CN114058646B - 一种表达PCV2d cap蛋白的载体及方法 - Google Patents
一种表达PCV2d cap蛋白的载体及方法 Download PDFInfo
- Publication number
- CN114058646B CN114058646B CN202111329159.4A CN202111329159A CN114058646B CN 114058646 B CN114058646 B CN 114058646B CN 202111329159 A CN202111329159 A CN 202111329159A CN 114058646 B CN114058646 B CN 114058646B
- Authority
- CN
- China
- Prior art keywords
- promoter
- pcv2d
- vector
- baculovirus
- protein
- 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.)
- Active
Links
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 128
- 102000004169 proteins and genes Human genes 0.000 title claims abstract description 79
- 239000013598 vector Substances 0.000 title claims abstract description 50
- 238000000034 method Methods 0.000 title claims abstract description 14
- 241000701447 unidentified baculovirus Species 0.000 claims abstract description 56
- 239000013612 plasmid Substances 0.000 claims abstract description 28
- 238000012546 transfer Methods 0.000 claims abstract description 20
- 108091093126 WHP Posttrascriptional Response Element Proteins 0.000 claims abstract description 19
- 230000001105 regulatory effect Effects 0.000 claims abstract description 14
- 230000001124 posttranscriptional effect Effects 0.000 claims abstract description 13
- 241000588724 Escherichia coli Species 0.000 claims abstract description 9
- 238000004113 cell culture Methods 0.000 claims abstract description 5
- 230000017105 transposition Effects 0.000 claims abstract description 5
- 230000006798 recombination Effects 0.000 claims abstract description 4
- 238000005215 recombination Methods 0.000 claims abstract description 4
- 229940031626 subunit vaccine Drugs 0.000 claims abstract description 4
- 239000000427 antigen Substances 0.000 claims abstract description 3
- 102000036639 antigens Human genes 0.000 claims abstract description 3
- 108091007433 antigens Proteins 0.000 claims abstract description 3
- 230000035755 proliferation Effects 0.000 claims abstract description 3
- 241000238631 Hexapoda Species 0.000 claims description 17
- 241001673669 Porcine circovirus 2 Species 0.000 claims description 12
- 239000002773 nucleotide Substances 0.000 claims description 12
- 125000003729 nucleotide group Chemical group 0.000 claims description 12
- 238000010367 cloning Methods 0.000 claims description 11
- 239000007788 liquid Substances 0.000 claims description 9
- 238000000746 purification Methods 0.000 claims description 7
- 230000001580 bacterial effect Effects 0.000 claims description 6
- 239000003623 enhancer Substances 0.000 claims description 6
- 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 claims description 6
- OPIFSICVWOWJMJ-AEOCFKNESA-N 5-bromo-4-chloro-3-indolyl beta-D-galactoside Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1OC1=CNC2=CC=C(Br)C(Cl)=C12 OPIFSICVWOWJMJ-AEOCFKNESA-N 0.000 claims description 5
- 108020004705 Codon Proteins 0.000 claims description 5
- 238000002360 preparation method Methods 0.000 claims description 5
- NWUYHJFMYQTDRP-UHFFFAOYSA-N 1,2-bis(ethenyl)benzene;1-ethenyl-2-ethylbenzene;styrene Chemical compound C=CC1=CC=CC=C1.CCC1=CC=CC=C1C=C.C=CC1=CC=CC=C1C=C NWUYHJFMYQTDRP-UHFFFAOYSA-N 0.000 claims description 4
- 239000004098 Tetracycline Substances 0.000 claims description 4
- 239000003729 cation exchange resin Substances 0.000 claims description 4
- 229930027917 kanamycin Natural products 0.000 claims description 4
- 229960000318 kanamycin Drugs 0.000 claims description 4
- 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 claims description 4
- 229930182823 kanamycin A Natural products 0.000 claims description 4
- 229960002180 tetracycline Drugs 0.000 claims description 4
- 229930101283 tetracycline Natural products 0.000 claims description 4
- 235000019364 tetracycline Nutrition 0.000 claims description 4
- 150000003522 tetracyclines Chemical class 0.000 claims description 4
- 229930182566 Gentamicin Natural products 0.000 claims description 3
- CEAZRRDELHUEMR-URQXQFDESA-N Gentamicin Chemical compound O1[C@H](C(C)NC)CC[C@@H](N)[C@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](NC)[C@@](C)(O)CO2)O)[C@H](N)C[C@@H]1N CEAZRRDELHUEMR-URQXQFDESA-N 0.000 claims description 3
- 230000009977 dual effect Effects 0.000 abstract description 23
- 241000201370 Autographa californica nucleopolyhedrovirus Species 0.000 abstract description 11
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 abstract description 7
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 abstract description 7
- 238000004519 manufacturing process Methods 0.000 abstract description 7
- 108091062157 Cis-regulatory element Proteins 0.000 abstract description 6
