CN104017814B - Be suitable to chicken infectivity bursa of Fabricius virus Viral structural protein VP2 and expression vector and application that silkworm middle division of silkgland is expressed - Google Patents
Be suitable to chicken infectivity bursa of Fabricius virus Viral structural protein VP2 and expression vector and application that silkworm middle division of silkgland is expressed Download PDFInfo
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Abstract
nullThe invention discloses and be suitable to chicken infectivity bursa of Fabricius virus Viral structural protein VP2 and expression vector and the application that silkworm middle division of silkgland is expressed，The gene order of chicken infectivity bursa of Fabricius virus Viral structural protein VP2 such as SEQ ID NO.1，The aminoacid sequence of its coding is as shown in SEQ ID NO.2，The sequence of coding chicken infectivity bursa of Fabricius virus Viral structural protein VP2 is constituted expression cassette with secreting type sericin 1 gene promoter and terminator，And with enhancer hr3 Enhanced expressing，It is connected into piggyBac swivel base arm and fluorescent screening marker gene construction of expression vector simultaneously，This expression vector can express chicken infectivity bursa of Fabricius virus Viral structural protein VP2 at silkworm middle division of silkgland，And there is immune prototype，Mice can be stimulated to produce antibody，Lay a good foundation for utilizing silkworm middle division of silkgland bioreactor to express animal virus subunit vaccine.
Description
Technical field
The invention belongs to genetic engineering field, particularly to being suitable to the infectious bursal disease that silkworm middle division of silkgland is expressed
Virus structural protein VP2, further relates to express the expression vector of chicken infectivity bursa of Fabricius virus Viral structural protein VP2 and utilize silkworm
Middle division of silkgland expresses the method for chicken infectivity bursa of Fabricius virus Viral structural protein VP2.
Background technology
Infectious bursal disease (Infectious Bursal Disease, IBD) is by infectious bursal disease virus
What (Infectious Bursal Disease virus, IBDV) caused encroaches on chickling central immune organ method with main
The infectious disease of family name's capsule.This disease may result in immunity of organism and suppresses even individual death, and causes the chicken group susceptibility to other cause of disease
Strengthening and immuning failure, provisions fowl industry brings huge economic loss.In recent years, multiple country, the world is all found that
Virulence is higher, can escape the IBDV variant of existing vaccine protection, makes the popular of IBD and prevention and control increasingly complex and difficult.Utilize
Technique for gene engineering produces the important directions and effective means that subunit vaccine is preventing and treating livestock and poultry, can be prevented effectively from because of tradition
Inactivation or the risk of weak poison IBDV vaccine None-identified.VP2 albumen is the major structural protein of IBDV, containing virulent main anti-
Former protectiveness site, is the first-selected target of exploitation subunit vaccine.At present, research worker utilizes escherichia coli, yeast, restructuring disease
Poison and the expression system, successful expression such as Oryza sativa L. chicken infectivity bursa of Fabricius virus Viral structural protein VP2, and confirm effectively to sting
Swash the generation of neutralizing antibody, IBDV is infected in chicken prevention and plays protected effect in various degree.
Silkworm is famous economic insects, is mainly used in so far taking silk weaving silk fabric, for the economy of China from being tamed by artificial rearing
Development is made that tremendous contribution.Domestic natural silk gland is synthesis and the organ of secretion fibroin, has powerful albumen synthesis and divides
Secreting ability, from newly-hatched silkworm to matured silkworm, the weight of sericterium adds 160,000 times, and its weight accounts for body weight of silkworm larva amount 5% below 4 ages, to 5
Mid-term in age increases rapidly, has reached 40%～45% during matured silkworm, organ maximum in becoming silkworm body.Silk after silkworm 5 mid-term in age
In gland, the aggregate velocity of fibroin albumen is up to 8mg/g h.In vertebrates, little chicken liver is that synthesis albumin speed is the highest
Organ, about 0.1mg/g h, the aggregate velocity of sericterium is its 80 times.Silkworm is with its superpower protein synthesis capacity, feeding cost
The advantages such as scale low, nontoxic to people and animals, easy and factorial praluction, it is counted as the excellent biology of a kind of great potential
Reactor.But there is no method and the phase utilizing transgenic bombyx mori to express chicken infectivity bursa of Fabricius virus Viral structural protein VP2 so far
Close report.
