CN106834352A - The polyhedrosis method of parcel carp herpesviral II type antigens is prepared based on baculovirus expression system - Google Patents

The polyhedrosis method of parcel carp herpesviral II type antigens is prepared based on baculovirus expression system Download PDF

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CN106834352A
CN106834352A CN201710109641.4A CN201710109641A CN106834352A CN 106834352 A CN106834352 A CN 106834352A CN 201710109641 A CN201710109641 A CN 201710109641A CN 106834352 A CN106834352 A CN 106834352A
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silkworm
polh
carp
polyhedral body
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贡成良
曹广力
胡小龙
薛仁宇
刘波
李坤
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Suzhou Peiente Biotechnology Co ltd
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Abstract

The present invention relates to antigen protein expression technology, the polyhedrosis method of parcel carp herpesviral II type antigens is specifically related to prepared based on baculovirus expression system, controlled by baculoviral P10 promoters by designing 1 186,993 1197,603 783,85 186 regional sequences of recombinant bombyx mori nuclear polyhedrosis virus BmNPV VP3 cyHV polh, ORF72, ORF66, ORF81 and ORF82 and the coded sequence fused in tandem sequence in the region of silkworm cytoplasmic polyhedrosis virus structural protein VP3 gene 1 279；Silkworm matter type polyhedron gene is controlled by the polyhedrin gene promoter of baculoviral.With the virus inoculation silkworm or silkworm cultured cell, the antigen protein of the carp herpesviral II types of expression of recombinant virus can be wrapped up into silkworm matter type polyhedral body.The polyhedral body of formation can be realized purifying by simple differential centrifugation；After the polyhedral body of purifying is cracked in the basic conditions, can precipitate polyhedrin by centrifugation, and carp herpesviral II type antigens are deposited in supernatant, such that it is able to quickly and easily obtain carp herpesviral II type antigens.

Description

The polygonal of parcel carp herpesviral II type antigens is prepared based on baculovirus expression system The method of body
Technical field
The present invention relates to viral genetic engineering technical field, and in particular to prepare parcel carp based on baculovirus expression system The polyhedrosis method of herpesviral II type antigens.
Background technology
Hybridized prussian carp gill hemorrhage is by carp herpesviral II types（Cyprinid herpesvirus II, CyHV-2）Infection Cause, the main immunocompetence from enhancing fish of the preventing and treating of the relevant disease at present, reduction stress reaction, raise together with, reduce cultivation density, Strengthen the aspects such as detection, the ill fish of removal in time of disease to be controlled.It is generally acknowledged that immunoprophylaxis is fishes virus disease Prevent and treat one of most efficient method.At present, the vaccine of fish mainly has inactivated vaccine（Killed vaccine）, attenuated vaccine, subunit's epidemic disease Seedling, DNA vaccination and protein subunit/DNA combined vaccines.Inactivated vaccine is prepared and is easier to relatively, but immunogenicity is poor, need to contain The inactivation of viruses of q.s can just cause effective immune response, and such vaccine can not enter major histocompatibility complex class I (MHC I) class antigen processing pathways, can not effective inducing cytotoxic T cell（CTL）Reaction, therefore, immune effect is relatively not Preferable, immunity persistence is poor.Attenuated vaccine immunity is strong, long action time, but attenuated vaccine production cost is high, has virulence to return Ancestral's phenomenon, security is poor, has and replys pathogenic potential danger.Subunit vaccine has that antigenicity is strong, guard time is long, peace The advantages of good perfection, preparation and convenient transportation, but there is the deficiency of epitope loss, therefore, the research and development of vaccine of new generation Direction is DNA vaccination；But so far without the DNA vaccination of effective CyHV-2.
The many antigen protein subunits combined vaccine produced by genetic engineering can be prevented and treated because caused by virus mutation Immunologic escape, immune protective effect is good.The current recombinant vaccine overwhelming majority is produced by escherichia expression system, to the greatest extent Pipe escherichia expression system expression foreign protein technology is relatively ripe, but the substantial amounts of pure recombinant protein of acquisition still suffers from lacking Fall into.Additionally, due to Escherichia coli without the modification system after translation, the immunogenicity for often leading to recombinant protein is poor and thin Contain miscellaneous endotoxin in born of the same parents' pericentral siphon.Therefore, it is necessary to find new expression system.
Immune formulation not only influences immune effect, also influences immune period.There are some researches show sodium alginate carries medicine Microballoon as new sustained-release preparation, can enhancement antigen immunocompetence.But the preparation technology of sodium alginate antigen microballoon is more multiple Miscellaneous, influence factor is more；Matter type polyhedral body is that one kind embedding that insect cytoplasmic polyhedrosis virus is formed in infection cell is ill The protein polyhedral crystalline of virion.