- 108091081062 Repeated sequence (DNA) Proteins 0.000 abstract description 5
- 210000004027 cell Anatomy 0.000 description 52
- 241000700605 Viruses Species 0.000 description 34
- 238000013320 baculovirus expression vector system Methods 0.000 description 11
- 108020004414 DNA Proteins 0.000 description 10
- 108020005202 Viral DNA Proteins 0.000 description 10
- 239000003153 chemical reaction reagent Substances 0.000 description 9
- 241000699670 Mus sp. Species 0.000 description 8
- 210000002966 serum Anatomy 0.000 description 8
- 238000001890 transfection Methods 0.000 description 8
- 108010077850 Nuclear Localization Signals Proteins 0.000 description 7
- 238000002474 experimental method Methods 0.000 description 7
- 101150027376 chiA gene Proteins 0.000 description 6
- 238000012217 deletion Methods 0.000 description 6
- 230000037430 deletion Effects 0.000 description 6
- 201000010099 disease Diseases 0.000 description 6
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 6
- 208000024191 minimally invasive lung adenocarcinoma Diseases 0.000 description 6
- 108091008146 restriction endonucleases Proteins 0.000 description 6
- 239000006228 supernatant Substances 0.000 description 6
- 238000005406 washing Methods 0.000 description 6
- 241000894006 Bacteria Species 0.000 description 5
- 244000292604 Salvia columbariae Species 0.000 description 5
- 235000012377 Salvia columbariae var. columbariae Nutrition 0.000 description 5
- 235000001498 Salvia hispanica Nutrition 0.000 description 5
- 235000014167 chia Nutrition 0.000 description 5
- 238000012258 culturing Methods 0.000 description 5
- 229940088598 enzyme Drugs 0.000 description 5
- 239000013604 expression vector Substances 0.000 description 5
- 239000012634 fragment Substances 0.000 description 5
- 239000002245 particle Substances 0.000 description 5
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- 241001533384 Circovirus Species 0.000 description 4
- 241000256251 Spodoptera frugiperda Species 0.000 description 4
- 241001492404 Woodchuck hepatitis virus Species 0.000 description 4
- 238000003776 cleavage reaction Methods 0.000 description 4
- 230000000295 complement effect Effects 0.000 description 4
- 208000015181 infectious disease Diseases 0.000 description 4
- 230000007017 scission Effects 0.000 description 4
- 238000012163 sequencing technique Methods 0.000 description 4
- 230000009466 transformation Effects 0.000 description 4
- 229960005486 vaccine Drugs 0.000 description 4
- NHBKXEKEPDILRR-UHFFFAOYSA-N 2,3-bis(butanoylsulfanyl)propyl butanoate Chemical compound CCCC(=O)OCC(SC(=O)CCC)CSC(=O)CCC NHBKXEKEPDILRR-UHFFFAOYSA-N 0.000 description 3
- 108010084457 Cathepsins Proteins 0.000 description 3
- 108010022172 Chitinases Proteins 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 3
- 108090000790 Enzymes Proteins 0.000 description 3
- 108700026244 Open Reading Frames Proteins 0.000 description 3
- 101710182846 Polyhedrin Proteins 0.000 description 3
- 241000202347 Porcine circovirus Species 0.000 description 3
- 239000007983 Tris buffer Substances 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 210000004369 blood Anatomy 0.000 description 3
- 239000008280 blood Substances 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 230000009089 cytolysis Effects 0.000 description 3
- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 238000010790 dilution Methods 0.000 description 3
- 239000012895 dilution Substances 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 230000006801 homologous recombination Effects 0.000 description 3
- 238000002744 homologous recombination Methods 0.000 description 3
- 230000003053 immunization Effects 0.000 description 3
- 238000002649 immunization Methods 0.000 description 3
- 239000012535 impurity Substances 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 238000012216 screening Methods 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 3
- 230000003612 virological effect Effects 0.000 description 3
- 241000255789 Bombyx mori Species 0.000 description 2
- 102000005600 Cathepsins Human genes 0.000 description 2
- 102000012286 Chitinases Human genes 0.000 description 2
- 102000053602 DNA Human genes 0.000 description 2
- 241000282414 Homo sapiens Species 0.000 description 2
- 101000833492 Homo sapiens Jouberin Proteins 0.000 description 2
- 101000651236 Homo sapiens NCK-interacting protein with SH3 domain Proteins 0.000 description 2
- 102100024407 Jouberin Human genes 0.000 description 2
- 101150009852 ORF2 gene Proteins 0.000 description 2
- 238000012408 PCR amplification Methods 0.000 description 2
- 229930040373 Paraformaldehyde Natural products 0.000 description 2