Summary of the invention
In view of this, an object of the present invention is to provide the infections chicken cloacal bursa being suitable to the expression of silkworm middle division of silkgland
Sick virus structural protein VP2；The two of the purpose of the present invention are to provide expression chicken infectivity bursa of Fabricius virus structural protein
The expression vector of VP2；The three of the purpose of the present invention are to provide expression vector to express avian infectious Fa Shi at silkworm middle division of silkgland
Application in bursal disease virus Viral structural protein VP2；The four of the purpose of the present invention are that offer utilizes described expression vector in silkworm
Portion's sericterium expresses the method for chicken infectivity bursa of Fabricius virus Viral structural protein VP2.
For achieving the above object, the present invention provides following technical scheme:
Be suitable to the chicken infectivity bursa of Fabricius virus Viral structural protein VP2 that silkworm middle division of silkgland is expressed, described avian infectious method
The gene order of family name's bursal disease virus Viral structural protein VP2 is if SEQ ID NO.1 the 7th is to shown in 1329.
Preferably, the aminoacid sequence such as SEQ ID NO.2 institute of described chicken infectivity bursa of Fabricius virus Viral structural protein VP2
Show.
2, in silkworm, express the expression vector of described chicken infectivity bursa of Fabricius virus Viral structural protein VP2.
Preferably, described expression vector contains the enhancer hr3 being linked in sequence, and secreting type sericin 1 gene promoter, chicken pass
Metachromia bursa of Fabricius virus Viral structural protein VP2 gene and the terminator of sericin 1 gene.
Preferably, described expression vector possibly together with fluorescent screening marker gene expression cassette and piggyBac swivel base arm sequence,
Described fluorescent screening marker gene expression cassette is positioned at described enhancer hr3 upstream, and described piggyBac swivel base arm sequence is positioned at glimmering
Between light riddled basins expression cassette and the terminator of sericin 1 gene.
Preferably, described expression vector is connected into same through AscI after AscI enzyme action by the sequence shown in SEQ ID NO.3
PBac [3 × p3-EGFPaf] transgene expression vector enzyme action, dephosphorylized prepares.
3, described expression vector answering in silkworm middle division of silkgland expresses chicken infectivity bursa of Fabricius virus Viral structural protein VP2
With.
4, described expression vector is utilized to express chicken infectivity bursa of Fabricius virus Viral structural protein VP2 at silkworm middle division of silkgland
Method, comprises the steps:, by described expression vector microinjection Eggs of Silkworm, to be placed on 25 DEG C, phase with nontoxic glue sealing
Hatch in the environment that humidity is 90%, screening transgenic positive moth circle, take positive transgenic Cocoon shell, pulverize in liquid nitrogen
Becoming powder, be then dissolved in the solution containing 20mM Tris-Cl, 8M Urea, pH8.0,15000rpm is centrifugal collects supernatant, warp
Purification, obtains chicken infectivity bursa of Fabricius virus Viral structural protein VP2.
Preferably, described purification, for supernatant is crossed nickel post, is first washed rinsing with the concentration imidazoles less than 30mM, is used
120mM imidazoles rinses, and collects eluent, finally crosses Q post,.