Therefore, the polyhedral body of parcel carp herpesviral II type antigens is prepared possible as good by shaft-like expression system Vaccine, but up to the present, also reported without correlative study.
The content of the invention
Goal of the invention of the invention is to provide a kind of preparation based on baculovirus expression system and wraps up carp herpesviral II types The polyhedrosis method of antigen；The Antigen Stability wrapped up with matter type polyhedrin is good, is difficult to be degraded；Purifying is wrapped in matter The method of the antigen protein in type polyhedral body is easy；Seedless acid pollution, biological safety is higher.
To reach above-mentioned purpose, the technical solution adopted by the present invention is：
A kind of polyhedrosis method that parcel carp herpesviral II type antigens are prepared based on baculovirus expression system, including it is following Step：
（1）Silkworm cytoplasmic polyhedrosis virus Cloning of the polyhedrin gene is entered into pFastBacDual plasmidsEcoRI and Between XbaI, restructuring transfer pFastBacDual-polh plasmids are built；
（2）Synthesis fusion dna sequence；The fusion dna sequence such as SEQ ID NO:Shown in 3；
（3）By step（2）Fusion dna sequence clone into step（1）PFastBacDual-polh plasmidsXhoI andKpnI Site, obtains pFast-VP3-cyHV-polh plasmids；
（4）By step（3）PFast-VP3-cyHV-polh plasmid transformed competence colibacillus cells, coat the agar containing culture medium On flat board, in 37 DEG C of cultures, picking white colony extracts restructuring Bacmid-VP3-cyHV-polh DNA；
（5）By step（4）Restructuring Bacmid-VP3-cyHV-polh DNA transfection silkworm cultured cell, 26~27 DEG C culture, After cell morbidity, cells and supernatant is collected, obtain recombinant virus BmNPV- VP3-cyHV-polh；
（6）By step（5）Recombinant virus BmNPV-VP3-cyHV-polh inoculation silkworm or silkworm cultured cell, after morbidity, receive Collection silkworm hemolymph or silkworm cultured cell, purify polyhedral body, obtain the polyhedral body of parcel carp herpesviral II type antigens.
In above-mentioned technical proposal, step（1）In, preferably with the geneome RNA of silkworm cytoplasmic polyhedrosis virus or silkworm matter The total serum IgE of the midgut tissue of type polyhedrosis virus infection is template, and reverse transcription is expanded by PCR and obtain silkworm matter into after cDNA Type polyhedrosis virus polyhedron gene.Can also be by being chemically synthesized, pFastBacDual plasmids therein are beautiful The product of Invitrogen companies of state, belongs to Bac-to-Bac（Bacteria to Baculovirus）Expression system carrier.
In above-mentioned technical proposal, step（4）In, the competent cell is DH10/Bac competent cells；The culture Base is obtained for LB culture mediums addition tetracycline, kanamycins, gentamicin, X-gal, IPTG；Tetracycline, kanamycins, celebrating are big The content of mycin, IPTG and X-gal on LB agar plates is respectively 10 μ g/ml, 50 μ g/ml, 7 μ g/ml, 40 μ g/ml With 100 μ g/ml.Beneficial to the screening of restructuring Bacmid.
Step（4）In, transfection is specifically as follows and Bacmid-VP3-cyHV-polh DNA is added to without hyclone In Insect culture medium, mix；Separately take transfection reagent to be added in the Insect culture medium without hyclone, mix；The former is dripped again It is added in the latter, mixes after placing 30 minutes, be added drop-wise to the Insect culture medium without hyclone, silkworm cultured cell is transfected thereafter BmN。
In above-mentioned technical proposal, step（6）In, first by step（5）Recombinant virus BmNPV-VP3-cyHV-polh again Secondary transfection silkworm cultured cell, 26~27 DEG C of cultures after cell morbidity, collect cells and supernatant, obtain two generation recombinant viruses BmNPV-VP3-cyHV-polh；Then two generation recombinant virus BmNPV-VP3-cyHV-polh are inoculated with silkworm or silkworm culture is thin Born of the same parents；The silkworm be 4~5 instar larvaes or silkworm chrysalis, it is cost-saved.Step（5）The recombinant virus BmNPV- VP3- of acquisition CyHV-polh can be expanded by being inoculated with silkworm cultured cell again, obtain two generation recombinant virus BmNPV- of high titre VP3-cyHV-polh.Being inoculated with silkworm larva or silkworm chrysalis with two generation recombinant viruses can improve the morbidity of success ratio of inoculation, raising silkworm Rate.
