观察细胞病变,待出现明显细胞病变后,用4%多聚甲醛,室温固定2h,弃固定液和低熔点琼脂糖凝胶,用结晶紫溶液染色5min,经去离子水洗涤后,观察病毒形成的噬斑形态,结果显示,rHuN4-F112-SNE株所形成的噬斑形态与亲本毒HuN4-F112相似,近似圆形(图7)。
1.7.6重组病毒多步生长曲线的绘制
预先将Marc-145细胞平铺于6孔板中,待形成单层后,按照0.01MOI的剂量将重组毒株rHuN4-F112-SNE接种于Marc-145细胞中,并分别在感染后12h、24h、36h、48h、60h和72h收取细胞培养上清,将各时间点采集的上清样品进行病毒滴度测定,按照Reed-Muench法计算病毒病毒TCID50,然后根据不同时间点病毒的TCID50绘制其增殖曲线。rHuN4-F112-SNE株多步生长曲线绘制结果显示,重组病毒rHuN4-F112-SNE在感染后60h达到复制峰值,其增殖趋势与亲本病毒HuN4-F112株相似,但略低于亲本毒(图8)。
1.8重组病毒遗传稳定性分析
将重组病毒rHuN4-F112-SNE株在Marc-145细胞中按照上述方法进行连续传代,其中,每间隔5代,进行一次噬斑纯化,纯化3次,然后将纯化的重组病毒连续传至30代。分别选择第10代、第20代和第30代次的重组病毒进行RT-PCR扩增和基因测序鉴定,分析插入的SNE基因的变化。同时,利用针对PRRSV N蛋白单抗和PEDV S蛋白单抗分别对不同代次的重组病毒进行IFA检测和Western-blot检测,检测SNE蛋白能否稳定表达。对第10代、第20代和第30代次的重组病毒进行RT-PCR扩增和基因测序鉴定,结果显示,第10代、第20代和第30代等不同代次的重组病毒rHuN4-F112-SNE均能够扩增出大小约2800bp的目标条带。测序结果显示,各代次重组毒株中的SNE基因与原始序列保持一致,核苷酸序列未发生缺失或突变。而且,用PRRSV N蛋白单抗和PEDV S蛋白单抗进行IFA检测结果显示,各代次重组病毒就能被抗PRRSV N蛋白单抗和PEDV S蛋白单抗识别(图9)。同样Western-blot检测结果显示,各代次重组病毒除了能够检测到PRRSV N蛋白的条带而外,还能够检测到大小约为100KDa的SNE蛋白条带,由此可见,重组病毒rHuN4-F112-SNE传至30代,引入的SNE基因仍然能够稳定表达蛋白(图10),表明获得的重组病毒rHuN4-F112-SNE具有遗传稳定性。
上述实验结果充分证明了,本研究成功获得了能够稳定表达PEDV S蛋白优势抗原区域的PRRSV重组病毒rHuN4-F112-SNE株,可以作为今后开发用于预防PRRS和PED的新型基因工程活载体疫苗的候选毒株。
前述内容出于解释目的描述了一些示例性实施方案。尽管前面的讨论已经给出了具体的实施方案,但是本领域技术人员将认识到,可以在形式和细节上做出改变,而不脱离本发明的更广泛的精神和范围。因此,说明书和附图应当以说明性的而不是限制性的意义考虑。因此,该详细描述不应以限制性意义理解,并且本发明的范围仅由所包括的权利要求书以及这些权利要求书所享有的等同方案的全部范围来限定。
Claims (10)
1.一种针对PEDV的S蛋白融合抗原表位肽,所述S蛋白融合抗原表位肽是选择S蛋白中的具有中和活性的抗原表位以及优势抗原区域进行串联组合和优化合成,并在前端引入S基因的信号肽序列,将合成后的基因命名为SNE,基因序列为1788bp,如SEQ ID NO.1所示。
2.如权利要求1所述的针对PEDV的S蛋白融合抗原表位肽,所述抗原表位肽N端或C端还可以偶联有多肽标记;所述多肽标记为生物素标记或荧光标记。
3.如权利要求1所述的针对PEDV的S蛋白融合抗原表位肽在制备针对PRRS和PED的二价基因工程活疫苗中的应用。
4.一种表达权利要求1所述PEDV S蛋白优势抗原区域的重组PRRSV rHuN4-F112-SNE疫苗株,其特征在于所述PEDV S蛋白优势抗原区域为上述S蛋白融合抗原表位肽。
5.如权利要求4所述的重组PRRSV rHuN4-F112-SNE疫苗株,其全长核苷酸序列为SEQID NO.10所示。
6.权利要求1所述的S蛋白融合抗原表位肽或权利要求4或5所述的重组PRRSV rHuN4-F112-SNE疫苗株在制备用于预防或治疗PRRS和PED的药物中的应用。
7.权利要求1所述的S蛋白融合抗原表位肽作为抗PRRS和PED感染药物筛选靶标的应用。
8.一种用于预防或治疗PRRS和PED感染的药物组合物,其包含权利要求1所述的S蛋白融合抗原表位肽和药学上可接受的辅料。
9.如权利要求8所述的药物组合物,所述药物组合物为经鼻施用的剂型;优选为喷雾剂、滴鼻剂、粉末剂、凝胶制剂或微球制剂。
10.一种用于PRRS和PED感染的诊断试剂,其包含权利要求1所述的S蛋白融合抗原表位肽,所述权利要求1所述的S蛋白融合抗原表位肽包被于检测载体上,所述检测载体选自聚苯乙烯微量反应板、胶体金试剂条、磁珠和微流控芯片中的任一种。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202210793115.5A CN116731195A (zh) | 2022-07-07 | 2022-07-07 | 表达pedv s蛋白优势抗原区域的重组prrsv疫苗株的构建及应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202210793115.5A CN116731195A (zh) | 2022-07-07 | 2022-07-07 | 表达pedv s蛋白优势抗原区域的重组prrsv疫苗株的构建及应用 |
Publications (1)
Publication Number | Publication Date |
---|---|
CN116731195A true CN116731195A (zh) | 2023-09-12 |
Family
ID=87908526