The beneficial effects of the present invention is: the present invention is suitable to, by codon optimized acquisition, the chicken that silkworm middle division of silkgland is expressed
The gene of infectious bursal disease virus Viral structural protein VP2, by chicken infectivity bursa of Fabricius virus Viral structural protein VP2 gene with point
Secrete type sericin 1 gene promoter and terminator constitute expression cassette and utilize enhancer hr3 Enhanced expressing, with fluorescent marker gene
Building efficient expression vector with piggyBac swivel base arm, this carrier can be at silkworm middle division of silkgland specific high-efficiency expression, in cocoon shell
Recombiant protein content is high, is 110 μ g/g, and the recombiant protein expressed purified after there is typical α, β spiral and random
Coiled structure, Heat stability is good consistent with natural VP2 protein structure, the most undegraded after processing 2h at 80 DEG C, it is 5.5-8 at pH
In the range of the most degradable, this is for utilizing transgenic bombyx mori large-scale production and isolated and purified particularly significant.RVP2 albumen passes through
Inoculation mice can produce anti-rVP2 antibody at its Immune inducing in vivo, and agarose is double expands test and ELISA experiment card further
The real rVP2 albumen utilizing transgenic bombyx mori middle division of silkgland to express has certain immunogenicity.Chicken disclosed by the invention is utilized to pass
The gene of metachromia bursa of Fabricius virus Viral structural protein VP2 and expression vector, just can be a large amount of by extensive raising transgenic silkworm
Acquisition has immunogenic VP2 albumen；This invention is single for utilizing silkworm middle division of silkgland bioreactor to produce animal virus Asia
Position vaccine provides new approaches and new method.
Accompanying drawing explanation
In order to make the purpose of the present invention, technical scheme and beneficial effect clearer, the present invention provides drawings described below:
Fig. 1 is that pBhSer1vp2 transgene carrier structure is intended to.
Fig. 2 is fluorescent screening figure (A:G1 embryo's white light figure of pBhSer1vp2 transgenic silkworm；B: Bombycis mori white light figure；C:G1
Embryo's green glow figure；D: Bombycis mori green glow figure).
Fig. 3 is the copy number of pBhSer1vp2 system positives moth circle vp2 gene.
Fig. 4 is the expression of different moth circle vp2 genes in fluorescent quantitation detection pBhSer1vp2 transgenic.
Fig. 5 is rVP2 albumen thermostability testing result at different temperatures.
Fig. 6 is testing result after rVP2 albumen processes under different pH.
Fig. 7 is that (A represents ni-sepharose purification result for the isolated and purified testing result of cocoon shell rVP2 albumen；M:marker；1:WT；
2: transgenic bombyx mori cocoon shell lysate；3: flow through liquid；4:50mM Tris-HcI, 8M Urea (pH8.0) rinsing liquid；5:10mM miaow
Azoles rinsing liquid；6:20mM imidazoles rinsing liquid；7:30mM imidazoles rinsing liquid；8:120mM imidazoles rinsing liquid；B represents that Q column purification is tied
Really, 9: ni-sepharose purification concentrated solution；10:Q column purification liquid).
Fig. 8 is that (A represents that rVP2 albumen is substrate to rVP2 albumen agarose double expansion experimental result；B represents that IBD antigen is the end
Thing；0:rVP2 albumen；1:PBS；2:rVP2 serum；3: vaccine serum；4: positive serum；5:IBD antigen).
Fig. 9 is that (A:rVP2 albumen is substrate to ELISA experimental result picture；B:IBD antigen is substrate).
Detailed description of the invention
Below in conjunction with accompanying drawing, the preferred embodiments of the present invention are described in detail.In embodiment unreceipted specifically
The experimental technique of condition, generally according to normal condition, such as Molecular Cloning: A Laboratory guide (third edition, J. Pehanorm Brooker etc. writes)
Described in condition, or according to the condition proposed by manufacturer.