- 101710159752 Poly(3-hydroxyalkanoate) polymerase subunit PhaE Proteins 0.000 description 2
- 101710130262 Probable Vpr-like protein Proteins 0.000 description 2
- 108020004682 Single-Stranded DNA Proteins 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 102000008579 Transposases Human genes 0.000 description 2
- 108010020764 Transposases Proteins 0.000 description 2
- 206010058874 Viraemia Diseases 0.000 description 2
- 238000000246 agarose gel electrophoresis Methods 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 230000003698 anagen phase Effects 0.000 description 2
- 230000004071 biological effect Effects 0.000 description 2
- 238000005341 cation exchange Methods 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000001976 enzyme digestion Methods 0.000 description 2
- 239000012530 fluid Substances 0.000 description 2
- 239000000499 gel Substances 0.000 description 2
- 229960002518 gentamicin Drugs 0.000 description 2
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 2
- 239000010931 gold Substances 0.000 description 2
- 229910052737 gold Inorganic materials 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 2
- 230000005847 immunogenicity Effects 0.000 description 2
- 238000003780 insertion Methods 0.000 description 2
- 230000037431 insertion Effects 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 229920002866 paraformaldehyde Polymers 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 230000010076 replication Effects 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 230000001131 transforming effect Effects 0.000 description 2
- 238000011144 upstream manufacturing Methods 0.000 description 2
- 239000012224 working solution Substances 0.000 description 2
- 101150039504 6 gene Proteins 0.000 description 1
- 229920000936 Agarose Polymers 0.000 description 1
- 241001367049 Autographa Species 0.000 description 1
- 101150044789 Cap gene Proteins 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 108090000565 Capsid Proteins Proteins 0.000 description 1
- 108010059892 Cellulase Proteins 0.000 description 1
- 102100023321 Ceruloplasmin Human genes 0.000 description 1
- 241001533399 Circoviridae Species 0.000 description 1
- 108020004638 Circular DNA Proteins 0.000 description 1
- 208000003322 Coinfection Diseases 0.000 description 1
- 241000537219 Deltabaculovirus Species 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 206010053177 Epidermolysis Diseases 0.000 description 1
- 108090000331 Firefly luciferases Proteins 0.000 description 1
- 102000053187 Glucuronidase Human genes 0.000 description 1
- 108010060309 Glucuronidase Proteins 0.000 description 1
- 101001001300 Human cytomegalovirus (strain Towne) 65 kDa phosphoprotein Proteins 0.000 description 1
- 102000003960 Ligases Human genes 0.000 description 1
- 108090000364 Ligases Proteins 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 108010036176 Melitten Proteins 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 241000699666 Mus <mouse, genus> Species 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 108700020497 Nucleopolyhedrovirus polyhedrin Proteins 0.000 description 1
- 101100382437 Porcine circovirus 2 Cap gene Proteins 0.000 description 1
- 108010076504 Protein Sorting Signals Proteins 0.000 description 1
- 101710088839 Replication initiation protein Proteins 0.000 description 1
- 101710203837 Replication-associated protein Proteins 0.000 description 1
- 101710172711 Structural protein Proteins 0.000 description 1
- 101000936049 Vibrio cholerae serotype O1 (strain ATCC 39315 / El Tor Inaba N16961) Outer membrane lipoprotein Blc Proteins 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 102000005421 acetyltransferase Human genes 0.000 description 1
- 108020002494 acetyltransferase Proteins 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 230000001464 adherent effect Effects 0.000 description 1
- 239000002671 adjuvant Substances 0.000 description 1
- 239000011543 agarose gel Substances 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 210000004436 artificial bacterial chromosome Anatomy 0.000 description 1
- 210000004507 artificial chromosome Anatomy 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 102000005936 beta-Galactosidase Human genes 0.000 description 1
- 108010005774 beta-Galactosidase Proteins 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- AIYUHDOJVYHVIT-UHFFFAOYSA-M caesium chloride Chemical compound [Cl-].[Cs+] AIYUHDOJVYHVIT-UHFFFAOYSA-M 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 238000006555 catalytic reaction Methods 0.000 description 1
- -1 cationic lipid Chemical class 0.000 description 1
- 230000021164 cell adhesion Effects 0.000 description 1
- 239000006143 cell culture medium Substances 0.000 description 1