Purifying polyhedral body be specifically as follows, take the hemolymph of the silkworm of infection morbidity, by 1000 revs/min and 3000 turns/ The differential centrifugation repeatedly for dividing, the matter type polyhedral body that can be purified, polyhedral body is parcel carp herpesviral II type antigens Polyhedral body；Or silkworm cultured cell is inoculated with two generation recombinant viruses, 26 DEG C of cultures collect silkworm culture thin to cell morbidity Born of the same parents, alternate freezing and thawing 3~5 times, by 1000 revs/min and 3000 revs/min of differential centrifugation repeatedly, can also be purified to matter type polygonal Body.
The matter type polyhedral body of parcel carp herpesviral II type antigens of the invention is molten with sodium carbonate-bicarbonate at 37 DEG C Liquid is cracked, and then adjusts pH to 7.12 or so with hydrochloric acid solution, after there is substantial amounts of flocculent deposit, with 12000 revs/min of centrifugations, institute Supernatant is obtained for carp herpesviral II type antigens.
Parcel carp herpesviral II type antigens are prepared based on baculovirus expression system more the invention also discloses above-mentioned The polyhedral body for wrapping up carp herpesviral II type antigens and wrap up many of carp herpesviral II type antigens prepared by the method for angle body Application of the angle body in hybridized prussian carp gill bleeding disease immunity of Chinese idle prophylactic agent is prepared.
Further, the invention discloses a kind of hybridized prussian carp gill bleeding disease immunity of Chinese idle prophylactic agent, including above-mentioned parcel carp The polyhedral body of herpesviral II type antigens.
The invention also discloses a kind of fusion dna sequence, DNA sequence dna such as SEQ ID NO:Shown in 3.The fusion dna sequence Row can be expressed as ORF72-ORF66-ORF81-ORF82-VP3 fusion dna sequences, wherein, carp herpesviral II types ORF72, The DNA sequence dna in 1-186,993-1197,603-783,85-186 region of ORF66, ORF81 and ORF82 press ORF72, The order series connection of ORF66, ORF81, ORF82, its expressing protein has many subunit antigen protein specificities；The password of the DNA sequence dna Son is matched with baculovirus expression vector system, so as to improve the expression of antigen protein；Especially in the fusion sequence The 1-279 held with silkworm cytoplasmic polyhedrosis virus structural protein VP3 gene 5 ' after ORF72, ORF66, ORF81, ORF82 series connection The coded sequence fusion in region, and add at 5 ' endsXhoI site, 3 ' end plus terminator codon TAA andKpnI site, so that table The antigen protein for reaching is wrapped up into matter type polyhedral body.According to Figure of description 4, the specific band of 35kDa is can detect, with reason It is consistent by value, illustrates that the recombinant antigen protein of expression is wrapped into polyhedral body.
Further, parcel carp blister is being prepared the invention discloses silkworm matter type polyhedral body or synthesis fusion dna sequence Application in the polyhedral body of exanthema virus II type antigens；The fusion dna sequence such as SEQ ID NO:Shown in 3.
The present invention clones into step the fusion dna sequence of ORF72-ORF66-ORF81-ORF82-VP3（1）'s PFastBacDual-polh plasmidsXhoI andKpnI site, obtains pFast-VP3-cyHV-polh.In pFast-VP3-cyHV- In polh, the coded sequence of matter type polyhedron gene is controlled by baculovirus polyhedrin gene promoter, ORF72- The fusion dna sequence of ORF66-ORF81-ORF82-VP3 is controlled by the p10 promoters of baculoviral.
The polyhedral body of the parcel carp herpesviral II type antigens obtained by above-mentioned technical proposal, through SDS-PAGE and Western blotting detect that the antigen of detectable carp herpesviral II types illustrates that the recombinant antigen of expression is polygonal Body protein crystallite is wrapped up.
Because above-mentioned technical proposal is used, the present invention has following advantages compared with prior art：
1. the polyhedral body for preparing parcel carp herpesviral II type antigens by rhabdovirus system disclosed by the invention is not appeared in the newspapers Road；Rod string design of the invention is a kind of eukaryotic expression system, the expression alien gene in silkworm, expresses water Flat high, product bioactivity is good, low production cost, and the natural host of baculoviral is strictly limited in insect, dynamic to vertebra Thing no pathogenicity, and polyhedral body prepared by the present invention does not embed virion, also virus-free DNA pollution, biological safety is high, from And can be used for the research and development of vaccine.
2. the present invention is based on rhabdovirus system, it is to avoid the antigen protein of conventional E. coli system expression it is many with The form of inclusion body is present, and the purifying process of antigen protein is complicated, isolate and purify the big defect of difficulty；And avoid due to big Enterobacteria without the modification system after translation, often lead to recombinant protein immunogenicity is poor and periplasmic in contain Miscellaneous endotoxic problem.