Embodiment 1, structure transgene expression vector pBac [3 × P3EGFP, hr3CQSer1-vp2-Ser1PA]
According to report IBDV highly virulent strain virus structural protein VP2 gene order (GenBank.JF907703.1) and
The codon preference of silkworm is to codon optimized, it is thus achieved that the vp2 gene order after optimization, then adds 6 His labels at C end
Nucleotide formed vp2-His6, connect BamHI and NotI restriction enzyme site respectively at the nucleotide sequence two ends of vp2-His6, so
Rear trust Shenzhen Hua Da genome company synthesizing ribonucleotide sequence sequence as shown in SEQ ID NO.1, coded by its coding region
Aminoacid sequence is as shown in SEQ ID NO.2.In the sequence of SEQ ID NO.1, the 1st to the 6th is BamHI enzyme action position
Point, the 7th to the 1329th is vp2 gene coded sequence, the 1330th to the 1350th 6 the His labels added for C end,
1351st to 1358 is NotI restriction enzyme site.Then the nucleotide of SEQ ID NO.2 is connected in PUC57 plasmid, shape
Become PUC57-vp2 plasmid.
PUC57-vp2 plasmid BamH I and Not I is carried out double digestion, reclaims through sepharose electrophoresis and obtain vp2 gene sheet
Section, is connected to that on psl1180 [hr3Pser1spRedSer1PA] carrier through same enzyme action, (construction method is shown in Publication No.
The Chinese patent of 102492692A), it is built into psl1180 [hr3Pser1vp2Ser1PA] carrier；Then with under Asc I enzyme action
Expression cassette hr3Pser1vp2Ser1PA containing vp2 gene, is then attached to equally through the dephosphorylized pBac of Asc I enzyme action
[3 × p3-EGFPaf] transgene expression vector (Carsten Horn.et al2000), is built into transgene expression vector pBac
[3 × p3-EGFP, hr3Pser1vp2Ser1PA], referred to as pBhSer1vp2 transgene carrier, structure is as shown in Figure 1.Wherein,
The nucleotide sequence of hr3Pser1vp2Ser1PA expression cassette is as shown in SEQ ID NO.3, and the 1st to 8 is Asc I enzyme action position
Point, the 31st to 36 is NcoI restriction enzyme site, and the 37th to 983 is enhancer hr3, and the 984th to 989 is NcoI enzyme
Cutting site, the 993rd to 1667 is secreting type sericin 1 gene promoter, and the 1668th to 1673 is BamHI enzyme action position
Point, the 1670th to 7996 is vp2 gene order, and the 2997th to 3017 is 6 His labels, and the 3018th to 3025
Position is NotI restriction enzyme site, and the 3026th to 3410 is the terminator of sericin 1 gene, and the 3411st to 3418 is AscI enzyme
Cut site.
Embodiment 2, transgenic microinjection and the screening of positive individuals and subculture
Polyvoltinism cultivated silkworm breed variety " D9L " is placed in standard environment and raises (temperature: 25 DEG C, humidity: 90%) with Folium Mori, change
By female male Bombycis mori copulation two-by-two about 4 hours after moth, the silkworm seed given birth to after separation of copulating moth is for next step microinjection.
By the silkworm seed proper alignment given birth on clean microscope slide, use in 1h～4h after silkworm seed is given birth to
PBhSer1vp2 transgene carrier and helper plasmid pHA3PIG equimolar are injected by Eppendorf microinjection instrument than after mixing
Enter in 400 " D9L " silkworm seeds, with nontoxic glue sealing be placed on 25 DEG C, the environment of relative humidity 90% hastens the hatching of silkworms 10 days left
239 G0 of hatching are raised to changing moth by right hatching for newly-hatched silkworm Folium Mori collection, and G0 passes through selfing or the common acquisition that backcrosses for Bombycis mori
110 moth circle G1, for silkworm seed, useElectronic macroscopic view fluorescence microscope G1 embryo screening, result is as in figure 2 it is shown, obtain
Obtain 16 positive moth circles, the i.e. transgenic bombyx mori of the external source vp2 gene expression of 16 Ser1 promoters driven, be called for short
PBhSer1vp2 transgenic system, transformation efficiency is 14.5%.6 pBhSer1vp2 positive moth circles of random choose carry out unilateral tonsillitis
Circle is raised, and subculture expands.