- 239000013553 cell monolayer Substances 0.000 description 1
- 210000003855 cell nucleus Anatomy 0.000 description 1
- 229940106157 cellulase Drugs 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 239000012149 elution buffer Substances 0.000 description 1
- 108010048367 enhanced green fluorescent protein Proteins 0.000 description 1
- 210000003527 eukaryotic cell Anatomy 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 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 1
- 238000000799 fluorescence microscopy Methods 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 102000037865 fusion proteins Human genes 0.000 description 1
- 108020001507 fusion proteins Proteins 0.000 description 1
- 229960002442 glucosamine Drugs 0.000 description 1
- 239000003102 growth factor Substances 0.000 description 1
- 238000003306 harvesting Methods 0.000 description 1
- 210000000087 hemolymph Anatomy 0.000 description 1
- 230000028996 humoral immune response Effects 0.000 description 1
- 238000010191 image analysis Methods 0.000 description 1
- 230000036737 immune function Effects 0.000 description 1
- 238000010166 immunofluorescence Methods 0.000 description 1
- 230000002163 immunogen Effects 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 230000002458 infectious effect Effects 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 101150066555 lacZ gene Proteins 0.000 description 1
- 230000001418 larval effect Effects 0.000 description 1
- 210000004324 lymphatic system Anatomy 0.000 description 1
- 210000004962 mammalian cell Anatomy 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- VDXZNPDIRNWWCW-JFTDCZMZSA-N melittin Chemical compound NCC(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(=O)N[C@@H](C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(=O)N[C@@H](CC(C)C)C(=O)N1CCC[C@H]1C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CO)C(=O)N[C@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(N)=O)C(N)=O)CC1=CNC2=CC=CC=C12 VDXZNPDIRNWWCW-JFTDCZMZSA-N 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000007918 pathogenicity Effects 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 230000004481 post-translational protein modification Effects 0.000 description 1
- 208000028489 postweaning multisystemic wasting syndrome Diseases 0.000 description 1
- 230000002062 proliferating effect Effects 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 229940023143 protein vaccine Drugs 0.000 description 1
- 239000012521 purified sample Substances 0.000 description 1
- 238000003753 real-time PCR Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 238000011076 safety test Methods 0.000 description 1
- 239000000523 sample Substances 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 238000013207 serial dilution Methods 0.000 description 1
- 239000012679 serum free medium Substances 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 238000003892 spreading Methods 0.000 description 1
- 230000007480 spreading Effects 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 238000004114 suspension culture Methods 0.000 description 1
- 208000011580 syndromic disease Diseases 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000010189 synthetic method Methods 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 230000000007 visual effect Effects 0.000 description 1
- 239000011534 wash buffer Substances 0.000 description 1
Classifications
-
- 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
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/86—Viral vectors
-
- 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
- A61P31/20—Antivirals for DNA viruses
-
- 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
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/525—Virus
- A61K2039/5258—Virus-like particles
-
- 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
- C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
- C12N2710/00011—Details
- C12N2710/14011—Baculoviridae
- C12N2710/14111—Nucleopolyhedrovirus, e.g. autographa californica nucleopolyhedrovirus
- C12N2710/14141—Use of virus, viral particle or viral elements as a vector
- C12N2710/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
-
- 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
- C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
- C12N2750/00011—Details
- C12N2750/10011—Circoviridae
- C12N2750/10022—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
- C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
- C12N2750/00011—Details
- C12N2750/10011—Circoviridae
- C12N2750/10023—Virus like particles [VLP]
-
- 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