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202210793115.5A Pending CN116731195A (zh) | 2022-07-07 | 2022-07-07 | 表达pedv s蛋白优势抗原区域的重组prrsv疫苗株的构建及应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN116731195A (zh) |
-
2022
- 2022-07-07 CN CN202210793115.5A patent/CN116731195A/zh active Pending
Similar Documents
Publication | Publication Date | Title |
---|---|---|
WO2021254327A1 (zh) | 一种包膜替换型病毒载体疫苗及其构建方法 | |
CN111088283B (zh) | mVSV病毒载体及其病毒载体疫苗、一种基于mVSV介导的新冠肺炎疫苗 | |
CN109762792B (zh) | 一种猪繁殖与呼吸综合征病毒嵌合毒株及其应用 | |
Aguirreburualde et al. | Efficacy of a BVDV subunit vaccine produced in alfalfa transgenic plants | |
Cruz et al. | Vectored vaccines to protect against PRRSV | |
CN109536461B (zh) | 一种o型口蹄疫病毒突变株及其制备方法和应用 | |
US20050002953A1 (en) | SARS-coronavirus virus-like particles and methods of use | |
CN113666990A (zh) | 一种诱导广谱抗冠状病毒的t细胞疫苗免疫原及其应用 | |
US20210401983A1 (en) | Arthrogenic alphavirus vaccine | |
CA2811243C (en) | Bvdv vaccine | |
Phillpotts et al. | Intranasal immunisation with defective adenovirus serotype 5 expressing the Venezuelan equine encephalitis virus E2 glycoprotein protects against airborne challenge with virulent virus | |
Sangewar et al. | Mosaic bovine viral diarrhea virus antigens elicit cross-protective immunity in calves | |
CN103555680A (zh) | 一种具有免疫原性的prrsv病毒样颗粒及其制备与应用 | |
Dai et al. | Research progress in the development of porcine reproductive and respiratory syndrome virus as a viral vector for foreign gene expression and delivery | |
Zakhartchouk et al. | Optimization of a DNA vaccine against SARS | |
CN116240222A (zh) | 一种密码子优化的牛病毒性腹泻病毒1型e2蛋白基因及其应用 | |
CN116731195A (zh) | 表达pedv s蛋白优势抗原区域的重组prrsv疫苗株的构建及应用 | |
TWI328039B (zh) | ||
Mondal et al. | Development and characterization of a synthetic infectious cDNA clone of the virulent Bucyrus strain of equine arteritis virus expressing mCherry (red fluorescent protein) | |
KR20230008707A (ko) | 코로나바이러스 치료용 백신 조성물 | |
JP2023526770A (ja) | 三量体を形成する新型コロナウイルス(covid-19、コロナウイルス感染症2019)の組換えスパイクタンパク質および植物における上記組換えスパイクタンパク質の大量生産方法と、これを基盤とするワクチン組成物の製造方法(植物における新型コロナウイルスの三量体スパイクタンパク質の生産方法およびワクチン接種のための使用) | |
Hernández et al. | Immunogenicity of a recombinant adenovirus expressing porcine reproductive and respiratory syndrome virus polyepitopes | |
CN107746848B (zh) | 重组猪瘟病毒e2蛋白及其表达细胞系、制备方法、应用及猪瘟病毒亚单位疫苗 | |
CN116535517A (zh) | 一种重组表达pedv s蛋白受体rbd结构域的prrsv活载体疫苗株的构建及应用 | |
Cai et al. | Construction and characterization of a recombinant canine adenovirus expressing GP5 and M proteins of porcine reproductive and respiratory syndrome virus |
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 |