The copy number of vp2 gene in embodiment 3, detection pBhSer1vp2 transgenic system
PBhSer1vp2 transgenic system embodiment 2 prepared and normal " D9L " are placed under standard conditions raising, respectively
Extract each 50 μ g of its Bombycis mori genomic DNA to detect for Southern Blot.During detection with restricted enzyme Xbal I and
BamHI digests overnight, after the agarose gel electrophoresis through 0.8% separates, uses vacuum wiped film instrument by the DNA in agarose gel
Be transferred on the nylon membrane of pretreatment, transferring film terminate after through prehybridization, hybridize, wash film and color developing detection, testing result such as Fig. 3 institute
Show.Result shows, in pBhSer1vp2 transgenic system, vp2 gene is mainly inserted into domestic silkworm gene group with single copy.
Above-mentioned hybridization probe is the EGFP coded sequence of digoxigenin labeled, and the EGFP coded sequence of digoxigenin labeled is by as follows
Prepared by method: the total length primer of design EGFP gene, EGFP southern-F:5 '-cagtgcttcagccgctaccc-3 '
(SEQ ID NO.4), EGFP southern-R:5 '-ggatgttgccgtcctccttg-3 ' (SEQ ID NO.5), then with
PBac [3xp3-EGFPaf] plasmid is that template carries out PCR amplification, is reclaimed by cutting glue by amplified production, and enters reclaiming product
Row digoxin random labelling, forms EGFP hybridization probe.
Embodiment 4, detection vp2 gene transcribe situation in pBhSer1vp2
PBhSer1vp2 embodiment 2 prepared and normal " D9L " are placed under standard conditions raisings, 7 days ages of extraction larva 5
Sericterium total serum IgE, reverse transcription becomes cDNA, is eventually adding the TE buffer cDNA to transcribing and makees 4 times of dilutions, the mould detected as lower step
Plate.
Design Ser1 gene and the fluorescence quantification PCR primer of vp2 gene, Ser1 gene by fluorescence quantitative primer is Ser1-F:
5 '-atctgaagacggtttctggtggt-3 ' (SEQ ID NO.6), Ser1-R:5 '-Aactgcctgaagtggttgtgc-3 '
(SEQ ID NO.7), vp2 gene by fluorescence quantitative primer is vp2-F:5 '-tggttactgtggcaggtgttt-3 ' (SEQ ID
NO.8), vp2-R:5 '-tctttaatggggagttgaggtc-3 ' (SEQ ID NO.9), design reference gene fluorescence is fixed simultaneously
Amount primer (probe number is sw22934), forward primer is: sw22934-F:5 '-ttcgtactggctcttctcgt-3 ' (SEQ
ID NO.10), downstream primer is sw22934-R:5 '-caaagttgatagcaattccct-3 ' (SEQ ID NO.11).Take
State each 2 μ L of the cDNA template after dilution, TaKaRa companyPremix EX TaqTMII is anti-as quantitative fluorescent PCR
The reagent answered carries out fluorescent quantitation on 7500Fast Real-Time PCR Syatem instrument (Applied Biosystems)
PCR detect, PCR reaction condition is: 95 DEG C of 1 circulations in 30 seconds, 95 DEG C 5 seconds, 60 DEG C of 40 circulations in 30 seconds.Treat program end of run
After, experimental data Excel form derives, and uses 2-ΔΔCtRelative quantification method carries out data analysis, and result is as shown in Figure 4.Knot
Fruit shows that the success of vp2 gene is expressed at sericterium transcription, and transcribing between different positive moth circle exists notable difference, expression
Be equivalent to the 13.9%～66.1% of Ser1 gene.