- C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
- C12N2750/00011—Details
- C12N2750/10011—Circoviridae
- C12N2750/10034—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
- C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
- C12N2750/00011—Details
- C12N2750/10011—Circoviridae
- C12N2750/10051—Methods of production or purification of viral material
-
- 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
- C12N2800/00—Nucleic acids vectors
- C12N2800/10—Plasmid DNA
- C12N2800/103—Plasmid DNA for invertebrates
- C12N2800/105—Plasmid DNA for invertebrates for insects
-
- 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
- C12N2800/00—Nucleic acids vectors
- C12N2800/22—Vectors comprising a coding region that has been codon optimised for expression in a respective host
-
- 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
- C12N2830/00—Vector systems having a special element relevant for transcription
- C12N2830/15—Vector systems having a special element relevant for transcription chimeric enhancer/promoter combination
-
- 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
- C12N2830/00—Vector systems having a special element relevant for transcription
- C12N2830/48—Vector systems having a special element relevant for transcription regulating transport or export of RNA, e.g. RRE, PRE, WPRE, CTE
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Virology (AREA)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Biotechnology (AREA)
- Molecular Biology (AREA)
- General Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Microbiology (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Veterinary Medicine (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Biomedical Technology (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- Mycology (AREA)
- Immunology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Epidemiology (AREA)
- Physics & Mathematics (AREA)
- Gastroenterology & Hepatology (AREA)
- Plant Pathology (AREA)
- Communicable Diseases (AREA)
- Oncology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明公开了一种表达PCV2d cap蛋白的载体及方法，使用Bac‑to‑Bac杆状病毒表达系统，通过对pFast Bac Dual载体进行改造，使用SV40启动子顺式作用元件、增加AcMNPV基因中同源重复序列(Hr1)、增加转录后调控元件(WPRE)改造pFast Bac Dual载体，PCV2d cap蛋白编码基因与pFast Bac Dual载体连接，将所得转移质粒转化DH10Bac大肠杆菌，通过转座重组，获得重组杆状病毒质粒，采用Sf9细胞培养和重组杆状病毒增殖，来提高PCV2cap重组蛋白的表达量。对Bac‑to‑Bac杆状病毒表达系统表达的目的蛋白浓度进一步提高，有助于提高制备PCV2d的抗原、抗体、或亚单位疫苗的生产效率和降低生产成本。
Description
技术领域
本发明属于杆状病毒表达载体系统技术领域，具体涉及一种表达PCV2d cap蛋白的载体及方法。
背景技术
1993年出现了Bacmid技术，Bacmid是组装有杆状病毒DNA的细菌人工染色体(Bacterial artificial chromosomes,BACs)，因此Bacmid可以维持于大肠杆菌细胞内并在大肠杆菌中增殖，Bac-to Bac具有“Bacteria”到“Baculovirus”之意。杆状病毒表达载体系统包含3个部分:转移质粒、杆状病毒表达载体和昆虫细胞。转移质粒由Ph或P10启动子、启动子下游的多克隆位点、polyA序列、需要可在外源基因N端加入GP64、蜂毒素或几丁质酶等信号肽促使蛋白分泌表达，在开放阅读框N端或C端加入6x组氨酸标签利于蛋白纯化，TEV酶切位点可用于后期组氨酸标签的移除。感受态大肠杆菌DH10Bac中包含两个质粒:穿梭质粒和辅助质粒。穿梭质粒(Bacmid)具有在细菌中低拷贝mini-F复制子、mini-attTn7转座位点、卡纳霉素抗性基因的杆状病毒基因组，不含有杆状病毒多角体蛋白；辅助质粒编码转座酶，可维持在大肠杆菌中，使转移质粒上的表达盒通过转座作用定向转移到杆状病毒表达载体上，含有四环素抗性基因。
转移质粒转化到感受态细胞中后，在转座酶催化作用下，将Tn7转座子左右臂定向转移到Bacmid的Tn7位点，形成具有外源基因的Bacmid。转移载体中携带有LacZ基因，其产生的a-肽与宿主菌的a-肽互补可编码β半乳糖苷酶，经过IPTG诱导分解底物X-gal形成蓝色噬菌斑。当外源基因插入到转移载体中LacZ的多克隆位点，导致LacZ失活，破坏了a.-肽的互补形成，在含有X-gaL和IPTG的平板中生长白色噬菌斑。因此重组质粒可在相应抗性、IPTG和X-gal(或Bluo-gal)底物的琼脂糖平板上通过蓝白斑筛选获得，然后经过碱裂解方法获得病毒DNA，纯化的杆粒DNA用于转染昆虫细胞，生产具有感染性的BV。
第二代商品化BEVS为Invitrogen公司生产的Bac-to-BacTM该表达系统，杆状病毒表达载体优势:(i)昆虫细胞是真核细胞，具有完备的翻译后加工修饰功能，重组蛋白与天然蛋白具有相似的生物活性；(ii)外源基因容量大，杆状病毒表达载体大小约为134kbp,适合较大的基因克隆和同时多个基因的克隆；(iii)生物安全性好，杆状病毒不感染包括人在内的哺乳动物；(iv)在强启动子Ph或P10的调控下，重组蛋白具有很高的表达水平。该系统生产的杆状病毒不需要蚀斑纯化过程，然而，需要通过抗性基因从亲代病毒DNA中筛选重组病毒DNA，含有重组病毒DNA的细菌增殖过程以及重组病毒DNA的抽提纯化过程，均可能会影响杆状病毒粒子的稳定性，甚至导致重组病毒表达丢失。
autographa California multiple nucleopolyhedrovirus,AcMNPV(苜蓿银纹夜蛾核型多角体病毒)是研究最广泛且应用较多的一种杆状病毒，以AcMNPV为基础的杆状病毒表达载体系统(baculovirus expression vector system,BEVS)发展迅速，已成为应用最普遍的表达系统之一。第一代商品化BEVS是Clontech生产的BacPAK6TM，该表达系统对AcMNPV基因组进行以下修饰，即经过Bsu361酶切，病毒DNA线性化，除去了lacZ基因和orf1629的部分基因，导致病毒在昆虫细胞中无法复制的病毒DNA与转移载体混合后转染到昆虫细胞进行同源重组，表达盒插入到病毒DNA,修复了orf1629基因，病毒DNA恢复环形，同时恢复了感染性。但由于Bsu361酶切效率并不是100％，转染出的病毒是亲代病毒和重组病毒的混合物，需要进一步的蚀斑纯化实验，将重组病毒从亲代病毒中筛选出来。
FlashBACTM BEVS对AcMNPV基因组进行了改造，AcMNPV病毒DNA缺失orf1629基因部分序列和BAC取代多角体蛋白基因。缺失了orf1629部分基因序列的病毒DNA不能在昆虫细胞内复制，BAC取代多角体蛋白基因后，病毒DNA能在细菌细胞内维持并进行低拷贝复制。改造后病毒DNA在细菌中增殖后,经碱裂解和氯化铯梯度纯化，获得用于共转染的flashBACMTM病毒DNA，与带有外源基因的转移质粒共转染于昆虫细胞，通过细胞内同源重组，重组病毒的orf1629基因完整，病毒恢复复制功能，外源基因同时插入到病毒DNA中，重组杆状病毒获得拯救。该系统中，去除了蓝白斑筛选过程，亲代病毒不可能在昆虫细胞中复制。
FlashBACTMGOLD BEVS是在flashBACTM基础上缺失了几丁质酶基因(chiA)和组织蛋白酶基因(v-cath)。ChiA是一个辅助基因，编码的酶能够促进细胞内切酶和外切酶的活性。对于感染的昆虫细胞，chiA表达于感染的晚期，与组织蛋白酶一起促进宿主表皮分解和组织溶解，从而释放病毒，感染更多的宿主细胞。已有文献报道，缺失病毒基因组中chiA和(或)v-cath基因，可提高昆虫细胞和昆虫幼虫蛋白稳定性。Possee等将chiA基因从杆状病毒基因组缺失后，分泌性重组蛋白表达水平提高；Lee等在B.mori幼虫中表达纤维素酶，表达量提高了17％；Suzuki等用缺失v-cath的杆状病毒表达萤火虫荧光素酶和人生长因子,发现蛋白稳定性大大增加；Park等使用缺失chiA/v-cath的病毒在B.mori中表达GFP融合蛋白，血淋巴中几丁质酶和组织蛋白酶的活性分别降低了95％和50％，与未修饰的病毒相比，表达的GFP(uv)-beta、3-N-葡萄糖胺乙酰转移酶降解量减少，活性增加了2.8倍。FlashBACTMGOLD杆状病毒表达载体不仅增加了分泌蛋白的表达量，同时提高了重组蛋白的稳定性。
