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 PDFInfo
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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
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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