The thermostability detection of rVP2 albumen in embodiment 5, pBhSer1vp2 transgenic system cocoon shell
Take pBhSer1vp2 transgenic cocoon shell and be broken into cocoon powder through low temperature, with 50mM Tris-HcI, 8M Urea (pH8.0)
Solution dissolve the albumen in cocoon shell, respectively the water-bath 25 DEG C, 40 DEG C, 60 DEG C and 80 DEG C process 30min, 1h, 1.5h and
2h, then detects rVP2 albumen with SDS-PAGE, and result is as shown in Figure 5.Result shows, cocoon shell rVP2 albumen has good resistance to
Hot, i.e. degrade not yet after 80 DEG C process 2h.
The Acidity of Aikalinity detection of rVP2 albumen in embodiment 6, pBhSer1vp2 transgenic system cocoon shell
Take pBhSer1vp2 transgenic cocoon shell and be broken into cocoon powder through low temperature, respectively with different pH value (1.5,2.5,5.5,
7.0,7.5,8.0,9.0 and 10.0) 50mM Tris-HcI, 8M Urea solution albumen in ambient temperature overnight dissolves cocoon shell.
Then using SDS-PAGE to detect rVP2 albumen, result is as shown in Figure 6.Result shows, cocoon shell rVP2 albumen is at highly acid
(pH1.5) and highly basic (pH12) is all unstable, more stable in the range of weak acid (pH5.5) to weak base (pH8.0).
The analysis purification of rVP2 albumen in embodiment 7, pBhSer1vp2 transgenic system cocoon shell
Take pBhSer1vp2 transgenic cocoon shell and in liquid nitrogen, be broken into cocoon powder, with 50mM Tris-HcI, 8M Urea
(pH8.0) solution dissolves the albumen in cocoon shell, makes its cocoon glutelin abundant in stirred overnight at room temperature after 80 DEG C of water-bath 30min
Dissolve, cross nickel column separating purification through the centrifugal supernatant of collecting of 15000rpm, collect respectively and flow through liquid, 50mM Tris-HCl, 8M
Urea (pH8.0) rinsing solution, 10mM imidazoles rinsing liquid, 20mM imidazoles rinsing liquid, 30mM imidazoles rinsing liquid, 120mM imidazoles float
Washing liquid, and with normal " D9L " cocoon shell solvent soln, pBhSer1vp2 transgenic cocoon shell lysate for comparison, then carry out SDS-
PAGE detects, and result is as shown in Figure 7 A.Result shows, 10mM, 20mM and 30mM degree imidazoles can with eluting major part foreign protein, and
RVP2 albumen can be eluted at 120mM, but still be about the foreign protein of 25KDa containing molecular weight.In order to obtain high-purity
RVP2 albumen, the rVP2 albumen of ni-sepharose purification is carried out Q column purification again, result is as shown in Figure 7 B.Result shows, by pure for Q post
The purity rVP2 albumen more than 90% is obtained after change.Then calculating the content of rVP2 albumen in cocoon shell, result shows in cocoon shell
The content of rVP2 albumen is 110 μ g/g.
Embodiment 8, the Analysis of Immunogenicity of rVP2 albumen
By highly purified rVP2 albumen and commercially available IBDV vaccine (Shanghai biologics) through abdominal part immunity 4 week old
SPF mice, after immunity terminates, collection serum carries out agarose double expansion test, and result is as shown in Figure 8.Result shows, rVP2 albumen
Antigen is only capable of and forms obvious precipitation line with rVP2 immune serum, it is impossible to vaccine serum (hole 3) and commercially available method
Family name's capsule virus fine jade expands positive serum (along vigorous biotechnology company limited) and forms precipitation line (Fig. 8 A), and IBDV virus fine jade expands
Antigen (along vigorous biotechnology company limited) can form obvious precipitation line with vaccine serum and positive serum, but can not be with
RVP2 immune serum forms precipitation line (Fig. 8 B).Again the serum of collection is carried out stepwise dilution for 1:4000 by volume, until
Being diluted to 1:2048000, carry out ELISA experiment using rVP2 albumen and IBDV virus agp antigen as substrate respectively, result is such as
Shown in Fig. 9.With rVP2 albumen generation enzyme linked immunoassay, and dosage effect can be presented from Fig. 9 A, rVP2 immune serum,
Further demonstrate that and create rVP2 protein antibodies in the Mice Body of immunity rVP2 albumen, but vaccine serum is only capable of highly concentrated
Certain enzyme linked immunoassay is there is with rVP2 albumen when spending.From Fig. 9 B, vaccine serum can occur with IBDV virus antigen
Immunoreation, and present dosage effect, and rVP2 immune serum can not produce immunoreation with IBDV virus antigen.In appearance
The reason stating result is, vp2 gene order makes a variation at IBDV camber, and determines virulence and the antigenic property of different strain.