FlashBACTMULTRABEVS是在flashTMBACGOLD基础上,缺失了p10、p26和p74基因。
Silvia等将BEVS的双表达盒载体进行了改造，polh启动子后克隆杆状病毒源激活因子ie0、iel；p6.9p10双启动子代替原来的p10启动子，与杆状病毒源的增强子hrl顺式连接，启动外源蛋白的表达，polhAc-ie-01/hr1p6.9p10称为TB表达盒，该表达盒延长了细胞感染后保持完整性的时间，提高了蛋白的完整性，且EGFP表达量是原表达盒的4倍，TB表达盒可显著提高BEVS的蛋白表达水平。随后,Javier等使用新的杆状病毒表达盒TB分别高效表达了PCV2Cap蛋白和RHDVVP60蛋白,两种蛋白的表达量比原表达盒相比均提高了300％，均组装形成了PCV2 VLPs和RHDVVLPs。
猪圆环病毒是一种无囊膜、单股、负链、环状的DNA病毒，它属于圆环病毒科圆环病毒属。猪圆环病毒主要有3种基因型，圆环病毒1型、圆环病毒2型和圆环病毒3型。PCV2致病性较强，可引起多种猪圆环病毒相关性疾病(Porcine circovirus-as sociateddiseases,PCVADs)，其中最主要的是断奶仔猪多系统衰弱综合征(Postweaningmultisystemic wasting syndrome,PMWS)，PCV2主要破坏淋巴系统，导致机体免疫功能缺陷，且容易与其它病原发生混合感染，该病在我国乃至世界猪群中广泛存在，已成为一种常见病和多发病，给养猪业带来了巨大的经济损失。PCV2又被分为PCV2a、PCV2b、PCV2c、PCV2d、PCV2e和PCV2f这6个基因亚型，其中PCV2d在中国的流行率最高，大部分的养猪场都能够检测到PCV2d病毒。
cap蛋白是PCV2d唯一的衣壳蛋白和主要的免疫原性蛋白，杆状病毒表达系统具有可插入较大的外源基因片段、目的蛋白表达量高和表达的蛋白生物学活性较好的优点被广泛应用。目前已知PCV2基因组中含有11个开放阅读框(ORFs),其中最主要的为ORF1和ORF2。ORF1位于单链DNA 51～985nt,编码病毒的复制相关蛋白(Rep和Rep')；ORF2位于单链DNA1034～1735nt,该区域的互补链编码病毒的结构蛋白(Cap蛋白)。Mahe等发现PCV2 Cap蛋白65-87aa、113-139aa、169-183aa和193-207aa 4个区域具有免疫原性。Cap蛋白前41个氨基酸为核定位信号肽(NLS)，关文献报道，缺失或突变NLS的Cap蛋白表达量高于未改变NLS的Cap蛋白。Liu等在大肠杆菌中表达了PCV2 ORF2编码的完整Cap蛋白,表达产量约为lmg/L；之后，Zhoul和Marial分别将完整的ORF2基因和缺失NLS的ORF2基因在大肠杆菌中表达，发现缺失NLS的重组菌表达量高达20mg/L,
吉林农业大学的张艳艳等人根据H5细胞对密码子的偏好性，对Cap蛋白密码子进行了优化并合成，同时突变了Cap蛋白的NLS序列，此外还在杆状病毒转移载体中克隆了增强子、双启动子，显著提高了目的蛋白在杆状病毒载体中表达水平，Cap蛋白的表达含量从20-50ug/ml提高到了150ug/ml左右。
虽然市面上已经有杆状病毒表达的PCV2d cap蛋白疫苗，但是运用新的技术和方法可以对其表达量进一步提高，有助于提高生产效率和降低生产成本。
发明内容
本发明使用Bac-to-Bac杆状病毒表达系统，通过对pFast Bac Dual载体进行改造，使用CAG、SV40启动子等顺式作用元件、增加AcMNPV基因中同源重复序列(Hr1)、增加转录后调控元件(WPRE)来提高PCV2 CAP重组蛋白的表达量。对Bac-to-Bac杆状病毒表达系统表达的目的蛋白浓度进一步提高，有助于提高生产效率和降低生产成本。
一种表达PCV2d cap蛋白的载体，由骨架载体和插入其启动子P10下游的多克隆位点的一个或多个PCV2d cap蛋白编码基因构成，所述骨架载体的启动子P10和其下游的多克隆位点之间插入了如SEQ ID.No.6所示的转录后调控序列WPRE，所述骨架载体的启动子Ph被替换为如SEQ ID.No.4所示的启动子SV40，或启动子Ph和启动子P10之间插入了如SEQID.No.5所示的增强子hr1。
进一步地，所述PCV2d cap蛋白的编码基因的核苷酸序列根据杆状病毒密码子优化，优化后的核苷酸序列如SEQ ID NO.3所示。
进一步地，所述的骨架载体为pFast Bac Dual。
一种表达PCV2d cap蛋白的方法，将上述PCV2d cap蛋白的编码基因，与所述骨架载体连接，将所得重组杆状病毒转移质粒转化DH10Bac大肠杆菌，通过转座重组，获得重组杆状病毒质粒，复苏后的菌液涂布卡那霉素、四环素、庆大霉素、IPTG、X-gal平板，37℃避光培养，选白色单一菌落扩大培养，转染Sf9昆虫细胞从而获得P0代重组杆状病毒，采用Sf9细胞培养和重组杆状病毒增殖，结合阳离子交换树脂纯化得到PCV2d cap蛋白；
所述骨架载体的核苷酸序列如SEQ ID NO.1所示，具有如下组件：启动子Ph被启动子SV40替换，启动子P10和其下游的多克隆位点之间插入了转录后调控序列WPRE；
或所述骨架载体的核苷酸序列如SEQ ID NO.2所示，具有如下组件：启动子Ph未被启动子SV40替换，所述转移质粒的启动子P10和Ph之间插入了增强子hrl，启动子P10和其下游的多克隆位点之间插入了转录后调控序列WPRE。
如上所述的方法在制备PCV2d的抗原、抗体、或亚单位疫苗中的应用。
SV40启动子顺式作用元件的序列如SEQ ID.No.4所示，SV40启动子序列通过合成的方式替换掉Ph启动子。
AcMNPV基因中同源重复序列(Homologous Region 1，简称Hr1)序列如SEQID.No.5所示，Hr1基因通过合成的方式插入P10和Ph启动子之间。
土拨鼠肝炎病毒转录后调控元件(Woodchuck hepatitis virus post-transcriptional regulatory elemem，简称WPRE)的序列如SEQ ID.No.6所示，WPRE基因通过合成的方式插入P10表达框的后面。
将上述的3个元件以联合作用构建到pFast Bac Dual载体上，得到具有SV40-WPRE、或HR1-WPRE的pFast Bac Dual载体，将2个拷贝的PCV2d cap基因构建到含有这些元件组合的pFast Bac Dual载体上，只含有2个拷贝的PCV2d cap的pFast Bac Dual载体。
改造后的SV40-WPRE、HR1-WPRE组PCV 2d cap蛋白的表达量明显增加。使用SV40启动子等顺式作用元件、增加AcMNPV基因中同源重复序列(Hr1)、增加转录后调控元件(WPRE)来提高PCV2 CAP重组蛋白的表达量。
附图说明
图1为pFast Bac Dual-cap的图谱；
图2为用引物扩增的cap蛋白编码基因的PCR鉴定结果图；
图3为重组的转移质粒转化后的单菌落的PCR鉴定结果图；
图4为转染重组杆状病毒前后sf9细胞的状态图，A为转染前的正常细胞，B为转染后的细胞；
图5为荧光显微镜观察7种重组杆状病毒液和空白对照在细胞孔发荧光的检测图；
图6为7种重组杆状病毒表达的cap蛋白的SDS-PAGE电泳结果；
图7为重组杆状病毒HR1-WPRE组表达蛋白的SDS-PAGE电泳结果；
图8为TEM观察重组杆状病毒HR1-WPRE组表达蛋白的形貌图；
图9为ELISA检测重组杆状病毒HR1-WPRE组表达蛋白和BAC-PCV2d组表达蛋白的特异性抗体水平结果对比图；
图10为攻毒后14天检测血清中的PCV2病毒载量结果对比图。
具体实施方式
下面结合具体实施例对本发明作进一步的详细说明。实施例中所述的实验方法无特别说明，即按常规分子生物学实验方法操作。
首先对本发明所设计的术语解释如下：
“pFastBacTM Dual载体”其特点是在单个载体上有两个启动子，用于在使用杆状病毒表达系统(/> Baculovirus Expression System)时在昆虫细胞中同时表达两个蛋白质。该载体具有两个强启动子，多角体蛋白启动子和p10启动子，因此可实现高水平表达。包含CAT和β-葡萄糖醛酸酶(gus)的表达对照。
“sf9”是Spodoptera frugiperda cell的缩写，即草地贪夜蛾细胞，草地贪夜蛾也称草地夜蛾。命名源于两篇文献：Hegedus et al.,1998；Pfeifer et al.,1997。生物实验中常用的昆虫细胞还有：LD652Y、sf21、High Five等。
“ II试剂”是一种阳离子脂质制剂，设计用于昆虫细胞的极佳转染。 II是原始/>试剂的改进版本。除提供与/>相同的出色性能外，/> II试剂的生产旨在确保批间一致性，并经过优化，可用于更快的方案。当使用BaculoDirectTM和InsectSelectTM表达系统时，使用/> II试剂进行转染可导致Sf9、Sf21和High FiveTM细胞的一致和高效转染。/> II试剂也可用于在含血清或无血清培养基中转染贴壁或悬浮哺乳动物细胞。
实施例1
1、转移载体的构建
1)CAG组载体
CAG启动子顺式作用元件的序列如SEQ ID.No.7所示，将CAG启动子序列通过合成的方式替换掉pFast Bac Dual载体中的Ph启动子。
2)SV40组载体
SV40启动子顺式作用元件的序列如SEQ ID.No.4所示，将SV40启动子序列通过合成的方式替换掉pFast Bac Dual载体中的Ph启动子。
3)WPRE组载体
土拨鼠肝炎病毒转录后调控元件(Woodchuck hepatitis virus post-transcriptional regulatory elemem，简称WPRE)的序列如SEQ ID.No.6所示，将WPRE基因通过合成的方式插入pFast Bac Dual载体中的P10表达框的后面。4)SV40-WPRE组载体
将WPRE基因通过合成的方式插入pFast Bac Dual载体中的P10表达框的后面，将SV40启动子序列通过合成的方式替换掉pFast Bac Dual载体中的Ph启动子。
5)CAG-WPRE组载体
将WPRE基因通过合成的方式插入pFast Bac Dual载体中的P10表达框的后面，将CAG启动子序列通过合成的方式替换掉pFast Bac Dual载体中的Ph启动子。
6)HR1-WPRE组载体
AcMNPV基因中同源重复序列(Homologous Region 1，简称Hr1)序列如SEQID.No.5所示，将Hr1基因通过合成的方式插入pFast Bac Dual载体中的P10和Ph启动子之间，将WPRE基因通过合成的方式插入pFast Bac Dual载体中的P10表达框的后面。