3. be wrapped in antigen protein in polyhedral body by the present invention；Polyhedral body is water insoluble, can by simple differential from The heart realizes purifying；After the polyhedral body of purifying is cracked in the basic conditions, pH is adjusted to polyhedrin isoelectric point with hydrochloric acid solution, can Polyhedrin is precipitated with by centrifugation, and carp herpesviral II type antigens are deposited in supernatant, such that it is able to quick, convenient Ground obtains carp herpesviral II type antigens, achieves unexpected technique effect.
4. in the polyhedral body of parcel carp herpesviral II type antigens of the invention, polyhedral body has to the antigen protein for wrapping up Protective effect, therefore the polyhedral body of parcel carp herpesviral II type antigens that the present invention is obtained is without specially treated, at normal temperatures Just can for a long time preserve, solve the antigen protein of existing acquisition because easily degrading, often need to be preserved at low temperature after freezing Defect.
5th, in the present invention, matter type polyhedral body can be used as a kind of good load medicine body, and polyhedral body is the albumen of 2-3 microns Crystallite, not only has stabilization to the albumen for wrapping up, and has slow-releasing and controlled-releasing action, therefore is wrapped up with matter type polyhedrin crystallite Protein medicaments can not only stablize medicine, and have slow-releasing and controlled-releasing action, so that present invention matter type polyhedral body coating antigen albumen Prepare the level of protection that microparticle sustained release vaccine is possible to improve vaccine immunity.
Brief description of the drawings
Fig. 1 is the sequencing result of the ORF72-ORF66-ORF81-ORF82-VP3 fusion sequences of the chemical synthesis of embodiment one Figure；
Fig. 2 is the electrophoresis detection figure of the recombinant virus BmNPV-VP3-cyHV-polh infected silkworm hemolymphs of embodiment one；
Fig. 3 is the polyhedrosis micrograph of the parcel carp herpesviral II type antigens that embodiment one is obtained；
After Fig. 4 is cracked for the polyhedral body purified in embodiment one, the electrophoresis detection figure of supernatant.
Specific embodiment
Below in conjunction with the accompanying drawings and embodiment the invention will be further described：
Embodiment one wraps up the polyhedrosis preparation method of carp herpesviral II type antigens
（1）Silkworm matter type polyhedral body virus gene group RNA is extracted with QIAGEN Viral RNA Mini Kit (Qiagen companies), Specification is recorded by product and viral RNA is converted into cDNA, with RNA PCR KitVer.3.0 (Qiagen companies) with SEQ ID NO:1 is forward primer CPV-PH-EI（EcoThe restriction enzyme sites of R I）, with SEQ ID NO:2 is reverse primer（XbaI digestions position Point）, 5 ' ends and 3 ' ends band respectively are synthesized by PCREcoRI andXbaThe coded sequence of the matter type polyhedron gene of I site, Clone into T- carriers, obtain pMD19T-PH plasmids.The plasmid is by after sequence verification, usingEcoRI andXbaI digestions pMD19T-PH Plasmid, reclaims matter type polyhedron gene fragment, clones into pFastBac Dual'sEcoRI andXbaI site, builds weight Group transferring plasmid pFastBac Dual-polh.
（2）Synthesize the fusion dna sequence of ORF72-ORF66-ORF81-ORF82-VP3, sequence information is shown in SEQ ID NO: 3；In the fusion dna sequence of ORF72-ORF66-ORF81-ORF82-VP3, carp herpesviral II types（GenBank accession number： KT387800）The DNA sequences in 1-186,993-1197,603-783,85-186 region of ORF72, ORF66, ORF81 and ORF82 Row, through codon optimization after, with silkworm cytoplasmic polyhedrosis virus structural protein VP3 gene（GenBank accession number： GU323606 ）The coded sequence fusion in the 1-279 regions at 5 ' ends, and add at 5 ' endsXhoI site, 3 ' ends plus terminator codon TAA andKpnI site；The fusion dna sequence of the ORF72-ORF66-ORF81-ORF82-VP3 of chemical synthesis by sequencing identification, Result is referring to Fig. 1, and synthesized sequence is consistent with theory, and in order to accompanying drawing is succinct, accompanying drawing only provides part and can accurately confirm to be closed Into sequence and theoretical consistent sequencing identification result.
（3）The fusion dna sequence of ORF72-ORF66-ORF81-ORF82-VP3 is cloned into step（1）PFastBac Dual-polh plasmidsXhoI andKpnI site, obtains pFast-VP3-cyHV-polh.
（4）PFast-VP3-cyHV-polh converts DH10/Bac competent cells, coats containing tetracycline（10 µg/ ml）, kanamycins（50 µg/ml）, gentamicin（7 µg/ml）、IPTG（40µg/ml）、X-gal（100 µg/ml）LB On agar plates；Cultivated 48 hours in 37 DEG C, picking white colony culture, extract restructuring Bacmid genomic DNAs, life Entitled Bacmid-VP3-cyHV-polh, with M13 forward primers (SEQ ID NO:4) with M13 reverse primers (SEQ ID NO:5) performing PCR is entered to restructuring Bacmid, the purpose bar consistent with theoretical molecular can be amplified from restructuring Bacmid DNA Band, illustrates correctly to construct restructuring Bacmid on request.
（5）Bacmid-VP3-cyHV-polh DNA(3μg）It is added to 98 μ L TC-100 culture mediums（Without hyclone, GIBCO BRL companies）Middle mixing, separately takes 10 μ L FuGENE HD transfection reagents (Roche companies) and is added to 90 μ L TC- 100 culture mediums（Without hyclone）Middle mixing, then the former is added drop-wise in the latter, mix after placing 30 minutes, it is added drop-wise to 800 μ L TC-100 culture mediums（Without hyclone）Middle mixing, transfection silkworm cultured cell BmN.After 4 is small, cell culture fluid is removed, used TC-100 culture mediums containing 10% hyclone are cultivated 3 days at 27 DEG C, receive transfecting cultured cells supernatant, obtain P1 for recombinant virus BmNPV-VP3-cyHV-polh.P1 generation virus infection individual layer BmN cells are taken, after cultivating 5 days, the culture for collecting virus infection is thin Born of the same parents' supernatant, obtains P2 for recombinant virus BmNPV- VP3-cyHV-polh, 4 DEG C keep in dark place it is standby.
（6）P2 is taken for recombinant virus, the silkworm from the age of percutaneous puncture-inoculation at coria 5,25 DEG C or so normal feedings with the insect needle libation at an ancient wedding ceremony Support, silkworm hemolymph was collected every 24 hours, Western blotting are carried out with the antibody of the ORF72 of carp herpesviral II types Detection, can be observed the specific band of 35kDa, see Fig. 2, illustrate that recombinant antigen protein is correctly expressed, wherein swimming lane 1：It is wild The hemolymph of raw type bombyx mori nuclear polyhydrosis virus infected silkworm；Swimming lane 2-4：Respectively recombinant virus BmNPV-VP3-cyHV- Polh infects the hemolymph of 24,48,72 hours;Swimming lane 5：Albumen Marker；Swimming lane 6-9：96th, the blood of 120,144,168 hours Lymph, the primary antibody of Western blotting is the anti-carp herpesviral II types ORF72 antibody of mouse；Secondary antibody is horseradish peroxidating The sheep anti-mouse igg of thing enzyme mark.
Sediments microscope inspection can be observed there is polyhedral body, the blood strangury of the silkworm of infection morbidity in the hemolymph of the silkworm of infection morbidity Bar can be purified to matter type polyhedral body by 1000 revs/min and 3000 revs/min of differential centrifugation repeatedly, see Fig. 3, illustrate that matter type is more Polyhedrin gene is correctly expressed, and polyhedral body is the albumen crystallite of 2-3 microns；From every milliliter of blood of the silkworm of infection morbidity About 10 can be obtained in lymph8Individual polyhedral body.Polyhedral body cracks 30 points in 37 DEG C of soda-sodium bicarbonate solutions with pH10.8 Clock, pH to 7.12 or so is adjusted with the hydrochloric acid solution of 1M, after there is substantial amounts of flocculent deposit, with 12000 revs/min of centrifugations, takes supernatant, Western blotting detections are carried out with the antibody of the ORF72 of carp herpesviral II types, the specific bar of 35kDa is can detect Band, is shown in Fig. 4, illustrates that the recombinant antigen protein of expression is wrapped into polyhedral body；Swimming lane 1：DNA Marker；Swimming lane 2-7：Respectively It is the polyhedral body purified in 72,96,120,144,168 hours hemolymphs of recombinant virus infected silkworm, swimming lane 7：Wild type silkworm Polyhedrosis.The primary antibody of Western blotting is the antibody of the ORF72 of the anti-carp herpesviral II types of mouse；Secondary antibody is The sheep anti-mouse igg of horseradish peroxidase-labeled.
Embodiment two wraps up the polyhedrosis preparation method of carp herpesviral II type antigens
Compared with embodiment one, wherein step（5）In, 26 DEG C are cultivated 4 days, collect the cultured cells supernatant of virus infection, obtain P1 For recombinant virus BmNPV-VP3-cyHV-polh.P1 is inoculated with silkworm cultured cell, 26 DEG C of cultures to cell hair for recombinant virus Disease, collects cell conditioned medium and obtains P2 generation viruses；Wherein step（6）In, take P2 with the insect needle libation at an ancient wedding ceremony and be inoculated with silkworm chrysalis for recombinant virus, 25 DEG C through 5 days or so, silkworm chrysalis morbidity.Remaining step is consistent, by 1000 revs/min and 3000 revs/min of differential centrifugation repeatedly, can be from Matter type polyhedral body is purified in the hemolymph of morbidity silkworm chrysalis, is the albumen crystallite of 2-3 microns.From every milliliter of infection morbidity About 1.5 × 10 can be obtained in the hemolymph of silkworm chrysalis8Individual polyhedral body.Microscopy images and electrophoresis result show that the present invention is clearly The polyhedral body of carp herpesviral II type antigens is obtained wrapping up.
Embodiment three wraps up the polyhedrosis preparation method of carp herpesviral II type antigens
Compared with embodiment one, wherein step（6）In, P2 is inoculated with 4 age silkworm larvas for recombinant virus, and remaining step is consistent, leads to 1000 revs/min and 3000 revs/min of differential centrifugation repeatedly is crossed, matter type polyhedral body can be purified to, be that the albumen of 2-3 microns is micro- It is brilliant；About 10 can be obtained from every milliliter of hemolymph of the silkworm of infection morbidity8Individual polyhedral body.Microscopy images and electrophoresis result are aobvious Show, the present invention has clearly obtained wrapping up the polyhedral body of carp herpesviral II type antigens.
Example IV wraps up the polyhedrosis preparation method of carp herpesviral II type antigens
Compared with embodiment one, wherein step（6）In, P2 is inoculated with silkworm cultured cell, 26 DEG C of cultures to cell for recombinant virus Morbidity（4 days）, collection cell, alternate freezing and thawing 3 times, then by 1000 revs/min and 3000 revs/min of differential centrifugation repeatedly, can Matter type polyhedral body is purified to, from 105About 10 can be obtained in individual infection cultured cells6Individual polyhedral body, is the albumen of 2-3 microns Crystallite.Microscopy images and electrophoresis result show that the present invention has clearly obtained wrapping up the polygonal of carp herpesviral II type antigens Body.
In the present invention, DNA sequence dna is：
SEQ ID NO:1：
GAATTCATGGCAGACGTAGCAGGAACA
SEQ ID NO:2：
TCTAGATCACTGACGGTTACTCAGAGC
SEQ ID NO:3：
CTCGAGATGTACGGTCTGAACAACGCTCAGGGTTTCATCGATACCGAGTGGATCGACAGACAGTCAATCGCTA TGACCGCTCAGGAAACAAGCAGACTGTTGAACCCGTACCTGGCTACAAAAGGTCAGAGAGTGGACCCTTCAAACCTG TACATCCCAGACGGTATCTTCGTGACATACACACCTACCGGTTCAAGACACCCTATGGTGGAATACGAACGCATCGA ATCACAACTGGAACCAGACACAGGAGCTTCAAGCTTCGTGGACAAACTGGTGGAAAAGTCAGACCTGTCAGCTTGCT TGTCACACACAGACGGTACAATCGAACTGCCTTCATCTTGGGTGGACCCTAGAGCTAAACAGTTCCACGATCTGGAC GACCTGATGATCTACGGTTCAGCAGATTACATGGACTCAGACGACGAAGATTACGATTCATACGCCGGAGCAGCAGA AACACTGCAAAGAAGCATGCGCGACTACGCTGACGAAGAAGATAGCGACATGGACGATAGCACATCACTGCTGAACA GCGTGAGAAAGATGAGCAGCAAGTTCAAGAGCACCGTGTACGAAGACGACCAAACACAGAGCAGCAACACAACAGCT ACAAGCGACCGCTACAAGTACTACACAAGCAGAGGTACAATCCAACGCGCTAACAGACCGGGTTTGATGTGGCACTA TACGAGTATCAACAATGACACGAGAGTAGCACTTGACCCCAAACCAAACCAAATTAGAACGATAACAAAACCAAACA CAGTACCTCAACTCGGCACAGACTACTTGTACACTTTCAACTCACAACGACGATCACACACGTTACGACTATTAGGA CCTTTTCAGTACTTCAACTCTTCCGAGACAGATAGAGGACATCCACTATTTCGCTTACCCCTTAAGTATCCGTCAAA AGCAATACCAGCGGATGAGTTAATTGATAACTTGCATTAAGGTACC
SEQ ID NO:4：
CCCAGTCACGACGTTGTAAAACG
SEQ ID NO:5：
AGCGGATAACAATTTCACACAGG
SEQUENCE LISTING
<110>University Of Suzhou
<120>The polyhedrosis method of parcel carp herpesviral II type antigens is prepared based on baculovirus expression system
<160>5
<210> 1
<211>27
<212> DNA
<213>It is artificial synthesized
<400> 1
GAATTCATGGCAGACGTAGCAGGAACA 27
<210> 2
<211> 27
<212> DNA
<213>It is artificial synthesized
<400> 2
TCTAGATCACTGACGGTTACTCAGAGC 27
<210> 3
<211> 966
<212> DNA
<213>It is artificial synthesized
<400> 3
CTCGAGATGTACGGTCTGAACAACGCTCAGGGTTTCATCG 40
ATACCGAGTGGATCGACAGACAGTCAATCGCTATGACCGC 80
TCAGGAAACAAGCAGACTGTTGAACCCGTACCTGGCTACA 120
AAAGGTCAGAGAGTGGACCCTTCAAACCTGTACATCCCAG 160
ACGGTATCTTCGTGACATACACACCTACCGGTTCAAGACA 200
CCCTATGGTGGAATACGAACGCATCGAATCACAACTGGAA 240
CCAGACACAGGAGCTTCAAGCTTCGTGGACAAACTGGTGG 280
AAAAGTCAGACCTGTCAGCTTGCTTGTCACACACAGACGG 320
TACAATCGAACTGCCTTCATCTTGGGTGGACCCTAGAGCT 360
AAACAGTTCCACGATCTGGACGACCTGATGATCTACGGTT 400
CAGCAGATTACATGGACTCAGACGACGAAGATTACGATTC 440
ATACGCCGGAGCAGCAGAAACACTGCAAAGAAGCATGCGC 480
GACTACGCTGACGAAGAAGATAGCGACATGGACGATAGCA 520
CATCACTGCTGAACAGCGTGAGAAAGATGAGCAGCAAGTT 560
CAAGAGCACCGTGTACGAAGACGACCAAACACAGAGCAGC 600
AACACAACAGCTACAAGCGACCGCTACAAGTACTACACAA 640
GCAGAGGTACAATCCAACGCGCTAACAGACCGGGTTTGAT 680
GTGGCACTATACGAGTATCAACAATGACACGAGAGTAGCA 720
CTTGACCCCAAACCAAACCAAATTAGAACGATAACAAAAC 760
CAAACACAGTACCTCAACTCGGCACAGACTACTTGTACAC 800
TTTCAACTCACAACGACGATCACACACGTTACGACTATTA 840
GGACCTTTTCAGTACTTCAACTCTTCCGAGACAGATAGAG 880
GACATCCACTATTTCGCTTACCCCTTAAGTATCCGTCAAA 920
AGCAATACCAGCGGATGAGTTAATTGATAACTTGCATTAA 960
GGTACC 966
<210> 4
<211>23
<212> DNA
<213>It is artificial synthesized
<400> 4
CCCAGTCACGACGTTGTAAAACG 23
<210> 5
<211> 23
<212> DNA
<213>It is artificial synthesized
<400> 5
AGCGGATAACAATTTCACACAGG 23

Claims (10)

1. it is a kind of that the polyhedrosis method for wrapping up carp herpesviral II type antigens, its feature are prepared based on baculovirus expression system It is to comprise the following steps：
（1）Silkworm cytoplasmic polyhedrosis virus Cloning of the polyhedrin gene is entered into pFastBacDual plasmidsEcoRI andXbaBetween I, restructuring transfer pFastBacDual-polh plasmids are built；
（2）Synthesis fusion dna sequence；The fusion dna sequence such as SEQ ID NO:Shown in 3；
（3）By step（2）Fusion dna sequence clone into step（1）PFastBacDual-polh plasmidsXhoI andKpnI Site, obtains pFast-VP3-cyHV-polh plasmids；
（4）By step（3）PFast-VP3-cyHV-polh plasmid transformed competence colibacillus cells, coat the agar containing culture medium On flat board, in 37 DEG C of cultures, picking white colony extracts restructuring Bacmid-VP3-cyHV-polh DNA；
（5）By step（4）Restructuring Bacmid-VP3-cyHV-polh DNA transfection silkworm cultured cell, 26~27 DEG C culture, After cell morbidity, cells and supernatant is collected, obtain recombinant virus BmNPV- VP3-cyHV-polh；
（6）By step（5）Recombinant virus BmNPV-VP3-cyHV-polh inoculation silkworm or silkworm cultured cell, after morbidity, receive Collection silkworm hemolymph or silkworm cultured cell, purify polyhedral body, obtain the polyhedral body of parcel carp herpesviral II type antigens.
2. the polyhedral body of parcel carp herpesviral II type antigens is prepared based on baculovirus expression system according to claim 1 Method, it is characterised in that step（1）In, with the geneome RNA of silkworm cytoplasmic polyhedrosis virus or silkworm cytoplasmic polyhedrosis The total serum IgE of the midgut tissue of poison infection is template, and reverse transcription is expanded by PCR and obtain silkworm cytoplasmic polyhedrosis into after cDNA Malicious polyhedron gene.
3. the polyhedral body of parcel carp herpesviral II type antigens is prepared based on baculovirus expression system according to claim 1 Method, it is characterised in that step（4）In, the competent cell is DH10/Bac competent cells；The culture medium is LB Culture medium addition tetracycline, kanamycins, gentamicin, X-gal, IPTG are obtained.
4. the polygonal of parcel carp herpesviral II type antigens is prepared based on baculovirus expression system according to claim 3 The method of body, it is characterised in that step（4）In, tetracycline, kanamycins, gentamicin, IPTG and X-gal are in LB agar Content on flat board is respectively 10 μ g/ml, 50 μ g/ml, 7 μ g/ml, 40 μ g/ml and 100 μ g/ml.
5. the polyhedral body of parcel carp herpesviral II type antigens is prepared based on baculovirus expression system according to claim 1 Method, it is characterised in that step（6）In, first by step（5）Recombinant virus BmNPV-VP3-cyHV-polh turn again Dye silkworm cultured cell, 26~27 DEG C of cultures after cell morbidity, collect cells and supernatant, obtain two generation recombinant virus BmNPV- VP3-cyHV-polh；Then two generation recombinant virus BmNPV-VP3-cyHV-polh are inoculated with silkworm or silkworm cultured cell；Institute Silkworm is stated for 4~5 instar larvaes or silkworm chrysalis.
6. the polyhedral body of parcel carp herpesviral II type antigens is prepared based on baculovirus expression system according to claim 1 Method prepare parcel carp herpesviral II type antigens polyhedral body.
7. the polyhedral body of carp herpesviral II type antigens is wrapped up described in claim 6 to prepare hybridized prussian carp gill bleeding disease immunity of Chinese idle pre- Application in anti-medicine.
8. a kind of hybridized prussian carp gill bleeding disease immunity of Chinese idle prophylactic agent, it is characterised in that the hybridized prussian carp gill bleeding disease immunity of Chinese idle is pre- Anti- medicine includes the polyhedral body of parcel carp herpesviral II type antigens described in claim 6.
9. a kind of fusion dna sequence, it is characterised in that the fusion dna sequence such as SEQ ID NO:Shown in 3.
10. silkworm matter type polyhedral body or synthesis fusion dna sequence are preparing the polyhedral body of parcel carp herpesviral II type antigens In application；The fusion dna sequence such as SEQ ID NO:Shown in 3.
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CN108728490A (en) * 2018-05-29 2018-11-02 苏州大学 A kind of carp herpesviral II types DNA vaccination and its construction method and application based on baculovirus vector
CN108743933A (en) * 2018-05-29 2018-11-06 苏州大学 The construction method of albumen crystallite embedding carp herpesviral II types-grass carp hemorrhage virus subunit vaccine based on Yeast expression carrier
CN108728490B (en) * 2018-05-29 2021-03-26 苏州大学 Baculovirus vector-based carp herpesvirus II type DNA vaccine and construction method and application thereof
CN110408655A (en) * 2019-07-31 2019-11-05 盐城工学院 A kind of carp herpesvirusⅡtype ORF66 Protein reconstitution rhabdovirus expression vector and preparation method thereof
WO2022236720A1 (en) * 2021-05-11 2022-11-17 苏州大学 Method for knocking out essential gene of baculovirus/bacmid virus
CN114181969A (en) * 2021-12-09 2022-03-15 苏州大学 Method for preparing polyhedra wrapping foreign proteins
WO2023102862A1 (en) * 2021-12-09 2023-06-15 苏州大学 Method for preparing polyhedra for wrapping foreign proteins
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CN114432435A (en) * 2022-01-25 2022-05-06 苏州大学 SARS-Cov-2 vaccine based on polyhedron nano structure and its preparing method and use
CN114432435B (en) * 2022-01-25 2024-05-17 苏州大学 SARS-Cov-2 vaccine based on polyhedra nano structure and its preparation method and application
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