And there are differences between vvIBDV strain and the IBDV strain sequence of the coding rVP2 protein sequence of synthetic of the present invention, lead
Causing rVP2 immune serum can not be with IBDV antigen None-identified.
Finally illustrate, preferred embodiment above only in order to technical scheme to be described and unrestricted, although logical
Cross above preferred embodiment the present invention to be described in detail, it is to be understood by those skilled in the art that can be
In form and it is made various change, without departing from claims of the present invention limited range in details.
Claims (8)
1. be suitable to the gene of the chicken infectivity bursa of Fabricius virus Viral structural protein VP2 that silkworm middle division of silkgland is expressed, it is characterised in that:
The gene of described chicken infectivity bursa of Fabricius virus Viral structural protein VP2 is if SEQ ID NO.1 the 7th is to shown in 1329.
2. contain and described in claim 1, be suitable to the chicken infectivity bursa of Fabricius virus Viral structural protein VP2 that silkworm middle division of silkgland is expressed
Gene in silkworm express expression vector.
Expression vector the most according to claim 2, it is characterised in that: described expression vector contains the enhancer being linked in sequence
Hr3, secreting type sericin 1 gene promoter, chicken infectivity bursa of Fabricius virus Viral structural protein VP2 gene and the end of sericin 1 gene
Only son.
Expression vector the most according to claim 3, it is characterised in that: described expression vector is possibly together with fluorescent screening labelling base
Because of expression cassette and piggyBac swivel base arm sequence, described fluorescent screening marker gene expression cassette is positioned at described enhancer hr3 upstream,
Described piggyBac swivel base arm sequence is between fluorescent screening marker gene expression cassette and the terminator of sericin 1 gene.
5. according to the expression vector described in any one of claim 2-4, it is characterised in that: described expression vector is by SEQ ID
Sequence shown in NO.3 is connected into after AscI enzyme action and turns through pBac [3 × p3-EGFPaf] AscI enzyme action, dephosphorylized equally
Expression vector prepares.
6. expression vector described in any one of claim 2-5 expresses chicken infectivity bursa of Fabricius virus structure at silkworm middle division of silkgland
Application in albumen VP2.
7. utilize expression vector described in any one of claim 2-5 to express chicken infectivity bursa of Fabricius virus at silkworm middle division of silkgland
The method of Viral structural protein VP2, it is characterised in that comprise the steps: described expression vector microinjection Eggs of Silkworm, by nothing
Poison glue sealing be placed on 25 DEG C, relative humidity be 90% environment in hatch, screening transgenic positive moth circle, take the positive and turn base
Because of Cocoon shell, in liquid nitrogen, be ground into powder, be then dissolved in containing 20mM Tris-Cl, 8M Urea, pH8.0 solution in,
The centrifugal supernatant of collecting of 15000rpm, purified, obtain chicken infectivity bursa of Fabricius virus Viral structural protein VP2.
Method the most according to claim 7, it is characterised in that: described purification, for supernatant is crossed nickel post, is first less than by concentration
The imidazoles rinsing of 30mM, the most again with 120 mM imidazoles rinsings, collects eluent, finally crosses Q post,.
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