7)BAC-PCV2d组载体
未改造。
2、目的基因的扩增
将PCV2d cap蛋白编码基因进行杆状病毒密码子优化,将优化后的核苷酸序列进行基因合成，以合成后的cap蛋白编码基因为模板,用两对引物分别进行扩增，用第一对引物使BamHI和SacI的限制性内切酶酶切位点分别引入到cap蛋白编码基因的上下游，用第二对引物使SphI和XhoI的限制性内切酶酶切位点分别引入到cap蛋白编码基因的上下游。引物序列如表1所示，划下横线处为酶切位点；
表1
用于扩增上述cap蛋白编码基因的PCR反应体系(总体积50μL)如下：
PCR扩增反应后，扩增产物于1％琼脂糖凝胶电泳，并凝胶回收目的片段，PCR扩增结果如图1所示，-20℃保存备用。构建好的质粒图谱如图所1示。
3、片段和载体的酶切
将第一对引物扩增的cap蛋白编码基因和步骤2构建的一种pFast Bac Dual载体分别用BamHI和SacI限制性内切酶进行双酶切，将第二对引物扩增的cap蛋白编码基因和步骤2构建的一种pFast Bac Dual载体分别用SphI和XhoI限制性内切酶进行双酶切。酶切体系如下表2：
表2
| 模板 | 1μL |
| 10×Buffer | 2μL |
| 限制性内切酶1 | 1μL |
| 限制性内切酶2 | 1μL |
| ddH2O | upto20μL |
37℃反应3小时，酶切产物用1％琼脂糖凝胶电泳后再用琼脂糖凝胶回收试剂盒回收DNA片段及pFast Bac Dual载体骨架。
4、连接和转化
将回收后的目的DNA片段和pFast Bac Dual载体骨架用T4连接酶连接进行同源重组，配好连接体系后16℃过夜，连接后的重组转移质粒转化大肠杆菌TOP10感受态。连接体系如下表3：
表3
5、重组质粒的鉴定
转化后的单菌落进行PCR检测，鉴定到正确条带后送武汉生工生物有限公司进行测序，结果正确后进行下一步实验。菌落PCR鉴定结果如图3所示，构建好的pFast BacDual-2cap质粒的图谱如图1所示。
6、筛选重组杆粒
将100-300ng测序正确的转移质粒转化DH10Bac感受态细胞,复苏后的菌液涂布卡那霉素、四环素、庆大霉素、IPTG、X-gal平板，37度避光培养48h。挑选白色单克隆菌落，使用鉴定引物M13-F/R进行菌落PCR鉴定，扩增到正确条带后送测序，测序结果正确后，菌液进行扩大培养，然后提取重组杆状病毒质粒。
7、重组杆状病毒的拯救
(1)按照一个孔1.2×106个细胞数量，将对数生长期的sf9细胞铺于6孔板，静置1小时后，细胞贴壁融合度约70％～80％；
(2)取Cellfectin II试剂3-5μL入100μL无抗无血清细胞培养液；取5μgBacmid加入100uL无抗无血清细胞培养液中混匀；
(3)将(2)中的两管混合液合并混匀，室温孵育15min：
(4)孵育结束后轻轻加入6孔细胞板中，27℃培养箱避光孵育4-6h后将其更换为正常培养基，注意做不改造的转移质粒的转染对照孔以及空白对照孔；
(5)27℃培养箱中继续培养，每24h观察一次与对照组相比直至出现细胞直径增大、细胞核增大、细胞呈圆形且边界清晰、细胞内出现小泡，收细胞上清即为P0代重组杆状病毒的病毒液。如图4所示。
8、重组杆状病毒的扩增
使用细胞摇瓶悬浮培养sf9细胞，待细胞生长至对数生长期，密度为2×106cell/mL，将P0代病毒液按l：100的体积比接毒，悬浮培养96h后，收获P1代病毒，4℃，1000rpm，离心15min将上清与细胞分离，上清4℃避光保存。用该方法继续扩增P2和P3代病毒液。
9、重组杆状病毒滴度的测定
使用间接性免疫荧光法测定重组杆状病毒的滴度，具体步骤如下：
(1)细胞单层制备将处于对数生长期的sf9细胞计数后，按照每孔6.5×104个细胞，接种96孔细胞培养板，27℃培养1h：
(2)将待测重组杆状病毒用昆虫细胞培养基作10倍系列稀释，混匀。取10-4、10-5、10-6共3个稀释度。
(3)将稀释好的病毒液加入细胞孔中，每孔加入100μL病毒液，一个稀释梯度做8个重复；
(4)置27℃温箱培养96小时。
(5)细胞固定：用4％多聚甲醛150μL每孔，4℃放置30min；
(6)洗涤：弃去多聚甲醛，用PBST洗涤3次，200μL每孔，每次5min；
(7)封闭：加入封闭液50μL每孔，37℃放置30min；
(8)加入一抗：加入cap蛋白鼠的单克隆抗体，50μL每孔，37℃放置60min；
(9)洗涤：弃去一抗，用PBST洗涤3次，200μL每孔，每次5min；
(10)加入二抗：加入FITC标记的羊抗鼠抗体，37℃放置60min；
(11)洗涤：弃弃去二抗，用PBST洗涤3次，200μL每孔，每次5min；
(12)荧光显微镜观察细胞孔发荧光情况。如图5，步骤2构建的7种载体与cap蛋白编码基因重组后病毒滴度的测定结果如下表4所示：
表4
从结果可以看到包装的重组杆状病毒的滴度在107-109之间，病毒滴度都较高。10、重组cap蛋白的表达和纯化分析
将传至P2代的重组杆状病毒按照0.3MOI接种Sf9细胞，接种96h左右收获细胞，离心获得的上清进行SDS-PAGE电泳。用BCA法测定表达的蛋白的总浓度：
1)配制工作液：根据标准品和样品数量，按50体积BCA试剂A加1体积BCA试剂B试剂(50:1)配制适量的BCA工作液。
2)稀释标准品：取10μLBSA标准品用PBS稀释至100μL，使标准品的终浓度为0.5mg/mL。将标准品按0，1，2，4，8，12，16，20μL加到96孔板的蛋白标准品孔中，加PBS补足至20μL；
3)将样品稀释至合适的浓度，总体积为20μL；
4)各孔加200μLBCA工作液，37℃放置15-30分钟。用酶标仪测定562nm下的吸光值，根据标准曲线计算出总蛋白浓度，如表5。
表5
参考图像分析系统分析目的蛋白占总蛋白的比例得出目的蛋白的具体表达量。
SDS-PAGE电泳结果如图6所示，相较于没有改造的BAC-PCV2d组，改造后的SV40、SV40-WPRE、HR1-WPRE组PCV 2d cap蛋白的表达量明显增加，而改造后的CAG、WPRE、CAG-WPRE组PCV 2d cap蛋白的表达量明显减少。将表达量最高的HR1-WPRE组表达的PCV2d cap蛋白用阳离子交换树脂进行纯化，将收获的培养混合物以15,000g离心10分钟来除掉细胞和细胞碎片，上清液通过0.45μm滤器以除掉细小的杂质。用平衡缓冲液(20mM Tris，50mMNaCl，pH 8.0)将填好的阳离子交换柱平衡，过滤后的上清与平衡好的阳离子交换柱进行结合过柱；洗杂缓冲液(20mM Tris，50mMNaCl，pH 8.0)过柱洗杂；洗脱缓冲液(20mMTris，500mM NaCl，pH 8.0)过柱洗脱。将重组杆状病毒HR1-WPRE组表达蛋白经上述阳离子交换树脂纯化,纯化后的样品跑SDS-PAGE胶，结果如图7所示，后续的试验均使用该纯化后的蛋白。
11、重组PCV2d cap蛋白VLP颗粒的形成
将纯化的PCV2d cap蛋白(HR1-WPRE组)用HITACHI的透射电镜观察，结果如图所示，视野可见大小为17nm左右的VLP颗粒，形态稳定，大小均一。结果如图8所示。
12、疫苗的制备
将纯化的PCV2d cap蛋白用0.22μm滤器过滤除菌后与MontanideTM ISA201VG佐剂以1:1(w/w)的比例混合，制备成每毫升体积含有终浓度40μg cap蛋白的疫苗。
13、动物试验
1)安全性试验：8周龄的BALB/c雌鼠10只，分为A、B两组，每组5只。A组每只小鼠皮下注射0.5mL，制备好的疫苗；B组每只小鼠注射0.5mLPBS溶液；连续观察14天，两组小鼠状态相同且均无异常反应，此结果表明亚单位疫苗对小鼠是安全的。
2)免疫原性试验：将制备好的疫苗按照每只200μL免疫剂量注射到8周龄的BALB/c雌鼠的腿部肌肉中，14天后进行第二次免疫。28天后收集血样分离血清并检测特异性抗体水平，采血后用PCV2d病毒进行攻毒试验，攻毒剂量为每只老鼠0.5mL，攻毒后连续观察小鼠的状态，攻毒后14天采血，分离样品的血清并提取核酸，用荧光定量PCR检测血清中PCV2d病毒的拷贝数，引物的信息如下表表6所示：
表6ELISA检测PCV2d特异性抗体水平结果如图9所示，实验组的抗体水平在
1.4左右，明显高于对照组，说明表达的PCV2 cap蛋白能够有效激发机体的体液免疫应答。PCV2感染会引发机体产生严重的病毒血症，本试验用BALB/c雌鼠免疫了PCV2 cap蛋白后再进行攻毒实验，攻毒后14天检测血清中的PCV2病毒载量，结果如图10所示，对照组血清中的PCV2病毒拷贝数较高，是免疫后再攻毒组的300倍左右，说明免疫PCV2d cap蛋白后能够大大减轻小鼠的病毒血症。
/>
/>
/>
/>
/>
/>
/>
/>
Claims (3)
1.一种表达PCV2d cap蛋白的载体，由骨架载体和插入其启动子P10下游的多克隆位点的一个或多个PCV2d cap蛋白编码基因构成，所述骨架载体的核苷酸序列如SEQ ID NO.1所示，具有如下组件：启动子Ph被启动子SV40替换，启动子P10和其下游的多克隆位点之间插入了转录后调控序列WPRE；
或所述骨架载体的核苷酸序列如SEQ ID NO.2所示，具有如下组件：启动子Ph未被启动子SV40替换，所述转移质粒的启动子P10和Ph之间插入了增强子hrl，启动子P10和其下游的多克隆位点之间插入了转录后调控序列WPRE；
所述PCV2d cap蛋白的编码基因的核苷酸序列根据杆状病毒密码子优化，优化后的核苷酸序列如SEQ ID NO.3所示。
2.一种表达PCV2d cap蛋白的方法，其特征在于，将权利要求1中所述PCV2d cap蛋白的编码基因，与骨架载体连接，将所得转移质粒转化DH10Bac大肠杆菌，通过转座重组，获得重组杆状病毒质粒，复苏后的菌液涂布卡那霉素、四环素、庆大霉素、IPTG、X-gal平板，37℃避光培养，选白色单一菌落扩大培养，转染Sf9昆虫细胞从而获得P0代重组杆状病毒，采用Sf9细胞培养和重组杆状病毒增殖，结合阳离子交换树脂纯化得到PCV2d cap蛋白 ；
所述骨架载体的核苷酸序列如SEQ ID NO.1所示，具有如下组件：启动子Ph被启动子SV40替换，启动子P10和其下游的多克隆位点之间插入了转录后调控序列WPRE；
或所述骨架载体的核苷酸序列如SEQ ID NO.2所示，具有如下组件：启动子Ph未被启动子SV40替换，所述转移质粒的启动子P10和Ph之间插入了增强子hrl，启动子P10和其下游的多克隆位点之间插入了转录后调控序列WPRE；
所述PCV2d cap蛋白的编码基因的核苷酸序列根据杆状病毒密码子优化，优化后的核苷酸序列如SEQ ID NO.3所示。
