CN110184243A - The method and its kit of Antheraea pernyi nuclear polyhedrosis virus genomic DNA linearisation - Google Patents
The method and its kit of Antheraea pernyi nuclear polyhedrosis virus genomic DNA linearisation Download PDFInfo
- Publication number
- CN110184243A CN110184243A CN201910517434.1A CN201910517434A CN110184243A CN 110184243 A CN110184243 A CN 110184243A CN 201910517434 A CN201910517434 A CN 201910517434A CN 110184243 A CN110184243 A CN 110184243A
- Authority
- CN
- China
- Prior art keywords
- virus
- apnpv
- tussah
- genomic dna
- seq
- 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
- 238000000034 method Methods 0.000 title claims abstract description 49
- 241000337033 Antheraea pernyi nucleopolyhedrovirus Species 0.000 title claims abstract description 13
- 241000700605 Viruses Species 0.000 claims abstract description 191
- 108091008146 restriction endonucleases Proteins 0.000 claims abstract description 78
- 230000014509 gene expression Effects 0.000 claims abstract description 47
- 238000012216 screening Methods 0.000 claims abstract description 23
- 238000001976 enzyme digestion Methods 0.000 claims abstract description 11
- 230000029087 digestion Effects 0.000 claims abstract description 10
- 108020004414 DNA Proteins 0.000 claims description 178
- 108090000623 proteins and genes Proteins 0.000 claims description 74
- 239000013612 plasmid Substances 0.000 claims description 41
- 238000012546 transfer Methods 0.000 claims description 41
- 208000015181 infectious disease Diseases 0.000 claims description 38
- 239000013598 vector Substances 0.000 claims description 27
- 239000013604 expression vector Substances 0.000 claims description 22
- 241000238631 Hexapoda Species 0.000 claims description 21
- 230000003612 virological effect Effects 0.000 claims description 17
- 238000010790 dilution Methods 0.000 claims description 15
- 239000012895 dilution Substances 0.000 claims description 15
- 241000701447 unidentified baculovirus Species 0.000 claims description 15
- 238000012408 PCR amplification Methods 0.000 claims description 12
- 238000001514 detection method Methods 0.000 claims description 11
- 238000000926 separation method Methods 0.000 claims description 11
- 108010043121 Green Fluorescent Proteins Proteins 0.000 claims description 9
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 9
- 210000001124 body fluid Anatomy 0.000 claims description 9
- 239000010839 body fluid Substances 0.000 claims description 9
- 238000002360 preparation method Methods 0.000 claims description 9
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 claims description 7
- 101000702488 Rattus norvegicus High affinity cationic amino acid transporter 1 Proteins 0.000 claims description 7
- 238000013461 design Methods 0.000 claims description 7
- 238000000246 agarose gel electrophoresis Methods 0.000 claims description 6
- 239000000284 extract Substances 0.000 claims description 5
- 108010067770 Endopeptidase K Proteins 0.000 claims description 4
- 238000005227 gel permeation chromatography Methods 0.000 claims description 4
- 239000012528 membrane Substances 0.000 claims description 4
- 201000010099 disease Diseases 0.000 claims description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 3
- 238000005199 ultracentrifugation Methods 0.000 claims description 2
- 102100033072 DNA replication ATP-dependent helicase DNA2 Human genes 0.000 claims 1
- 102100031780 Endonuclease Human genes 0.000 claims 1
- 108010042407 Endonucleases Proteins 0.000 claims 1
- 101000927313 Homo sapiens DNA replication ATP-dependent helicase DNA2 Proteins 0.000 claims 1
- 206010027476 Metastases Diseases 0.000 claims 1
- 238000010276 construction Methods 0.000 claims 1
- 230000009401 metastasis Effects 0.000 claims 1
- 238000005192 partition Methods 0.000 claims 1
- 241000255789 Bombyx mori Species 0.000 abstract description 13
- 238000012827 research and development Methods 0.000 abstract description 3
- 210000001519 tissue Anatomy 0.000 description 22
- 241000382353 Pupa Species 0.000 description 20
- 239000007788 liquid Substances 0.000 description 19
- 238000001890 transfection Methods 0.000 description 17
- 239000001963 growth medium Substances 0.000 description 12
- 239000000047 product Substances 0.000 description 12
- 230000009385 viral infection Effects 0.000 description 11
- 102000004169 proteins and genes Human genes 0.000 description 9
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 8
- 239000003153 chemical reaction reagent Substances 0.000 description 8
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 7
- 238000004321 preservation Methods 0.000 description 7
- 238000004659 sterilization and disinfection Methods 0.000 description 7
- 239000012096 transfection reagent Substances 0.000 description 7
- 108091026890 Coding region Proteins 0.000 description 6
- 102000004144 Green Fluorescent Proteins Human genes 0.000 description 6
- 239000000872 buffer Substances 0.000 description 6
- 238000005119 centrifugation Methods 0.000 description 6
- 230000000694 effects Effects 0.000 description 6
- 239000012634 fragment Substances 0.000 description 6
- 239000005090 green fluorescent protein Substances 0.000 description 6
- 238000002156 mixing Methods 0.000 description 6
- 239000006228 supernatant Substances 0.000 description 6
- 238000011144 upstream manufacturing Methods 0.000 description 6
- 229920000936 Agarose Polymers 0.000 description 5
- 108090000790 Enzymes Proteins 0.000 description 5
- 102000004190 Enzymes Human genes 0.000 description 5
- 101150077103 TPO gene Proteins 0.000 description 5
- 239000012930 cell culture fluid Substances 0.000 description 5
- 239000006143 cell culture medium Substances 0.000 description 5
- 230000006798 recombination Effects 0.000 description 5
- 230000001954 sterilising effect Effects 0.000 description 5
- 238000004113 cell culture Methods 0.000 description 4
- 238000010367 cloning Methods 0.000 description 4
- 238000001914 filtration Methods 0.000 description 4
- 238000000799 fluorescence microscopy Methods 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 101710182846 Polyhedrin Proteins 0.000 description 3
- 108091081024 Start codon Proteins 0.000 description 3
- 239000007984 Tris EDTA buffer Substances 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 230000008901 benefit Effects 0.000 description 3
- 238000012790 confirmation Methods 0.000 description 3
- 239000012228 culture supernatant Substances 0.000 description 3
- 230000005058 diapause Effects 0.000 description 3
- 238000001962 electrophoresis Methods 0.000 description 3
- 229960004756 ethanol Drugs 0.000 description 3
- 235000019441 ethanol Nutrition 0.000 description 3
- 238000000605 extraction Methods 0.000 description 3
- 239000000499 gel Substances 0.000 description 3
- 239000011521 glass Substances 0.000 description 3
- 238000003119 immunoblot Methods 0.000 description 3
- 238000005215 recombination Methods 0.000 description 3
- 238000012163 sequencing technique Methods 0.000 description 3
- 238000009366 sericulture Methods 0.000 description 3
- 238000007711 solidification Methods 0.000 description 3
- 230000008023 solidification Effects 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 230000009182 swimming Effects 0.000 description 3
- 238000012549 training Methods 0.000 description 3
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 2
- 208000035143 Bacterial infection Diseases 0.000 description 2
- 208000003322 Coinfection Diseases 0.000 description 2
- 102000002265 Human Growth Hormone Human genes 0.000 description 2
- 108010000521 Human Growth Hormone Proteins 0.000 description 2
- 239000000854 Human Growth Hormone Substances 0.000 description 2
- 108020005202 Viral DNA Proteins 0.000 description 2
- 108700005077 Viral Genes Proteins 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 208000022362 bacterial infectious disease Diseases 0.000 description 2
- 230000003139 buffering effect Effects 0.000 description 2
- 239000006285 cell suspension Substances 0.000 description 2
- 238000010835 comparative analysis Methods 0.000 description 2
- 238000005520 cutting process Methods 0.000 description 2
- 230000007423 decrease Effects 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 230000007071 enzymatic hydrolysis Effects 0.000 description 2
- 238000006047 enzymatic hydrolysis reaction Methods 0.000 description 2
- 238000010195 expression analysis Methods 0.000 description 2
- 238000001502 gel electrophoresis Methods 0.000 description 2
- 230000006801 homologous recombination Effects 0.000 description 2
- 238000002744 homologous recombination Methods 0.000 description 2
- 238000007689 inspection Methods 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 230000010355 oscillation Effects 0.000 description 2
- 238000012856 packing Methods 0.000 description 2
- 230000001717 pathogenic effect Effects 0.000 description 2
- 230000001376 precipitating effect Effects 0.000 description 2
- 238000004064 recycling Methods 0.000 description 2
- 229940016590 sarkosyl Drugs 0.000 description 2
- 108700004121 sarkosyl Proteins 0.000 description 2
- 210000003625 skull Anatomy 0.000 description 2
- KSAVQLQVUXSOCR-UHFFFAOYSA-M sodium lauroyl sarcosinate Chemical compound [Na+].CCCCCCCCCCCC(=O)N(C)CC([O-])=O KSAVQLQVUXSOCR-UHFFFAOYSA-M 0.000 description 2
- 238000012409 standard PCR amplification Methods 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 1
- 241000255978 Antheraea pernyi Species 0.000 description 1
- 241000208340 Araliaceae Species 0.000 description 1
- 241001367049 Autographa Species 0.000 description 1
- 108020004705 Codon Proteins 0.000 description 1
- 238000007400 DNA extraction Methods 0.000 description 1
- 241000537219 Deltabaculovirus Species 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- ZGTMUACCHSMWAC-UHFFFAOYSA-L EDTA disodium salt (anhydrous) Chemical compound [Na+].[Na+].OC(=O)CN(CC([O-])=O)CCN(CC(O)=O)CC([O-])=O ZGTMUACCHSMWAC-UHFFFAOYSA-L 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- 235000005035 Panax pseudoginseng ssp. pseudoginseng Nutrition 0.000 description 1
- 235000003140 Panax quinquefolius Nutrition 0.000 description 1
- 238000012356 Product development Methods 0.000 description 1
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 1
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 108020005038 Terminator Codon Proteins 0.000 description 1
- 241000625014 Vir Species 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 230000003698 anagen phase Effects 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000005336 cracking Methods 0.000 description 1
- 229960000935 dehydrated alcohol Drugs 0.000 description 1
- 238000000432 density-gradient centrifugation Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 230000005611 electricity Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 238000011010 flushing procedure Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 235000008434 ginseng Nutrition 0.000 description 1
- 239000006451 grace's insect medium Substances 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 238000002844 melting Methods 0.000 description 1
- 230000008018 melting Effects 0.000 description 1
- 101150009602 mesh gene Proteins 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 229940126619 mouse monoclonal antibody Drugs 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
- 239000002574 poison Substances 0.000 description 1
- 231100000614 poison Toxicity 0.000 description 1
- 230000002062 proliferating effect Effects 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000009738 saturating Methods 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 239000013049 sediment Substances 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 230000029305 taxis Effects 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 239000004753 textile Substances 0.000 description 1
- 238000003151 transfection method Methods 0.000 description 1
- 238000000870 ultraviolet spectroscopy Methods 0.000 description 1
- 230000029812 viral genome replication Effects 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 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
-
- 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
-
- 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
- C12N2710/14121—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
- 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
- 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
- C12N2710/14151—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
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- General Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- Virology (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- Medicinal Chemistry (AREA)
- Immunology (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Molecular Biology (AREA)
- Plant Pathology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses the methods and its kit of a kind of linearisation of Antheraea pernyi nuclear polyhedrosis virus genomic DNA, belong to field of biotechnology.The invention discloses tools there are two restriction enzyme digestion sites, can express the ApNPV mutant virus ApNPV- Δ ph/Avr II of egfp+/egfp+, using restriction enzyme enzymolysis, digestion, realize the linearisation of ApNPV genomic DNA.Virus genom DNA after linearisation generates the ability of newly-built recombinant virus than using the virus genom DNA not linearized to improve 36 times or more in tussah silkworm chrysalis body, creates recombinant virus ratio and is greater than 99%.By the fluorescence signal difference of recombinant virus and mutant virus, the screening efficiency of recombinant virus is significantly improved.The invention also discloses prepare tussah virus expression systems application kit using the ApNPV genomic DNA of linearisation.The present invention linearizes ApNPV genomic DNA recombinant virus yield height, and quick and precisely, kit has practical value in terms of research and development of products, the scientific basic of field of biotechnology for screening.
Description
Technical field
The invention belongs to field of biotechnology, and in particular to a kind of Antheraea pernyi nuclear polyhedrosis virus genomic DNA linearisation
Method and its kit.
Background technique
Tussah is important one of the economic insects in China, and tussah cocoon annual output is up to 70,000 tons or so.Tussah cocoon shell is mainly used
Make textile raw material, though and tussah chrysalis in food, biological control and medicine and other fields obtains certain application, it is potential to be worth
Wait to further investigate and develop.Tussah because overwintering with pupal diapause, can long-term preservation, and pupal cell is larger, is considered can be developed into
For efficient bioreactor [Chinese invention patent: ZL 92103655.8], to further increase the exploitation value of tussah chrysalis
Value, is of great significance to tussah industry future high benefit, sustainable development.We utilize tussah caryogram in the research of early period
Polyhedrosis virus (Antheraea pernyi multinucleocapsid nucleopolyhedrovirus, ApNPV) is established
Tussah virus expression systems, and successfully express the genes [king such as green fluorescent protein, human growth hormone (HGH) in tussah silkworm chrysalis body
Lin Mei etc., " sericulture science ", 2010,36 (5): p792-799；Ye Bo etc., " sericulture science ", 2014,40 (2): p277-283],
Make it possible to carry out associated biomolecule product development using tussah chrysalis.But this expression system be not yet converted into it is mature can business
The product of change, main problem are that the recombinant virus efficiency that existing technical method generates is lower.
Insect baculovirus expression system is one of currently used four kinds of protein expression systems, wherein having been commercialized and obtaining
To being widely used that with baculoviral AcNPV (Autographa califomica nuclear polyhedrosis
Vires, AcNPV) it is carrier, it is the expression system that virus host is established using Insect cells Sf9, Sf21 or Tn-High Five
System.The key technique that recombinant virus is baculovirus expression system application is screened and obtains, traditional method is will be wild
Type virus genom DNA carries out homologous recombination with transfer expression vector dna co-transfecting host cells to realize, but genetic recombination
Low efficiency (0.1~1%), and lack selection markers, recombinant virus needs to obtain through excessive wheel plaque screening, and the duty cycle is long,
It is cumbersome.For the efficiency for improving baculoviral recombination and screening, and Kitts PA etc. [Nucleic Acids Res., 1990,18
(19): 5667-5672 for virus weight after] linearizing AcNPV genomic DNA by the single Bsu36I restriction enzyme site of insertion
Group, the ratio of recombinant virus increase (30%).
Although thinking that ApNPV and AcNPV belong to insect baculovirus on virus taxis, their cell host has very
Big difference.AcNPV does not infect tussah chrysalis and larva, and ApNPV does not infect Sf9, Sf21 cell, existing to have been commercialized
AcNPV expression system cannot be used directly for tussah field.Existing research show ApNPV genomic DNA be about 126Kb [Fan Q,
Et al., Virology, 2007,366 (2): 304-315], there is certain infectivity to Tn-High Five insect cell.
Thus, the building of presently disclosed tussah virus expression systems recombinant virus uses wild type ApNPV genomic DNA and transfer table
It transfects Tn-High Five cell jointly up to carrier DNA, then infects the strategy [Chinese invention patent: ZL of tussah chrysalis
201310562776.8.].But since Tn-High Five insect cell is not the specific host cell of ApNPV, genetic recombination
Inefficient, it is long that recombinant virus screens the period.Overcome above-mentioned technological difficulties to tussah virus expression systems using most important.
AcNPV genomic dna sequence is different from ApNPV genomic dna sequence, and linearization technique is not particularly suited for ApNPV.ApNPV
For the technical method of genomic DNA linearisation there is not yet open, market also has no related tussah virus expression systems application kit.
Summary of the invention
The technological difficulties of recombinant virus are obtained in view of above-mentioned tussah virus expression systems, the present invention provides a kind of ApNPV
The method of genomic DNA linearisation and the ApNPV genomic DNA preparation tussah virus expression systems application reagent for utilizing linearisation
Box, for the research and development of products of field of biotechnology, scientific basic research etc..
A kind of method that primary and foremost purpose of the invention is to provide Antheraea pernyi nuclear polyhedrosis virus genomic DNA linearisation.Tool
The technical solution of body are as follows:
(1) design three pairs have restriction enzyme digestion sites primers, building with corresponding restriction enzyme site, can table
Tussah virus up to green fluorescence protein gene (egfp) shifts expression vector；
(2) transfer expression vector plasmid DNA is extracted, and thin with ApNPV genomic DNA cotransfection Tn-High Five insect
Born of the same parents；
(3) by fluorescence microscope screening separation with step (1) described two restriction enzyme sites, can express egfp's
ApNPV mutant strain；
(4) the ApNPV mutant virus obtained in step (3) is infected into tussah chrysalis, takes the tussah chrysalis body fluid of infection morbidity,
Mutant virus is separated by ultracentrifugation separation method, then is extracted through protease K digesting, phenol, mutant virus is largely prepared
Genomic DNA；
(5) use digestion with restriction enzyme ApNPV mutant virus genomic DNA, through agarose gel electrophoresis separation or
Gel chromatography or UF membrane obtain linearisation ApNPV genomic DNA.
Specifically, the design is with restriction enzyme A vr II (Bln I) restriction enzyme site in above-mentioned technical proposal
Primer, specifically: by comparative analysis ApNPV genomic dna sequence (GenBank:EF207986) and it is known it is restricted in
Enzyme cutting restriction enzyme site sequence, screening go out in ApNPV genomic dna sequence without Avr II (Bln I) enzyme restriction enzyme site sequence.First
Avr II (Bln I) enzyme restriction enzyme site sequence is placed in the code area egfp both ends, and the upstream egfp is polygonal in ApNPV genome
Body protein gene (Polyhedron) promoter sequence and its upstream gene partial sequence, and the downstream egfp is ApNPV genome
Middle 3 ' end section coding region sequence of polyhedron gene and downstream gene partial sequence.Preferably, according to ApNPV genome
Middle polyhedron gene (Polyhedron) promoter sequence and its upstream gene partial sequence, design have restriction enzyme
The primer sequence in enzyme Hind III digestion site is SEQ ID NO:1:5 '-GGAAGCTTGCGCAAGAACGAGTCGTACTTG-
3 ', the primer sequence with restriction enzyme BamH I restriction enzyme site and Avr II (Bln I) restriction enzyme site is SEQ ID
NO:2: According to ApNPV gene
3 ' end section coding region sequence of polyhedron gene and downstream gene partial sequence in group, design have restriction enzyme
The primer sequence of Kpn I restriction enzyme site and Avr II (Bln I) restriction enzyme site is SEQ ID NO:3:With restriction enzyme
The primer sequence of EcoR I restriction enzyme site is SEQ ID NO:4:5 '-CCGAATTCTAAGCACAAAAACGGC TCCC-3'.Root
The primer of restriction enzyme digestion sites is had according to the design of egfp gene order, it is preferred that design has restriction enzyme
The primer sequence of BamH I restriction enzyme site and egfp gene start codon ATG are SEQ ID NO:5:With restriction enzyme Kpn I digestion
The primer sequence of site and egfp gene end codon TAA are SEQ ID NO:6: Above-mentioned primer sequence is as follows:
SEQ ID NO:1:5 '-GGAAGCTTGCGCAAGAACGAGTCGTACTTG-3'；
SEQ ID NO:2:
SEQ ID NO:3:
SEQ ID NO:4:5 '-CCGAATTCTAAGCACAAAAACGGCTCCC-3'；
SEQ ID NO:5:
SEQ ID NO:6:
Wherein SEQ ID NO:1 has restriction enzyme Hind III digestion site (underscore part), SEQ ID
NO:2 has restriction enzyme BamH I restriction enzyme site (underscore part) and Avr II (Bln I) restriction enzyme site (italics
Matrix shows), SEQ ID NO:3 has restriction enzyme Kpn I restriction enzyme site (underscore part and Avr II (Bln I) enzyme
Enzyme site (tilted letter expression), SEQ ID NO:4 have restriction enzyme EcoR I restriction enzyme site (underscore part),
SEQ ID NO:5 has restriction enzyme BamH I restriction enzyme site (underscore part) and egfp gene start codon ATG
(tilted letter expression), SEQ ID NO:6 have restriction enzyme Kpn I restriction enzyme site (underscore part and egfp gene
Terminator codon TAA (tilted letter expression).
Further, in above-mentioned technical proposal, restriction enzyme A vr II (Bln I) restriction enzyme site is not limited only to
It is inserted in ApNPV genome near polyhedron gene (Polyhedron) code area initiation codon ATG, Huo Zhe
It further include that may be inserted in ApNPV genomic dna sequence to appoint in ApNPV genome in the sequence of polyhedrin gene coding region
It anticipates in site, and still ensures that in virus replication the accuracy of Avr II (Bln I) restriction enzyme site sequence, and do not influence
The duplication and viral infectivity of ApNPV genome in host.
Further, in above-mentioned technical proposal, the restriction enzyme is not limited only to Avr II (Bln I), further includes
May on any site of ApNPV genomic dna sequence there are any one restriction enzyme of single restriction enzyme site sequence,
And any one restriction enzyme without single restriction enzyme site sequence on any site of ApNPV genomic dna sequence.
Further, in above-mentioned technical proposal, the restriction enzyme includes Avr II (Bln I), Sse8387I.
Further, in above-mentioned technical proposal, it is described building with restriction enzyme digestion sites, egfp can be expressed
Tussah virus transfer expression vector method, include the following steps:
(1) using ApNPV genomic DNA as template, PCR amplification is carried out with primer SEQ ID NO:1 and SEQ ID NO:2,
Obtain segment 1；PCR amplification is carried out with primer SEQ ID NO:3 and SEQ ID NO:4, obtains segment 2；
(2) segment 1 and segment 2 are cloned into respectively on pMD18-T carrier and obtain plasmid, then successively by segment 1 and segment 2
It is cloned on Hind III-BamH I and Kpn the I-EcoR I site of carrier Puc19 respectively, building obtains transfer vector；
(3) it is template by the Plasmid DNA with egfp gene, is carried out with primer SEQ ID NO:5 and SEQ ID NO:6
PCR amplification obtains segment and is cloned on pMD18-T carrier to obtain plasmid, then is cloned into the transfer vector that step (2) obtains
On BamH I-Kpn I site, obtain with restriction enzyme site, can expressing green fluorescent protein gene egfp tussah virus turn
Move expression vector.
In above-mentioned technical proposal, wherein restriction enzyme digestion sites sequence is located at the code area egfp both ends.It is described
The source of ApNPV genomic DNA can be to extract from the wild strain that nature separates and obtain, and be also possible to from by gene
It is obtained in the Antheraea pernyi nuclear polyhedrosis virus of recombination.
Specifically, in above-mentioned technical proposal, the extraction shift expression vector dna and with ApNPV genomic DNA cotransfection
Tn-High Five insect cell, specifically: pApPh-Avr II/egfp Plasmid DNA is extracted, uses ultraviolet spectrometry after aseptic process
Photometric quantification.PApPh-Avr II/egfp Plasmid DNA and the appropriate ratio of wild type ApNPV genomic DNA are mixed, then with
Transfection reagent Cellfectin mixing, room temperature was added in Tn-High Five cell after 30~50 minutes, in 27 DEG C of incubators
Culture 4~5 hours, replaces complete TNM-FH culture medium, sets in 27 DEG C of incubators 5 days, the cell culture fluid transfected.It will turn
Cell culture fluid infectable infection tussah chrysalis after dye is set 20~22 DEG C and is cultivated 10~12 days.Infection morbidity tussah chrysalis body fluid is taken, is used
The screening of virus mutation strain is carried out in infection Tn-High Five insect cell.
Specifically, in above-mentioned technical proposal, the screening separation band there are two Avr II (Bln I) restriction enzyme site, can
The ApNPV mutant strain of egfp is expressed, specifically: mutant virus is to pass through fluorescence microscopy in Tn-High Five insect cell
Mirror screening separation, and by infection tussah chrysalis obtain expanding it is numerous.It is obtained by two stages of cell and tussah chrysalis, 1~2 wheel screening
ApNPV mutant strain ApNPV- Δ ph/Avr II+/egfp+。
Specifically, in above-mentioned technical proposal, it is described to use ApNPV mutant infection tussah chrysalis, largely prepare mutant virus
Genomic DNA, specifically: mutant virus ApNPV- Δ ph/Avr II will be contained+/egfp+Cell culture fluid, with 100 μ L/
The amount infectable infection tussah chrysalis of grain pupa is set 20~22 DEG C and is cultivated 10~12 days.Take infection morbidity tussah chrysalis body fluid, by hypervelocity from
Heart method separates mutant virus, then extracts through protease K digesting, phenol, largely prepares mutant virus genomic DNA.
Specifically, in above-mentioned technical proposal, it is described to realize the linear of ApNPV genomic DNA using Avr II (Bln I)
Change, specifically: Avr II (Bln I) enzyme digestion ApNPV mutant virus genomic DNA is used, is separated through agarose gel electrophoresis
Preparation.Also using gel chromatography method or the ApNPV genomic DNA of membrane separating method recycling linearisation.
It is a further object of the present invention to provide prepare the application of tussah virus expression systems with linearisation ApNPV genomic DNA
Kit.The kit includes the ApNPV genomic DNA of linearisation, tussah Baculovirus transfer vector (pApM748MC1) plasmid
DNA, tussah chrysalis, PCR detection primer, viral dilution and kit operation instructions composition.The specific scheme is that linearisation
2~5 μ g of ApNPV genomic DNA, tussah Baculovirus transfer vector (pApM748MC1) Plasmid DNA 5~10 μ g, PCR detection is with drawing
Object is a pair of, 10 μM of each 50 μ L, primer sequence as described in SEQ ID NO:7 and SEQ ID NO:8,4~8mL of viral dilution and
Kit operation instructions.
Further, in above-mentioned technical proposal, the tussah chrysalis is 4~8.
Specifically, linearisation ApNPV genomic DNA described in above scheme is with Avr II (Bln I) enzyme digestion ApNPV
Mutant virus genomic DNA is separated through agarose gel electrophoresis and is prepared.Or it is returned using gel chromatography method or membrane separating method
Receive the ApNPV genomic DNA of linearisation.The ApNPV genomic DNA of linearisation is dissolved in sterile TE buffer (10mM Tris-
HCl, 1mM EDTA, pH 8.0) in, concentration is 0.1~0.2 μ g/ μ L, and packing 2~5 μ g/ pipe is stored in -80 DEG C, for assembling
Kit.Preferably, each kit uses the ApNPV genomic DNA of 2 μ g/ pipelines, for carrying out four cell transfectings
Experiment is used.
Further, in above-mentioned technical proposal, the preparation method of the tussah Baculovirus transfer vector Plasmid DNA, including such as
Lower step:
(1) using transfer vector pApM748BE Plasmid DNA as template, with primer SEQ ID NO:9 and SEQ ID NO:10 into
Row PCR amplification obtains segment 1, length 1462bp；PCR amplification is carried out with primer SEQ ID NO:11 and SEQ ID NO:12,
Segment 2, length 1418bp are obtained, segment 1 includes polyhedrin gene promoter sequence and upstream gene partial sequence, piece
Section 2 includes 3 ' end section coding region sequence of polyhedron gene and downstream part gene order；
(2) segment 1 and segment 2 are cloned into respectively on pMD18-T carrier, segment 2 is first cloned into transfer vector
Segment 1 after obtaining positive colony, then is cloned into Pst I-EcoR I by the corresponding Hind III-EcoR I site of pApM748BE
The building of transfer vector pApM748MC1 is completed in site.
Further, it in above-mentioned technical proposal, is introduced in tussah Baculovirus transfer vector (pApM748MC1) Plasmid DNA
A variety of restriction enzyme digestion sites, comprising: BamH I, Sal I, Kpn I, Sma I, EcoR I, Xho I, Avr II
(Bln I) and XbaI, convenient for the clone of target gene.Transfer vector pApM748MC1 Plasmid DNA is extracted, it is slow to be dissolved in sterile TE
In fliud flushing (pH8.0), concentration is 0.4~0.5 μ g/ μ L, and packing 5~10 μ g/ pipe is stored in -80 DEG C, for assembling kit.
Preferably, each kit uses 5 μ g/ pipe pApM748MC1 Plasmid DNA, the clone for target gene.
Above-mentioned primer sequence is as follows:
SEQ ID NO:9:GTCTGCAGGCTAAACCGAACAAAGCG；
SEQ ID NO:10:
SEQ ID NO:11:
SEQ ID NO:12:CTAAGCTTGACGACTTGCTGCTAGATGC。
Wherein SEQ ID NO:9 has restriction enzyme Pst I restriction enzyme site (underscore part), SEQ ID NO:10
With restriction enzyme EcoR I restriction enzyme site (underscore part) and Sma I, Kpn I, Sal I and BamH I digestion position
Point (tilted letter expression), SEQ ID NO:11 with restriction enzyme EcoR I restriction enzyme site (underscore part) and
Xho I, Avr II (Bln I) and XbaI enzyme cutting site (tilted letter expression), SEQ ID NO:12 have restriction enzyme
Hind III digestion site (underscore part).
Specifically, tussah chrysalis involved in kit described in above scheme be by inspection without pathogenic bacterial infection,
Termination of diapause and pupa calvarium plate is transparent or translucent, fresh and alive tussah chrysalis that pupa Full size is full.Each reagent
Box is attached to 4~8 tussah chrysalis, and the expansion for recombinant virus after DNA transfection cell is numerous.Before use, tussah chrysalis should be stored in 0~
4℃.It needs to carry out disinfection to tussah chrysalis body surface with 75% ethyl alcohol before virus infection.The tussah chrysalis of infection need to be placed in by virus when expanding numerous
18~24 DEG C of incubations.It preferably, is 22 DEG C.
Specifically, PCR detection involved in kit described in above scheme is SEQ ID NO:7 with primer sequence:
TTAACGCTTAGCCAGCAGCAG and SEQ ID NO:8:TTCTTTACCGCTCCAGTTGAC.10 μM of primer concentration, each 50 μ L.
The tussah chrysalis tissue gene group DNA for having virus infection is extracted, the PCR for recombinant virus is detected.Pure newly-built recombinant virus
PCR product size be 400bp+ target gene fragment.
Specifically, involved kit operation instructions, content include: cell transfecting in kit described in above scheme
When the linearisation dosage of ApNPV genomic DNA, transfection method, virus expand numerous, recombinant virus screening and identification.It is specific: used thin
Born of the same parents are insect cell Tn-High Five.Transfection is carried out in 12 holes or 6 porocyte plates, it is preferred that is 6 porocyte plates.Often
The dosage that ApNPV genomic DNA is linearized when the cell transfecting of hole is 0.2~0.5 μ g, it is preferred that is 0.5 μ g.When transfection and toothed oak
The ratio of silkworm virus transfer expression vector plasmid DNA is 1:1~2, it is preferred that is 1:2.Transfection reagent is Cellfectin, ginseng
Transfection procedure is carried out according to reagent operation instruction.It is numerous to carry out virus expansion by infection tussah chrysalis for cell transfecting liquid.Extraction has viral sense
The tussah chrysalis tissue gene group DNA of dye, the PCR for recombinant virus are identified.Recombinant virus screening is thin in Tn-High Five
It is carried out in born of the same parents, and realizes amplification in tussah chrysalis.
The present invention has the advantages that
1. present invention firstly discloses a kind of technical methods of ApNPV genomic DNA linearisation.This method constructs first
Tool there are two Avr II (Bln I) restriction enzyme site, the ApNPV mutant virus ApNPV- Δ ph/Avr II of egfp can be expressed+/
egfp+, the line of ApNPV genomic DNA can be realized by Avr II (Bln I) enzyme enzymolysis, digestion mutant virus genomic DNA
Property.On the one hand, cell transfecting is carried out using the ApNPV genomic DNA of linearisation, newly-built recombination is generated in tussah silkworm chrysalis body
The ability of virus generates the ability for creating recombinant virus than using the virus genom DNA not linearized to transfect in tussah silkworm chrysalis body
36 times or more are improved, recombinant virus ratio is created and is greater than 99%.Recombinant virus generation efficiency greatly improves, and is significantly better than tussah
The original technical method of virus expression systems.On the other hand, the ApNPV genomic DNA of linearisation will not carry egfp gene,
The new recombinant virus (unstressed configuration) for not expressing egfp gene will be generated for transfecting when homologous recombination occurs for cell, it can be with
It is come with the ApNPV mutant virus (having fluorescence) for the expression egfp gene not linearized by fluorescence signal significant difference, it is real
The quick and precisely screening of existing recombinant virus.
2. the present invention is also provided for the first time with linearisation ApNPV genomic DNA preparation tussah virus expression systems application examination
Agent box can be used for the commercial development of tussah virus expression systems.It can be used for infecting using the recombinant virus that the kit generates
Insect cell or tussah chrysalis produce recombinant protein, have in terms of research and development of products, the scientific basic of field of biotechnology
Practical value.
Detailed description of the invention
Fig. 1 is the transfer expression vector pApPh-Avr II/egfp signal of building tussah virus described in the embodiment of the present invention 1
Figure.
Fig. 2 be described in the embodiment of the present invention 1 with Avr II (Bln I) restriction enzyme site ApNPV mutant virus and
(M is albumen Marker to the PCR detection agarose gel electrophoresis figure of wild type ApNPV virus genom DNA in figure, and 1,2 swimming lane is
Mutant virus；3,4 swimming lanes are wild-type virus).
Fig. 3 is the ApNPV genomic DNA of linearisation described in the embodiment of the present invention 1 and the viral genome that does not linearize
DNA agarose gel electrophoresis figure.
Fig. 4 is the ApNPV genomic DNA of linearisation described in the embodiment of the present invention 2 and the viral genome that does not linearize
The application effect of mutant virus is generated after DNA transfection in tussah chrysalis.Ordinate is defined as the tussah of every microgram infection morbidity
Copy number containing mutant virus in pupa tissue gene group DNA.
Fig. 5 is the ApNPV genomic DNA of linearisation described in the embodiment of the present invention 3 and the viral genome that does not linearize
DNA generates the application of mutant virus after shifting expression vector plasmid DNA cotransfection with tussah virus respectively in tussah chrysalis
Effect.Ordinate is defined as the copy number containing mutant virus in the tussah chrysalis tissue gene group DNA of every microgram infection morbidity.
Fig. 6 is the ApNPV genomic DNA of linearisation described in the embodiment of the present invention 3 and the viral genome that does not linearize
DNA generates newly-built recombinant virus after shifting expression vector plasmid DNA cotransfection with tussah virus respectively in tussah chrysalis
ApNPV-Δph/Δegfp/htpo+Application effect.Ordinate is defined as the tussah chrysalis tissue gene of every microgram infection morbidity
Copy number containing newly-built recombinant virus in group DNA.
Fig. 7 is to detect pure recombinant virus ApNPV- Δ ph/ Δ egfp/ described in the embodiment of the present invention 4 with PCR method
htpo+As a result.
Fig. 8 is that recombinant virus ApNPV- Δ ph/ Δ egfp/htpo is created described in the embodiment of the present invention 4+The table in tussah chrysalis
The protein immunoblotting method testing result of intelligent's tpo gene.
Fig. 9 is building tussah Baculovirus transfer vector pApM748MC1 schematic diagram described in the embodiment of the present invention 5.
Specific embodiment
Below with reference to specific example, the present invention will be further described, can make those skilled in the art more comprehensively
Ground understands the present invention, but do not limit the invention in any way.Test method involved in example unless otherwise specified, is
Method proposed by conventional method or manufacturer；Used reagent, material etc. can pass through commercial sources unless otherwise specified
It obtains.
Wild type ApNPV Strain, which separates for this laboratory and passes through infection tussah chrysalis, is stored in -80 DEG C of [Fan Q, et
al.,Virology,2007,366(2):304-315].Researcher in this field can also be from the tussah of natural infection ApNPV
Separation obtains the virus in pupa.Tussah chrysalis is commercially available.Restriction enzyme A vr II (Bln I) and its enzymatic hydrolysis buffering
Liquid (K buffer) is purchased from TaKaRa company (1022A).Tn-High FiveTMCell (B855-02), transfection reagent
Cellfectin (10362-010), serum-free cell culture medium SF-900TMII SFM (10902-088) can be from Invitrogen
Company's purchase, complete TNM-FH culture medium is by Insect culture medium Grace ' s Insect Medium, Supplemented
(11605-094, Invitrogen) adds 10%FBS and is formulated, and FBS (35-010-CV) is Corning company product.TPO
Reference is GST-TPO, molecular weight 65.0kDa (Cloud-Clone Corp. product).
The tool of embodiment 1 there are two Avr II (Bln I) restriction enzyme site, can expressing green fluorescent protein gene (egfp)
ApNPV mutant strain ApNPV- Δ ph/Avr II+/egfp+Preparation
(1) tussah virus shifts expression vector establishment
Using ApNPV genomic DNA as template, with primer SEQ ID NO:1 and SEQ ID NO:2, SEQ ID NO:3 and
SEQ ID NO:4 carries out standard PCR amplification respectively, and it is long to obtain segment 1 (ApNPV genomic DNA, 125168nt-126244nt)
Degree is 1078bp, and (ApNPV genomic DNA, the 250nt-1288nt) length of segment 2 is 1041bp.Segment 1 includes polyhedrin
Gene promoter sequence and upstream gene partial sequence, segment 2 include 3 ' end section coding region sequence of polyhedron gene
And downstream part gene order.Two segments are by pMD18-T carrier cloning and after sequencing confirmation, successively by segment 1 and piece
Section 2 is cloned into respectively on Hind III-BamH I and Kpn the I-EcoR I site of carrier Puc19, and building obtains transfer vector
pApPh-Avr II.Using the Plasmid DNA with egfp gene as template, carried out with primer SEQ ID NO:5 and SEQ ID NO:6
Standard PCR amplification obtains 720bp segment, and is cloned into pMD18-T carrier.Sequencing confirmation rear clone is being obtained to carrier
The BamH I-Kpn I cloning site of pApPh-Avr II completes tussah virus and shifts expression vector pApPh-Avr II/egfp
Building, as shown in Figure 1.Egfp gene two sides have Avr II (Bln I) restriction enzyme site, have Avr II (Bln for constructing
I) restriction enzyme site, the ApNPV mutant virus of egfp gene can be expressed.
(2) cell transfecting and mutant virus screening
Tn-High Five cell carries out secondary culture with complete TNM-FH culture medium.By the Tn-High of logarithmic growth phase
Five attached cell is gently blown and beaten with suction pipe, is made about 0.5~1 × 106/ mL cell suspension, is added to 6 porocyte culture plates
In, make every hole cell density 80% or so, sets in 27 DEG C of incubators 1 hour, it is spare.
PApPh-Avr II/egfp Plasmid DNA is extracted, it is quantitative with ultraviolet specrophotometer after aseptic process, for transfection
With.Referring to Cellfectin reagent operation instruction, by pApPh-Avr II/egfp Plasmid DNA and wild type ApNPV genome
The appropriate ratio mixing of DNA, then mixed with suitable transfection reagent Cellfectin, room temperature is added to after 30~50 minutes to be ready to
Tn-High Five cell in, cultivated 4 hours in 27 DEG C of incubators.The complete TNM-FH culture medium of 1~2mL is replaced, sets 27 DEG C
5 days in incubator.The expression for having green fluorescent protein in cell can be observed under fluorescence microscope.
The cell culture fluid of above-mentioned transfection, infectable infection tussah chrysalis are taken, every 100 μ L of pupa sets 22 DEG C and cultivates 10~12 days.
With fluorescence microscope and confirm the expression for having green fluorescence protein gene in tussah chrysalis haemocyte.
The tussah chrysalis body fluid for taking mutant virus and wild virus mixed infection, with insect serum-free cell culture medium
SF-900TM II SFM 1:100 (v:v) dilution, filtration sterilization.Again by this 10 times of gradient dilution of sterile virus liquid, it is configured to
10-1、10-2、10-3、10-4、10-5With 10-6Various concentration virus liquid.1mL various concentration virus liquid is taken to infect Tn-High Five
Cell sets 27 DEG C of incubator cultures, takes out virus liquid after 1 hour, is gently eluted carefully with a small amount of insect serum-free cell culture medium
Born of the same parents are primary, and the low melting-point agarose of 1mL 2% is added, and after agarose solidification, the complete TNM-FH culture medium of 1mL are added, sets 27
DEG C incubator continues culture 5 days, fluorescence microscopy microscopic observation Green Fluorescent Protein Gene Expression situation.Take out culture supernatants,
The cell hole for selecting minimum virus concentration infection will have individually the virus of Green Fluorescent Protein Gene Expression to feel with glass pipette fixed point
The cell of dye is chosen, and is suspended in about 100 μ L serum-free cell culture mediums, vibrates and takes supernatant after being centrifuged, infectable infection tussah chrysalis,
A pupa every time is set 22 DEG C and is cultivated 10~12 days, and mutant virus is made to obtain breeding amplification.Extract the tussah chrysalis group of infection morbidity
Genomic DNA is knitted, with the purity of PCR detection method Analysis and Screening virus, detection primer sequence such as SEQ ID NO:7:
Described in TTAACGCTTAGCCAGCAGCAG and SEQ ID NO:8:TTCTTTACCGCTCCAGTTGAC.
As shown in Fig. 2, a single 980bp segment can be obtained through PCR amplification in pure mutant virus, and it is wild
A single 491bp segment can be obtained through PCR amplification in type ApNPV.
If desired, repeat this step carries out wheel virus screening again.It is final to obtain ApNPV mutant strain ApNPV- Δ ph/
Avr II+/egfp+。
(3) mutant virus extracting genome DNA
The mutant virus ApNPV- Δ ph/Avr II that screening is obtained+/egfp+Batch infection tussah chrysalis, sets 22 DEG C of trainings
It supports 10~12 days.Take viral pupal cell liquid, at 4 DEG C, 5000rpm is centrifuged 20 minutes.Take supernatant, at 4 DEG C, 25,000rpm centrifugations 30
Minute.Supernatant is abandoned, precipitating is suspended in a small amount of 0.1X TE (pH=8.0) buffer, then carries out sucrose density gradient centrifugation
(25%:55%), at 4 DEG C, 25,000rpm centrifugations 90 minutes.The viral sample for taking 25%:55% interfacial separation, with 0.1X TE
(pH=8.0) buffer dilutes, at 4 DEG C, 25,000rpm centrifugations 30 minutes.Supernatant is abandoned, precipitating is suspended in a small amount of cracking buffering
Liquid (0.1M Tris-HCl, pH=8.0；0.12M EDTA-2Na, 0.2M KCl) in, appropriate Proteinase K and the ten of 10% is added
Sarkosyl sodium (Sarkosyl), 50 DEG C of warm bath are stayed overnight.It is respectively extracted once through phenol, phenol/chloroform, chloroform, it is upper after centrifugation
It is precipitated with dehydrated alcohol clearly.DNA sample is finally dissolved in sterile TE buffer, quantitative, spare.
(4) mutant virus ApNPV- Δ ph/Avr II+/egfp+Identification and linearize ApNPV genomic DNA system
It is standby
In the reaction system of 100 μ L, the above-mentioned mutant virus ApNPV- Δ ph/Avr II of about 6 μ g is added+/egfp+
Genomic DNA, Avr II (Bln I) restriction endonuclease (TaKaRa, 10u/ μ L) of 9 μ L, the enzymatic hydrolysis buffer (K of 10 μ L 10x
Buffer, TaKaRa company, 1022A), it is mended with sterile water to 100 μ L.37 DEG C of warm bath are stayed overnight.0.8% Ago-Gel of sample
It is separated by electrophoresis, the small fragment of a 720bp should occurs after the mutant virus genome dna electrophoresis digested completely and one is greater than
The large fragment of 120kb.
The Ago-Gel of ApNPV genomic DNA with large fragment is cut down, is fitted into bag filter, is added few
0.5xTAE buffer is measured, the ApNPV genomic DNA of electrophoresis recycling linearisation, through phenol, phenol/chloroform, chloroform, ethyl alcohol is heavy
It forms sediment.It is dissolved in sterile TE buffer after drying, it is quantitative, it is spare.
As a result illustrate: the ApNPV genomic DNA of linearisation and the virus genom DNA Ago-Gel electricity not linearized
There is band see Fig. 3, in 720bp or so in result of swimming, and display genomic DNA is linearized.
The technical method of embodiment explanation, ApNPV genomic DNA linearisation disclosed by the invention is effective.
Embodiment 2 linearizes ApNPV genomic DNA cell transfecting and virus multiplication
The mutant virus ApNPV- Δ ph/Avr II that 0.5 μ g is linearized+/egfp+Genomic DNA and the 0.5 non-line of μ g
The ApNPV- Δ ph/Avr II of property+/egfp+Genomic DNA is mixed with suitable transfection reagent Cellfectin respectively, room temperature
It is respectively added to after 30~50 minutes in ready Tn-High Five cell, is cultivated 4 hours in 27 DEG C of incubators.Replacement 1
The complete TNM-FH culture medium of~2mL, sets in 27 DEG C of incubators 5~6 days.Take cell culture fluid, infectable infection tussah chrysalis, every pupa
100 μ L set 22 DEG C and cultivate 10~12 days.Take tussah chrysalis tissue fluid in fluorescence microscopy microscopic observation, linearized viral gene
It is barely perceivable egfp gene expression in the tussah chrysalis cell of group DNA transfection liquid infection, and in the viral gene not linearized
Egfp gene can be observed in the tussah chrysalis cell of group DNA transfection liquid infection and obtain great expression.Take the tussah chrysalis of infection morbidity
Tissue extraction genomic DNA analyzes and identifies virus multiplication situation with fluorescence quantifying PCR method.
As a result illustrate: in tussah after the ApNPV genomic DNA of linearisation and the virus genom DNA not linearized transfection
The application effect that mutant virus is generated in pupa is as shown in Figure 4, wherein ordinate is defined as the tussah chrysalis of every microgram infection morbidity
Copy number containing mutant virus in tissue gene group DNA.As can be seen from Fig. 4, it is transfected using the virus genom DNA of linearisation
And the ability (2.5 × 10 of mutant virus is regenerated in tussah silkworm chrysalis body6Copy number/μ g genomic DNA) it is more not linear than use
The virus genom DNA transfection of change regenerates the ability (8.0 × 10 of mutant virus in tussah silkworm chrysalis body9Copy number/μ g base
Because of a group DNA) 3000 times of decline or more.
The advantages of ApNPV genomic DNA disclosed by the invention using linearization process carries out cell transfecting first is that pole
The ability that former mutant virus generates is reduced greatly, will significantly improve the generation efficiency of newly-built recombinant virus.The implementation seen below
Example 3.
Embodiment 3 linearizes ApNPV genomic DNA for creating recombinant virus
Tn-High Five cell prepares according to method described in embodiment 1.Transfection makes referring to Cellfectin reagent
With explanation, the mutant virus ApNPV- Δ ph/Avr II that 0.5 μ g is linearized+/egfp+Genomic DNA and 1 μ g have external source
Gene tussah virus transfer expression vector plasmid (such as pApM748BE/htpo) DNA mixing, then with suitable transfection reagent
Cellfectin mixing, room temperature was added in ready Tn-High Five cell after 30~50 minutes, in 27 DEG C of incubators
Culture 4 hours.The replacement complete TNM-FH culture medium of 1~2mL is set in 27 DEG C of incubators 5~6 days.The ApNPV- Δ not linearized
ph/Avr II+/egfp+Genomic DNA is also used for and pApM748BE/htpo Plasmid DNA cotransfection Tn-High as control
Five cell, comparative analysis recombinant virus generation efficiency.Take the culture solution of transfection cell, infectable infection tussah chrysalis, every pupa 100
μ L sets 22 DEG C and cultivates 10~12 days.The tussah chrysalis tissue gene group DNA for extracting virus infection, is distinguished with fluorescence quantifying PCR method
Identify mutant virus (ApNPV- Δ ph/Avr II+/egfp+) and newly-built recombinant virus (ApNPV- Δ ph/ Δ egfp/
htpo+) proliferative conditions.
As a result illustrate: the ApNPV genomic DNA of linearisation and the virus genom DNA not linearized respectively with tussah
Virus transfer expression vector plasmid DNA cotransfection after in tussah chrysalis generate mutant virus application effect as shown in figure 5, its
In, ordinate is defined as the copy number containing mutant virus in the tussah chrysalis tissue gene group DNA of every microgram infection morbidity；Linearly
The ApNPV genomic DNA of change and the virus genom DNA not linearized are shifting expression vector plasmid with tussah virus respectively
Newly-built recombinant virus ApNPV- Δ ph/ Δ egfp/htpo is generated after DNA cotransfection in tussah chrysalis+Application effect such as Fig. 6 institute
Show, wherein ordinate is defined as the copy containing newly-built recombinant virus in the tussah chrysalis tissue gene group DNA of every microgram infection morbidity
Number.From Fig. 5 it is not difficult to find out that, using the virus genom DNA transfection of linearisation and mutant virus is generated in tussah silkworm chrysalis body
Ability (4.1 × 105Copy number/μ g genomic DNA) than being transfected in tussah silkworm chrysalis body using the virus genom DNA not linearized
Generate the ability (3.8 × 10 of mutant virus9Copy number/μ g genomic DNA) 9000 times of decline or more；And use linearisation
Virus genom DNA transfection simultaneously generates newly-built recombinant virus (ApNPV- Δ ph/ Δ egfp/htpo in tussah silkworm chrysalis body+) ability
(1.2×109Copy number/μ g genomic DNA) than being generated in tussah silkworm chrysalis body using the virus genom DNA transfection not linearized
The ability (3.2 × 10 of newly-built recombinant virus7Copy number/μ g genomic DNA) improve 36 times or more.Newly-built recombinant virus ratio
Greater than 99%.
Embodiment explanation, ApNPV genomic DNA linearization technique recombinant virus generation efficiency disclosed by the invention is significantly
It improves, is significantly better than the original technical method of tussah virus expression systems.
Embodiment 4 creates recombinant virus screening and gene expression analysis
The tussah chrysalis body fluid for taking mutant virus and newly-built recombinant virus mixed infection, with insect serum-free cell culture
Base SF-900TM II SFM 1:100 (v:v) dilution, filtration sterilization.Again by this 10 times of gradient dilution of sterile virus liquid, it is configured to
10-1、10-2With 10-3Various concentration virus liquid.It takes 1mL various concentration virus liquid to infect Tn-High Five cell, sets 27 DEG C of trainings
Case culture is supported, takes out virus liquid after 1 hour, it is primary gently to elute cell with a small amount of insect serum-free cell culture medium, and 1mL is added
2% low melting-point agarose is added the complete TNM-FH culture medium of 1mL, sets 27 DEG C of incubators and continue to cultivate after agarose solidification
5~6 days, fluorescence microscopy microscopic observation Green Fluorescent Protein Gene Expression situation.Culture supernatants are taken out, are pinpointed with glass pipette
The cell of redgreen fluorescence protein gene expression is drawn, in the about 100 μ L cell culture mediums that suspend, supernatant is taken after oscillation centrifugation, infuses
Infection tussah chrysalis is penetrated, every time a pupa, sets 22 DEG C and cultivate 10~12 days.Tussah chrysalis tissue gene group DNA is extracted, PCR method is used
Tussah chrysalis virus infection situation is analyzed, the purity of virus is screened.PCR identification primer sequence such as SEQ ID NO:7 and SEQ ID
Described in NO:8.It is general to can be obtained new recombinant virus by a wheel screening.If desired, using Tn-High Five cell-
Tussah chrysalis carries out wheel virus screening again, obtains pure recombinant virus ApNPV- Δ ph/ Δ egfp/htpo+。
By newly-built recombinant virus ApNPV- Δ ph/ Δ egfp/htpo+Tussah chrysalis is infected, 22 DEG C is set and cultivates 10~12 days, point
It does not take viral pupa body fluid and tissue to carry out gel electrophoresis of protein, and uses protein immunoblotting method, it is small with anti-human HTPO
The expression of mouse monoclonal antibody detection people tpo gene.
As a result illustrate: detecting pure recombinant virus ApNPV- Δ ph/ Δ egfp/htpo with PCR method+Gel electrophoresis
As a result as shown in fig. 7, the PCR product of pure newly-built recombinant virus is single, about 1.2kb segment (right side), and disease is mixed
The PCR product of poison has two, one be about 1.2kb segment, another be 980bp segment (left side).Newly-built recombinant virus
ApNPV-Δph/Δegfp/htpo+The protein immunoblotting method testing result of people tpo gene is expressed such as in tussah chrysalis
Shown in Fig. 8, the expression of people's tpo gene can be can be detected after recombinant virus infection 10 days in tussah chrysalis in tussah chrysalis tissue,
And reach a high level behind 11 days of infection；And tussah chrysalis has no people's tpo gene in tussah after wild virus infection 11 days
It is expressed in pupa tissue.
Embodiment explanation, can be in tussah silkworm chrysalis body using the new recombinant virus that linearisation ApNPV genomic DNA obtains
Successful expression foreign gene, the present invention are opened with linearisation ApNPV genomic DNA preparation tussah virus expression systems application kit
Hair has broad application prospects, it can be achieved that commercial applications, then fill up China in insect baculovirus expression system business
The blank of application field.
Embodiment 5 constructs tussah Baculovirus transfer vector pApM748MC1
It is with transfer vector pApM748BE [Wang Linmei etc., " sericulture science ", 2010,36 (5): p792-799] Plasmid DNA
Template carries out routine with primer SEQ ID NO:9 and SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12 respectively
PCR amplification obtains segment 1, length 1462bp；Segment 2, length 1418bp.Segment 1 starts comprising polyhedron gene
Subsequence and upstream gene partial sequence, segment 2 include 3 ' end section coding region sequence of polyhedron gene and downstream portion
Divide gene order.Segment 2 is first cloned into transfer vector by pMD18-T carrier cloning and after sequencing confirmation by two segments
Segment 1 after obtaining positive colony, then is cloned into Pst I-EcoR I by the corresponding Hind III-EcoR I site of pApM748BE
The building of transfer vector pApM748MC1 is completed, as shown in Figure 9 in site.Gram contained in newly-built transfer vector pApM748MC1
Grand site includes: BamH I, Sal I, Kpn I, Sma I, EcoR I, Xho I, Avr II (Bln I) and XbaI.
Embodiment explanation, the carrier disclosed by the invention provided using kit, cloning site is abundant, is convenient for different mesh
Gene clone, have wide range of applications.
The preparation tussah virus expression systems application kit of the linearisation ApNPV genomic DNA of embodiment 6
(1) kit 1: the dosage of linearisation ApNPV genomic DNA is 2 μ g, tussah Baculovirus transfer vector pApM748MC1
5 μ g of Plasmid DNA, tussah chrysalis 4 (each 2 of male and female pupa), PCR detection primer SEQ ID NO:7 and SEQ ID NO:8, primer
10 μM of concentration, each 50 μ L, viral dilution 4mL, kit operation instructions.Linearisation viral DNA, Plasmid DNA and primer set-
20 DEG C of preservations, tussah chrysalis and viral dilution set 0~4 DEG C of preservation.
(2) kit 2: the dosage of linearisation ApNPV genomic DNA is 5 μ g, tussah Baculovirus transfer vector pApM748MC1
10 μ g of Plasmid DNA, tussah chrysalis 8 (each 4 of male and female pupa), PCR detection primer SEQ ID NO:7 and SEQ ID NO:8 draw
10 μM of object concentration, each 100 μ L, viral dilution 8mL, kit operation instructions.Linearize viral DNA, Plasmid DNA and primer
- 20 DEG C of preservations are set, tussah chrysalis and viral dilution set 0~4 DEG C of preservation.
7 tussah virus expression systems application kit operation instructions of embodiment
(1) cell prepares: Tn-High Five cell carries out secondary culture with complete TNM-FH culture medium.By logarithmic growth
The Tn-High Five attached cell of phase is gently blown and beaten with suction pipe, is made about 0.5~1 × 106/ mL cell suspension, is added to 6 holes
In tissue culture plate, makes every hole cell density 80% or so, set in 27 DEG C of incubators 1 hour, it is spare.
(2) tussah virus transfer expression vector plasmid DNA prepares: the target gene expressed will be needed to be cloned into embodiment 5
Described on the corresponding restriction enzyme site of transfer vector pApM748MC1, building tussah virus transfer expression vector plasmid is (such as
PApM748MC1/ target gene).Plasmid DNA is extracted, is quantified after aseptic process spare.
(3) it transfects: referring to Cellfectin reagent operation instruction, the ApNPV genomic DNA and 1 μ g that 0.5 μ g is linearized
Transfer expression vector plasmid (such as pApM748MC1/ target gene) the DNA mixing of above-mentioned tussah virus, then with suitable transfection reagent
Cellfectin mixing, room temperature was added in ready Tn-High Five cell after 30~50 minutes, in 27 DEG C of incubators
Culture 4~5 hours.The complete TNM-FH culture medium of 1.5~2mL is replaced, is set in 27 DEG C of incubators 5~6 days.
(4) tussah chrysalis prepares: tussah chrysalis be by inspection without pathogenic bacterial infection, termination of diapause and pupa calvarium plate be in saturating
Fresh and alive tussah chrysalis bright or translucent, pupa Full size is full.Tussah chrysalis should be stored in 0~4 DEG C.It is needed before virus infection
It is carried out disinfection with 75% ethyl alcohol to tussah chrysalis body surface.
(5) virus expands numerous: taking out the culture solution of transfection cell, infectable infection tussah chrysalis, every 100 μ L of pupa sets 22 DEG C of trainings
It supports 10~12 days.It is a small amount of to extract tussah chrysalis tissue, egfp gene expression has been seen whether under fluorescence microscope.Recombinant virus will not
Egfp gene is expressed, green cells appearance is not observed under fluorescence microscope.
(6) recombinant virus PCR is identified: it is a small amount of to extract tussah chrysalis tissue, it is extracted using tissue gene group DNA extraction kit
Tussah chrysalis tissue gene group DNA carries out Standard PCR identification with primer SEQ ID NO:7 and SEQ ID NO:8 as template.
Recombinant virus PCR product size is 400bp+ target gene fragment, and the PCR product for the virus not recombinated is the piece of 980bp
Section.There is its PCR product of the tussah chrysalis tissue gene group DNA of hybrid virus infection that may occur above-mentioned two situations simultaneously.Such as
There is this to happen, recombinant virus needs further screening.
(7) recombinant virus screens: the tussah chrysalis body fluid for taking hybrid virus to infect, dilute with viral dilution 1:100 (v:v)
It releases, filtration sterilization.Again by this 10 times of gradient dilution of sterile virus liquid, it is configured to 10-1、10-2With 10-3Various concentration virus liquid.It takes
1mL various concentration virus liquid infects preprepared Tn-High Five cell, sets 27 DEG C of incubator cultures, takes after 1 hour
Virus liquid out, gently elutes that cell is primary with a small amount of insect serum-free cell culture medium, adds the low melting point agar of 1mL 2%
Sugar is added the complete TNM-FH culture medium of 1~2mL, sets 27 DEG C of incubators and continue culture 5~6 days after agarose solidification, fluorescence
Microscopically observation Green Fluorescent Protein Gene Expression situation.Culture supernatants are taken out, is pinpointed with glass pipette and draws redgreen
The cell of fluorescence protein gene expression, is suspended in about 100 μ L insect serum-free cell culture mediums, takes supernatant after oscillation centrifugation,
Infectable infection tussah chrysalis, a pupa, sets 22 DEG C and cultivates 10~12 days every time.Observe tussah chrysalis Infection Status.Extract the toothed oak of infection
Silkworm chrysalis tissue genomic DNA, with the purity of the PCR detection method analysis recombinant virus of step (4).Pure newly-built recombinant virus
PCR product be single 400bp+ target gene fragment.It is general to can be obtained pure recombinant virus by a wheel screening.
(8) expression of recombinant virus analysis and preservation: the tussah chrysalis body fluid of identified pure recombinant virus infection is taken, with disease
Malicious dilution 1:100 (v:v) dilution, filtration sterilization.Virus liquid is for infecting tussah chrysalis, every 100 μ L of pupa.Set 22 DEG C of cultures 10
~12 days, observe tussah chrysalis Infection Status.It can also be identified again with PCR method.The tussah chrysalis of infection can use protein expression analysis,
Or -80 DEG C of postposition preservations of sealing.
Note: (1) suggest: the cell and tussah chrysalis of virus infection should need before discarded by conventional high-pressure sterilization treatment.
(2) primer sequence:
SEQ ID NO:7:TTAACGCTTAGCCAGCAGCAG
SEQ ID NO:8:TTCTTTACCGCTCCAGTTGAC
(3) difference is little between individuals for protein expression, related with virus infection dosage and infection time, therefore reagent of the present invention
The tussah chrysalis inter-individual difference provided in box is on result without influence.
This example demonstrates that tussah virus expression systems application kit has perfect teachings file, convenient for making
User carries out related application according to specification.
Sequence table
<110>Research Institute of Ocean Fishery Science, Liaoning Province
<120>method and its kit of Antheraea pernyi nuclear polyhedrosis virus genomic DNA linearisation
<160> 12
<170> SIPOSequenceListing 1.0
<210> 1
<211> 30
<212> DNA
<213>artificial sequence (Artificial)
<400> 1
ggaagcttgc gcaagaacga gtcgtacttg 30
<210> 2
<211> 35
<212> DNA
<213>artificial sequence (Artificial)
<400> 2
ccggatccta ggttatagga aattttacta caaag 35
<210> 3
<211> 35
<212> DNA
<213>artificial sequence (Artificial)
<400> 3
acggtaccta ggaaacttat tgtcaactgg agcgg 35
<210> 4
<211> 28
<212> DNA
<213>artificial sequence (Artificial)
<400> 4
ccgaattcta agcacaaaaa cggctccc 28
<210> 5
<211> 30
<212> DNA
<213>artificial sequence (Artificial)
<400> 5
ccggatccat ggtgagcaag ggcgaggagc 30
<210> 6
<211> 30
<212> DNA
<213>artificial sequence (Artificial)
<400> 6
ccggtacctt acttgtacag ctcgtccatg 30
<210> 7
<211> 21
<212> DNA
<213>artificial sequence (Artificial)
<400> 7
ttaacgctta gccagcagca g 21
<210> 8
<211> 21
<212> DNA
<213>artificial sequence (Artificial)
<400> 8
ttctttaccg ctccagttga c 21
<210> 9
<211> 26
<212> DNA
<213>artificial sequence (Artificial)
<400> 9
gtctgcaggc taaaccgaac aaagcg 26
<210> 10
<211> 47
<212> DNA
<213>artificial sequence (Artificial)
<400> 10
atgaattccc ggggtaccgt cgacggatcc tctggaatag ttatagg 47
<210> 11
<211> 47
<212> DNA
<213>artificial sequence (Artificial)
<400> 11
cggaattcct cgagcctagg attctagaag atcctttaga caattac 47
<210> 12
<211> 28
<212> DNA
<213>artificial sequence (Artificial)
<400> 12
ctaagcttga cgacttgctg ctagatgc 28
Claims (9)
1. a kind of method of Antheraea pernyi nuclear polyhedrosis virus genomic DNA linearisation, which comprises the steps of:
(1) design three pairs have restriction enzyme digestion sites primers, building with corresponding restriction enzyme site, can express it is green
The tussah virus of color fluorescence protein gene egfp shifts expression vector；
(2) extract transfer expression vector plasmid DNA, and with ApNPV genomic DNA cotransfection Tn-High Five insect cell；
(3) by fluorescence microscope screening separation with step (1) described two restriction enzyme sites, the ApNPV of egfp can be expressed
Mutant strain；
(4) the ApNPV mutant virus obtained in step (3) is infected into tussah chrysalis, takes the tussah chrysalis body fluid of infection morbidity, passes through
Ultracentrifugation separation method separates mutant virus, then extracts through protease K digesting, phenol, largely prepares mutant virus gene
Group DNA；
(5) digestion with restriction enzyme ApNPV mutant virus genomic DNA is used, is separated, linearisation ApNPV gene is obtained
Group DNA；
Primer sequence such as SEQ ID NO.1 and the SEQ ID of restriction enzyme digestion sites is had in the step (1)
NO.2；SEQ ID NO.3 and SEQ ID NO.4；Shown in SEQ ID NO.5 and SEQ ID NO.6.
2. the method for Antheraea pernyi nuclear polyhedrosis virus genomic DNA linearisation according to claim 1, which is characterized in that
The restriction enzyme includes that there are any of single restriction enzyme site sequence on any site of ApNPV genomic dna sequence
A kind of restriction enzyme or on any site of ApNPV genomic dna sequence without any one of single restriction enzyme site sequence
Restriction enzyme.
3. the method for Antheraea pernyi nuclear polyhedrosis virus genomic DNA linearisation according to claim 2, which is characterized in that
The restriction enzyme includes Avr II (Bln I), Sse8387I.
4. the method for Antheraea pernyi nuclear polyhedrosis virus genomic DNA linearisation according to claim 1, which is characterized in that
Separation method in the step (5) includes agarose gel electrophoresis partition method, gel chromatography or membrane separation process.
5. the method for Antheraea pernyi nuclear polyhedrosis virus genomic DNA linearisation according to claim 1, which is characterized in that
The construction method of tussah virus transfer expression vector in the step (1), includes the following steps:
(1) using ApNPV genomic DNA as template, PCR amplification is carried out with primer SEQ ID NO.1 and SEQ ID NO.2, is obtained
Segment 1 carries out PCR amplification with primer SEQ ID NO.3 and SEQ ID NO.4, obtains segment 2；
(2) segment 1 and segment 2 are cloned into respectively on pMD18-T carrier and obtain plasmid, then successively distinguish segment 1 and segment 2
It is cloned on Hind III-BamH I and Kpn the I-EcoR I site of carrier Puc19, building obtains transfer vector；
(3) it is template by the Plasmid DNA with egfp gene, carries out PCR expansion with primer SEQ ID NO.5 and SEQ ID NO.6
Increase, obtains segment and be cloned on pMD18-T carrier to obtain plasmid, then be cloned into the BamH for the transfer vector that step (2) obtains
On I-Kpn I site, obtain with restriction enzyme digestion sites, can expressing green fluorescent protein gene egfp tussah
Virus transfer expression vector.
6. the method for Antheraea pernyi nuclear polyhedrosis virus genomic DNA linearisation according to claim 1 linearizes ApNPV base
Because of a group kit for DNA preparation tussah virus expression systems, which is characterized in that the kit includes linearisation ApNPV gene
Group DNA2~5g, 5~10g of tussah Baculovirus transfer vector Plasmid DNA, 10 μM of PCR detection primer SEQ ID NO.7 and SEQ
Each 50 μ L of ID NO.8,4~8mL of viral dilution and kit operation instructions.
7. kit according to claim 6, which is characterized in that the kit includes tussah chrysalis 4~8.
8. kit according to claim 6, which is characterized in that the preparation of the tussah Baculovirus transfer vector Plasmid DNA
Method includes the following steps:
(1) it using transfer vector pApM748BE Plasmid DNA as template, is carried out with primer SEQ ID NO.9 and SEQ ID NO.10
PCR amplification obtains segment 1, carries out PCR amplification with primer SEQ ID NO.11 and SEQ ID NO.12, obtains segment 2；
(2) segment 1 and segment 2 are cloned into respectively on pMD18-T carrier and obtain plasmid, segment 2 is first cloned into transfer vector
The corresponding Hind III-EcoR I site of pApM748BE, then segment 1 is cloned into Pst I-EcoR I site, complete tussah disease
The building of malicious metastasis transplanting physique grain DNA.
9. kit according to claim 6, which is characterized in that the limitation of the tussah Baculovirus transfer vector Plasmid DNA
Property endonuclease digestion site include BamH I, Sal I, Kpn I, Sma I, EcoR I, Xho I, Avr II (Bln I) and
XbaI。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910517434.1A CN110184243A (en) | 2019-06-14 | 2019-06-14 | The method and its kit of Antheraea pernyi nuclear polyhedrosis virus genomic DNA linearisation |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910517434.1A CN110184243A (en) | 2019-06-14 | 2019-06-14 | The method and its kit of Antheraea pernyi nuclear polyhedrosis virus genomic DNA linearisation |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN110184243A true CN110184243A (en) | 2019-08-30 |
Family
ID=67721900
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910517434.1A Pending CN110184243A (en) | 2019-06-14 | 2019-06-14 | The method and its kit of Antheraea pernyi nuclear polyhedrosis virus genomic DNA linearisation |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110184243A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110592139A (en) * | 2019-09-26 | 2019-12-20 | 辽宁省海洋水产科学研究院 | Construction method and application of tussah nuclear polyhedrosis virus shuttle vector |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1637138A (en) * | 2004-12-09 | 2005-07-13 | 江苏大学 | Silkworm nuclear polyhedron virus and its prepn process and application in gene expression |
| CN105296538A (en) * | 2015-11-16 | 2016-02-03 | 杭州普莱柯生物技术有限公司 | In-fusion cloning method for recombinant baculovirus |
-
2019
- 2019-06-14 CN CN201910517434.1A patent/CN110184243A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1637138A (en) * | 2004-12-09 | 2005-07-13 | 江苏大学 | Silkworm nuclear polyhedron virus and its prepn process and application in gene expression |
| CN105296538A (en) * | 2015-11-16 | 2016-02-03 | 杭州普莱柯生物技术有限公司 | In-fusion cloning method for recombinant baculovirus |
Non-Patent Citations (3)
| Title |
|---|
| PAUL A.KITTS: "A method for producing recombinant baculovirus expression vectors at high frequency", BIOTECHNIQUES, vol. 14, no. 5, pages 810 - 815 * |
| 李青兵;: "杆状病毒表达载体系统研究进展", 广东蚕业, vol. 32, no. 04, pages 60 - 64 * |
| 王林美;张波;叶博;赵振军;李树英;李文利;岳冬梅;范琦;: "利用柞蚕核型多角体病毒表达载体系统在柞蚕蛹中表达增强型绿色荧光蛋白", 蚕业科学, no. 05, pages 798 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110592139A (en) * | 2019-09-26 | 2019-12-20 | 辽宁省海洋水产科学研究院 | Construction method and application of tussah nuclear polyhedrosis virus shuttle vector |
| CN110592139B (en) * | 2019-09-26 | 2021-09-03 | 辽宁省海洋水产科学研究院 | Construction method and application of tussah nuclear polyhedrosis virus shuttle vector |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Van Oers et al. | Thirty years of baculovirus–insect cell protein expression: from dark horse to mainstream technology | |
| JP7595371B2 (en) | Expression of recombinant proteins in Trichoplusia ni pupae | |
| Summers | Milestones leading to the genetic engineering of baculoviruses as expression vector systems and viral pesticides | |
| CN111234036B (en) | African swine fever virus p72 fusion protein and preparation method and application thereof | |
| CN102245777B (en) | Baculovirus vectors | |
| NL2025015B1 (en) | Method for Efficiently Expressing PCV2 Cap and PCV3 Cap Fusion Protein | |
| Shen et al. | BM61 of Bombyx mori nucleopolyhedrovirus: its involvement in the egress of nucleocapsids from the nucleus | |
| CN110184243A (en) | The method and its kit of Antheraea pernyi nuclear polyhedrosis virus genomic DNA linearisation | |
| CN105002132B (en) | It is a kind of in II group of A type baculoviral Bacmid of various insects cell expresses exogenous albumen and application | |
| Liu et al. | Autographa californica multiple nucleopolyhedrovirus ac53 plays a role in nucleocapsid assembly | |
| Fang et al. | The Autographa californica multiple nucleopolyhedrovirus Ac132 plays a role in nuclear entry | |
| CN110129362B (en) | Method for inserting tag into turnip mosaic virus P3 protein, recombinant vector and application thereof | |
| CN106497974A (en) | Rhabdovirus expression vector and its structure and the application of titre can quickly be determined | |
| CN107304432A (en) | It is a kind of while expressing the method and its dedicated system of n albumen or protein protomer | |
| CN114058646B (en) | Vector and method for expressing PCV2d cap protein | |
| CN107653266A (en) | The application of recombinant expression carrier expressing protein in dipteral insect cell of the virus enhancer containing Lepidoptera | |
| CN102286534A (en) | Insect bioreactor capable of expressing multiple exogenous genes, and construction method and application thereof | |
| HUANG et al. | Construction of the Bac-to-Bac system of Bombyx mori nucleopolyhedroviru | |
| Zhao et al. | Construction of a Baculovirus derivative to produce linearized antheraea pernyi (Lepidoptera: Saturniidae) multicapsid nucleopolyhedrovirus genomic DNA | |
| CN103555675B (en) | Application of Insect Cell High Five in Culturing Tussah Mori Karyopolyhedrosis Virus | |
| CN106754896B (en) | A kind of synthetic method of Baculovirus Gene group and its application in recombinant baculovirus building | |
| CN103215309A (en) | Method for expressing polypeptide | |
| CN109457016A (en) | A kind of quantitative approach of huichun viremia virus | |
| JP7145477B2 (en) | baculovirus genome | |
| Correia | Check for updates Chapter 6 Adaptive Laboratory Evolution to Improve Recombinant Protein Production Using Insect Cells Ricardo Correia, Bárbara Fernandes, Paula M. Alves, and António Roldão |
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 | ||
| WD01 | Invention patent application deemed withdrawn after publication | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20190830 |