3.如权利要求2所述的方法在制备PCV2d 的抗原、抗体、或亚单位疫苗中的应用。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111329159.4A CN114058646B (zh) | 2021-11-10 | 2021-11-10 | 一种表达PCV2d cap蛋白的载体及方法 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111329159.4A CN114058646B (zh) | 2021-11-10 | 2021-11-10 | 一种表达PCV2d cap蛋白的载体及方法 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN114058646A CN114058646A (zh) | 2022-02-18 |
| CN114058646B true CN114058646B (zh) | 2023-11-03 |
Family
ID=80274838
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111329159.4A Active CN114058646B (zh) | 2021-11-10 | 2021-11-10 | 一种表达PCV2d cap蛋白的载体及方法 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114058646B (zh) |
Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102586326A (zh) * | 2012-01-20 | 2012-07-18 | 杭州贝尔塔生物技术有限公司 | 在哺乳动物细胞中高效表达猪圆环病毒2型核衣壳蛋白的方法 |
| CN102839195A (zh) * | 2012-07-18 | 2012-12-26 | 华南农业大学 | 利用pFast Bac Dual杆状病毒表达PCV 2 Cap蛋白的方法 |
| WO2014086973A1 (en) * | 2012-12-07 | 2014-06-12 | Alternative Gene Expression S.L. | Enhanced production of the porcine circovirus capsid protein by a baculovirus vector expression system |
| CN107446895A (zh) * | 2017-07-24 | 2017-12-08 | 西北农林科技大学 | 分泌型猪圆环病毒2型重组腺病毒及其构建方法 |
| CN107574182A (zh) * | 2017-10-12 | 2018-01-12 | 西北农林科技大学 | 跨膜型猪圆环病毒2型重组腺病毒及其构建方法 |
| CN110606873A (zh) * | 2019-09-09 | 2019-12-24 | 武汉科前生物股份有限公司 | 猪圆环病毒2d型与3型Cap蛋白二联亚单位疫苗及其制备方法与应用 |
| CN110964749A (zh) * | 2018-09-29 | 2020-04-07 | 普莱柯生物工程股份有限公司 | 一种在杆状病毒表达系统中高效表达外源蛋白的方法及其应用 |
| CA3116701A1 (en) * | 2018-10-15 | 2020-04-23 | Voyager Therapeutics, Inc. | Expression vectors for large-scale production of raav in the baculovirus/sf9 system |
-
2021
- 2021-11-10 CN CN202111329159.4A patent/CN114058646B/zh active Active
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102586326A (zh) * | 2012-01-20 | 2012-07-18 | 杭州贝尔塔生物技术有限公司 | 在哺乳动物细胞中高效表达猪圆环病毒2型核衣壳蛋白的方法 |
| CN102839195A (zh) * | 2012-07-18 | 2012-12-26 | 华南农业大学 | 利用pFast Bac Dual杆状病毒表达PCV 2 Cap蛋白的方法 |
| WO2014086973A1 (en) * | 2012-12-07 | 2014-06-12 | Alternative Gene Expression S.L. | Enhanced production of the porcine circovirus capsid protein by a baculovirus vector expression system |
| CN107446895A (zh) * | 2017-07-24 | 2017-12-08 | 西北农林科技大学 | 分泌型猪圆环病毒2型重组腺病毒及其构建方法 |
| CN107574182A (zh) * | 2017-10-12 | 2018-01-12 | 西北农林科技大学 | 跨膜型猪圆环病毒2型重组腺病毒及其构建方法 |
| CN110964749A (zh) * | 2018-09-29 | 2020-04-07 | 普莱柯生物工程股份有限公司 | 一种在杆状病毒表达系统中高效表达外源蛋白的方法及其应用 |
| CA3116701A1 (en) * | 2018-10-15 | 2020-04-23 | Voyager Therapeutics, Inc. | Expression vectors for large-scale production of raav in the baculovirus/sf9 system |
| CN110606873A (zh) * | 2019-09-09 | 2019-12-24 | 武汉科前生物股份有限公司 | 猪圆环病毒2d型与3型Cap蛋白二联亚单位疫苗及其制备方法与应用 |
Non-Patent Citations (5)
| Title |
|---|
| Modified recombinant adenoviruses increase porcine circovirus 2 capsid protein expression and induce enhanced immune responses in mice;Li, DL等;ACTA VIROLOGICA;第60卷(第3期);第271-280页 * |
| 兔出血症病毒VP60蛋白研究进展;申识川等;养殖与饲料(第8期);第7-9页 * |
| 杆状病毒系统可溶性高效表达PCV2Cap蛋白和PCV3 Cap蛋白及其免疫原性研究;王硕;中国优秀硕士学位论文全文数据库（电子期刊）基础科学辑(第8期);A006-528 * |
| 猪圆环病毒2型重组腺病毒载体疫苗的构建及其免疫原性研究;李德龙;中国博士学位论文全文数据库（电子期刊）农业科技辑(第11期);D050-50 * |
| 重组杆状病毒高效表达猪圆环病毒2型Cap蛋白及免疫原性分析;张艳艳;中国优秀硕士学位论文全文数据库（电子期刊）基础科学辑(第2期);D050-337 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN114058646A (zh) | 2022-02-18 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US11236307B2 (en) | Baculovirus-based production of biopharmaceuticals free of contaminating baculoviral virions | |
| CN110204598B (zh) | 一种猪圆环病毒ⅲ型病毒样颗粒及其制备方法 | |
| US10829784B2 (en) | Method for preparing recombinant adeno-associated virus | |
| CN111676248A (zh) | 表达新型冠状病毒S基因与流感M1基因嵌合SARS-CoV-2 VLP的构建 | |
| EP4079861A1 (en) | Preparation method and system for recombinant adeno-associated virus and recombinant bacmid | |
| CN109207441B (zh) | 重组杆状病毒表达猪圆环病毒3型Cap蛋白及其构建方法与引物 | |
| EP2350292B1 (en) | Insect-derived promoters for foreign proteins expression in insect cells | |
| CN114058646B (zh) | 一种表达PCV2d cap蛋白的载体及方法 | |
| WO2014086973A1 (en) | Enhanced production of the porcine circovirus capsid protein by a baculovirus vector expression system | |
| CN111349621A (zh) | 一种重组杆状病毒及其在制备新城疫病毒样颗粒中的应用 | |
| CN111378689A (zh) | 一种用于对虾的假型昆虫杆状病毒基因转移系统、病毒及构建方法和应用 | |
| HUANG et al. | Construction of the Bac-to-Bac system of Bombyx mori nucleopolyhedroviru | |
| EP4198135A1 (en) | Nucleic acid construct for increasing adeno-associated virus yield, and construction method therefor | |
| CN111549067A (zh) | Pcv2/pcv3二价重组病毒样颗粒及其应用 | |
| CN115850397A (zh) | 一种ii型草鱼呼肠孤病毒样颗粒及其制备方法和应用 | |
| Jin-shan et al. | Construction of the Bac-to-Bac system of Bombyx mori nucleopolyhedroviru | |
| JP2019180300A (ja) | バキュロウイルスゲノム |
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 | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |