CN104017814A - Chicken infectious bursal disease virus structural protein VP2 applicable to silkworm middle silk gland expression as well as expression vector and application thereof - Google Patents
Chicken infectious bursal disease virus structural protein VP2 applicable to silkworm middle silk gland expression as well as expression vector and application thereof Download PDFInfo
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Abstract
The invention discloses a chicken infectious bursal disease virus structural protein VP2 applicable to silkworm middle silk gland expression as well as an expression vector and application thereof. A gene sequence of the chicken infectious bursal disease virus structural protein VP2 is as shown in SEQ ID NO.1; a coded amino acid sequence of the chicken infectious bursal disease virus structural protein VP2 is as shown in SEQ ID NO.2; the sequence for coding the chicken infectious bursal disease virus structural protein VP2, and a promoter and a terminator of an excretion sericin 1 gene form an expression frame, and expression is enhanced by an enhancer hr3; meanwhile, a piggyback transposition arm and a fluorescence-screening marker gene constitute the expression vector; the expression vector can express the chicken infectious bursal disease virus structural protein VP2 in the silkworm middle silk gland, has immunogenicity, and can stimulate a mouse to generate antibodies, so that a foundation is laid up for expressing an animal virus subunit vaccine by utilizing a silkworm middle silk gland biological reactor.
Description
Technical field
The invention belongs to genetically engineered field, be particularly related to and be suitable for the chicken infectivity bursa of Fabricius virus structural protein VP2 that silkworm middle division of silkgland is expressed, also relate to and express the expression vector of chicken infectivity bursa of Fabricius virus structural protein VP2 and utilize silkworm middle division of silkgland to express the method for chicken infectivity bursa of Fabricius virus structural protein VP2.
Background technology
Infectious bursal disease (Infectious Bursal Disease, IBD) be by infectious bursal disease virus (Infectious Bursal Disease virus, IBDV), caused mainly to encroach on chick central immune organ---the transmissible disease of the fabricius bursa.This disease can cause immunity of organism to suppress even individual death, and causes chicken group to strengthen and immuning failure the susceptibility of other cause of disease, to aviculture, has brought huge financial loss.In recent years, the world has all found a plurality of countries the IBDV variant that virulence is stronger, can escape existing vaccine protection, makes the popular and prevention and control of IBD more complicated and difficult.Utilize genetic engineering technique to produce important directions and the effective means that subunit vaccine is control livestock and poultry, can effectively avoid the risk because of traditional deactivation or weak malicious IBDV vaccine None-identified.VP2 albumen is the major structural protein of IBDV, containing virulent major antigen protectiveness site, is the first-selected target of exploitation subunit vaccine.At present; researchist utilizes the expression systems such as intestinal bacteria, yeast, recombinant virus and paddy rice; successful expression chicken infectivity bursa of Fabricius virus structural protein VP2, and confirm can effective stimulus neutralizing antibody generation, chicken preventing infection IBDV is played to protection effect in various degree.
Silkworm is famous economic insects, from being tamed and be mainly used in so far getting silk weaving silk fabric by artificial rearing, for huge contribution has been made in the Economic development of China.Domestic natural silk gland is synthetic and the organ of secretion fibroin, having powerful albumen synthesizes and secretion capacity, from newly-hatched silkworm to matured silkworm, the weight of sericterium has increased by 160,000 times, its weight accounts for body weight of silkworm larva amount from 5% below 4 ages, to 5 mid-terms in age, increase rapidly, during matured silkworm, reached 40%～45%, become organ maximum in silkworm body.Silkworm after 5 mid-terms in age in sericterium the resultant velocity of silk fibroin up to 8mg/g ﹒ h.In vertebrates, little chicken liver is the synthetic the highest organ of albumin speed, about 0.1mg/g ﹒ h, and the resultant velocity of sericterium is its 80 times.Silkworm is low with its superpower protein synthesis capacity, feeding cost, to advantages such as nontoxic, the easy mass-producing of people and animals and batch production productions, it is counted as a kind of good bio-reactor that has potentiality.But there is no so far, utilize transgenic bombyx mori to express method and the relevant report of chicken infectivity bursa of Fabricius virus structural protein VP2.
Summary of the invention
In view of this, one of object of the present invention is to provide the chicken infectivity bursa of Fabricius virus structural protein VP2 that is suitable for the expression of silkworm middle division of silkgland; Two of object of the present invention is to provide the expression vector of expressing chicken infectivity bursa of Fabricius virus structural protein VP2; Three of object of the present invention is to provide expression vector to express the application in chicken infectivity bursa of Fabricius virus structural protein VP2 at silkworm middle division of silkgland; Four of object of the present invention is to provide utilizes described expression vector at silkworm middle division of silkgland, to express the method for chicken infectivity bursa of Fabricius virus structural protein VP2.
For achieving the above object, the invention provides following technical scheme:
Be suitable for the chicken infectivity bursa of Fabricius virus structural protein VP2 that silkworm middle division of silkgland is expressed, the gene order of described chicken infectivity bursa of Fabricius virus structural protein VP2 is if the 7th of SEQ ID NO.1 is to as shown in the of 1329.
Preferably, the aminoacid sequence of described chicken infectivity bursa of Fabricius virus structural protein VP2 is as shown in SEQ ID NO.2.
2, in silkworm, express the expression vector of described chicken infectivity bursa of Fabricius virus structural protein VP2.
Preferably, described expression vector contains the enhanser hr3 being linked in sequence, the terminator of secretor type sericin 1 gene promotor, chicken infectivity bursa of Fabricius virus structural protein VP2 gene and sericin 1 gene.
Preferably, described expression vector also contains fluorescent screening marker gene expression cassette and piggyBac swivel base arm sequence, described fluorescent screening marker gene expression cassette is positioned at described enhanser hr3 upstream, and described piggyBac swivel base arm sequence is between fluorescent screening marker gene expression cassette and the terminator of sericin 1 gene.
Preferably, described expression vector is connected into same cut through AscI enzyme, dephosphorylized pBac[3 * p3-EGFPaf by the sequence shown in SEQ ID NO.3 after AscI enzyme is cut] transgene expression vector makes.
3, described expression vector is expressed the application in chicken infectivity bursa of Fabricius virus structural protein VP2 at silkworm middle division of silkgland.
4, utilize described expression vector at silkworm middle division of silkgland, to express the method for chicken infectivity bursa of Fabricius virus structural protein VP2, comprise the steps: described expression vector microinjection Eggs of Silkworm, with nontoxic glue sealing, be placed on 25 ℃, relative humidity is to hatch in 90% environment, the positive moth circle of screening transgenic, get positive transgenic bombyx mori cocoon shell, in liquid nitrogen, be ground into powder, then be dissolved in the Tris-Cl containing 20mM, 8M Urea, in the solution of pH8.0, the centrifugal collection supernatant of 15000rpm, purified, obtain chicken infectivity bursa of Fabricius virus structural protein VP2.
Preferably, described purifying, for supernatant is crossed to nickel post, is first washed rinsing by concentration lower than the imidazoles of 30mM, and then with the rinsing of 120mM imidazoles, collects elutriant, finally crosses Q post.
Beneficial effect of the present invention is: the present invention is suitable for the gene of the chicken infectivity bursa of Fabricius virus structural protein VP2 of silkworm middle division of silkgland expression by codon optimized acquisition, chicken infectivity bursa of Fabricius virus structural protein VP2 gene and secretor type sericin 1 gene promotor and terminator are formed to expression cassette and utilize enhanser hr3 to strengthen and express, building efficient expression vector with fluorescent mark gene and piggyBac swivel base arm, this carrier can be at silkworm middle division of silkgland specific high-efficiency expression, in cocoon shell, recombinant protein content is high, be 110 μ g/g, and there is typical α after the recombinant protein of expressing is purified, β spiral and random coil structure, consistent with natural VP2 protein structure, Heat stability is good, degraded yet after 80 ℃ of processing 2h, at pH, be within the scope of 5.5-8, to be difficult for degraded, this is very important for utilizing transgenic bombyx mori scale operation and separation and purification.RVP2 albumen can be induced and be produced anti-rVP2 antibody by immunization mouse in its body, and the two expansion tests of agarose and ELISA experiment have further confirmed that the rVP2 albumen that utilizes transgenic bombyx mori middle division of silkgland to express has certain immunogenicity.Utilize gene and the expression vector of chicken infectivity bursa of Fabricius virus structural protein VP2 disclosed by the invention, by extensive raising transgenic silkworm, just can obtain in a large number and there is immunogenic VP2 albumen; This invention provides new approaches and novel method for utilizing silkworm middle division of silkgland bio-reactor to produce animal virus subunit vaccine.
Accompanying drawing explanation
In order to make object of the present invention, technical scheme and beneficial effect clearer, the invention provides following accompanying drawing:
Fig. 1 is pBhSer1vp2 transgene carrier structure intention.
Fig. 2 is fluorescent screening figure (the A:G1 embryo white light figure of pBhSer1vp2 transgenic silkworm; B: silkworm moth white light figure; C:G1 embryo green glow figure; D: silkworm moth green glow figure).
Fig. 3 is the copy number of positive moth circle vp2 gene in pBhSer1vp2 system.
Fig. 4 is the expression amount that fluorescent quantitation detects different moth circle vp2 genes in pBhSer1vp2 transgenosis.
Fig. 5 is rVP2 albumen thermotolerance detected result under differing temps.
Fig. 6 is detected result after rVP2 albumen is processed under different pH.
Fig. 7 is that (A represents ni-sepharose purification result for the separation and purification detected result of cocoon shell rVP2 albumen; M:marker; 1:WT; 2: transgenic bombyx mori cocoon shell lysate; 3: stream is worn liquid; 4:50mM Tris-HcI, 8M Urea (pH8.0) rinsing liquid; 5:10mM imidazoles rinsing liquid; 6:20mM imidazoles rinsing liquid; 7:30mM imidazoles rinsing liquid; 8:120mM imidazoles rinsing liquid; B represents Q column purification result, 9: ni-sepharose purification concentrated solution; 10:Q column purification liquid).
Fig. 8 is that (A represents that rVP2 albumen is substrate to the two expansion of rVP2 albumen agarose experimental result; B represents that IBD antigen is substrate; 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).
Embodiment
Below in conjunction with accompanying drawing, the preferred embodiments of the present invention are described in detail.The experimental technique of unreceipted actual conditions in embodiment, conventionally according to normal condition, for example condition described in molecular cloning experiment guide (third edition, the work such as J. Pehanorm Brooker), or the condition of advising according to manufacturer.
Embodiment 1, structure transgene expression vector pBac[3 * P3EGFP, hr3CQSer1-vp2-Ser1PA]
According to report the gene order (GenBank.JF907703.1) of IBDV highly virulent strain virus structural protein VP2 and the codon preference of silkworm to codon optimized, obtain the vp2 gene order after optimizing, at C end, add again the Nucleotide formation vp2-His6 of 6 His labels, at the nucleotide sequence two ends of vp2-His6, connect respectively BamHI and NotI restriction enzyme site, then entrust the sequence of Shenzhen Hua Da genome company synthesizing ribonucleotide sequence as shown in SEQ ID NO.1, the coded aminoacid sequence in its coding region is as shown in SEQ ID NO.2.In the sequence of SEQ ID NO.1, the 1st to the 6th is BamHI restriction enzyme site, and the 7th to the 1329th is vp2 gene coded sequence, the 1330th to the 1350th 6 His labels for the interpolation of C end, and the 1351st to 1358 is NotI restriction enzyme site.Then the Nucleotide of SEQ ID NO.2 is connected in PUC57 plasmid, form PUC57-vp2 plasmid.
PUC57-vp2 plasmid is carried out to double digestion by BamH I and Not I, through agarose electrophoresis, reclaim and obtain vp2 gene fragment, be connected to the psl1180[hr3Pser1spRedSer1PA cutting through same enzyme] on carrier (construction process is shown in that publication number is the Chinese patent of 102492692A), be built into psl1180[hr3Pser1vp2Ser1PA] carrier; Then with Asc I enzyme, cut the expression cassette hr3Pser1vp2Ser1PA containing vp2 gene, then be connected to the dephosphorylized pBac[3 * p3-EGFPaf cutting through Asc I enzyme equally] transgene expression vector (Carsten Horn.et al2000), be built into transgene expression vector pBac[3 * p3-EGFP, hr3Pser1vp2Ser1PA], referred to as pBhSer1vp2 transgene carrier, structure as shown in Figure 1.Wherein, the nucleotide sequence of hr3Pser1vp2Ser1PA expression cassette is as shown in SEQ ID NO.3, the 1st to 8 is Asc I restriction enzyme site, the 31st to 36 is NcoI restriction enzyme site, the 37th to 983 is enhanser hr3, the 984th to 989 is NcoI restriction enzyme site, the 993rd to 1667 is secretor type sericin 1 gene promotor, the 1668th to 1673 is BamHI restriction enzyme site, the 1670th to 7996 is vp2 gene order, the 2997th to 3017 is 6 His labels, the 3018th to 3025 is NotI restriction enzyme site, the 3026th to 3410 terminators that are sericin 1 gene, the 3411st to 3418 is AscI restriction enzyme site.
Screening and the subculture of embodiment 2, transgenosis microinjection and positive individuals
By polyvoltinism cultivated silkworm breed variety " D9L " be placed in mulberry leaf for standard environment raise (temperature: 25 ℃, humidity: 90%), change after moth female male Bombycis mori mating between two approximately 4 hours, the silkworm seed of giving birth to after separation of copulating moth is for next step microinjection.
By the silkworm seed proper alignment of giving birth on clean slide glass, in 1h～4h after silkworm seed is given birth to, with Eppendorf microinjection instrument, will after pBhSer1vp2 transgene carrier and the mixing of helper plasmid pHA3PIG equimolar ratio, be injected in 400 " D9L " silkworm seeds, with nontoxic glue sealing to be placed on the hatching about 10 days of hastening the hatching of silkworms in 25 ℃, the environment of relative humidity 90%, 239 G0 of hatching are collected and raised to changing moth with mulberry leaf for newly-hatched silkworm, G0 for silkworm moth by selfing or the common acquisition 110 moth circle G1 that backcross for silkworm seed, use
electronic macroscopical fluorescence microscope G1 embryo screening, result as shown in Figure 2, obtains 16 positive moth circles, and the transgenic bombyx mori of the external source vp2 genetic expression of 16 Ser1 promoters driven, is called for short pBhSer1vp2 transgenosis system, and transformation efficiency is 14.5%.Random choose the positive moth circles of 6 pBhSer1vp2 carry out single moth circle and raise, subculture expands.
The copy number of vp2 gene in embodiment 3, detection pBhSer1vp2 transgenosis system
By the pBhSer1vp2 transgenic lines of embodiment 2 preparation unify normally " D9L " be placed under standard conditions and raise, extract respectively its silkworm moth genomic dna respectively 50 μ g for Southern Blot, detect.During detection, with restriction enzyme Xbal I and BamHI digestion, spend the night, after 0.8% agarose gel electrophoresis separation, adopt vacuum transferring film instrument that the DNA in sepharose is transferred on pretreated nylon membrane, transferring film finishes by prehybridization, hybridizes, washes film and color developing detection, and detected result as shown in Figure 3.Result shows, in pBhSer1vp2 transgenosis system, vp2 gene is mainly inserted into domestic silkworm gene group with single copy.
Above-mentioned hybridization probe is the EGFP encoding sequence of digoxigenin labeled, the EGFP encoding sequence of digoxigenin labeled is prepared by the following 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 take pBac[3xp3-EGFPaf] plasmid carries out pcr amplification as template, amplified production is reclaimed by cutting glue, and carry out digoxin random labelling to reclaiming product, form EGFP hybridization probe.
Embodiment 4, the transcribe situation of detection vp2 gene in pBhSer1vp2
The pBhSer1vp2 of embodiment 2 preparations is placed under standard conditions and is raised with normal " D9L ", extract the total RNA of larva sericterium in 7 days 5 ages, reverse transcription becomes cDNA, finally adds TE damping fluid to do 4 times of dilutions to the cDNA transcribing, the template detecting as lower step.
The fluorescence quantification PCR primer of design Ser1 gene and 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 fluorescent quantitation primer (probe number is sw22934) simultaneously, upstream primer is: sw22934-F:5 '-ttcgtactggctcttctcgt-3 ' (SEQ ID NO.10), downstream primer is sw22934-R:5 '-caaagttgatagcaattccct-3 ' (SEQ ID NO.11).Get each the 2 μ L of cDNA template after above-mentioned dilution, TaKaRa company
premix EX Taq
tMiI carries out fluorescence quantitative PCR detection as the reagent of quantitative fluorescent PCR reaction on 7500Fast Real-Time PCR Syatem instrument (Applied Biosystems), PCR reaction conditions is: 95 ℃ of 1 circulations in 30 seconds, 95 ℃ 5 seconds, 60 ℃ of 40 circulations in 30 seconds.After program end of run, experimental data derives by Excel form, and adopts 2
-Δ Δ Ctrelative quantification method is carried out data analysis, and result as shown in Figure 4.Result shows that the success of vp2 gene is in the expression of sericterium transcription, and transcribing between different positive moth circle exists notable difference, expression amount to be equivalent to 13.9%～66.1% of Ser1 gene.
In embodiment 5, pBhSer1vp2 transgenosis system cocoon shell, the thermotolerance of rVP2 albumen detects
Get pBhSer1vp2 transgenosis cocoon shell and be broken into cocoon powder through low temperature, with the solution of 50mM Tris-HcI, 8M Urea (pH8.0), dissolve the albumen in cocoon shell, at the water-bath of 25 ℃, 40 ℃, 60 ℃ and 80 ℃, process 30min, 1h, 1.5h and 2h respectively, then with SDS-PAGE, detect rVP2 albumen, result as shown in Figure 5.Result demonstration, cocoon shell rVP2 albumen has good thermotolerance, after 80 ℃ are processed 2h, does not degrade yet.
In embodiment 6, pBhSer1vp2 transgenosis system cocoon shell, the acid-basicity of rVP2 albumen detects
Get pBhSer1vp2 transgenosis cocoon shell and be broken into cocoon powder through low temperature, with 50mM Tris-HcI, the 8M Urea solution of different pH values (1.5,2.5,5.5,7.0,7.5,8.0,9.0 and 10.0), in ambient temperature overnight, dissolve the albumen in cocoon shell respectively.Then adopt SDS-PAGE to detect rVP2 albumen, result as shown in Figure 6.Result demonstration, cocoon shell rVP2 albumen is all unstable at strongly-acid (pH1.5) and highly basic (pH12), and more stable in weak acid (pH5.5) arrives weak base (pH8.0) scope.
The analysis purifying of rVP2 albumen in embodiment 7, pBhSer1vp2 transgenosis system cocoon shell
Get pBhSer1vp2 transgenosis cocoon shell and in liquid nitrogen, be broken into cocoon powder, use 50mM Tris-HcI, the solution of 8M Urea (pH8.0) dissolves the albumen in cocoon shell, after 80 ℃ of water-bath 30min, in ambient temperature overnight, stir its cocoon glutelin is fully dissolved, through the centrifugal collection supernatant of 15000rpm, cross nickel column separating purification, collect respectively stream and wear liquid, 50mM Tris-HCl, 8M Urea (pH8.0) rinsing solution, 10mM imidazoles rinsing liquid, 20mM imidazoles rinsing liquid, 30mM imidazoles rinsing liquid, 120mM imidazoles rinsing liquid, and with normal " D9L " cocoon shell solvent soln, pBhSer1vp2 transgenosis cocoon shell lysate is contrast, then carry out SDS-PAGE detection, result as shown in Figure 7 A.Result shows, 10mM, 20mM and 30mM degree imidazoles can the most of foreign proteins of wash-out, and rVP2 albumen can be eluted at 120mM, but still contain the foreign protein that molecular weight is about 25KDa.In order to obtain highly purified rVP2 albumen, the rVP2 albumen of ni-sepharose purification is carried out to Q column purification again, result is as shown in Figure 7 B.Result shows, by the rVP2 albumen that obtains purity after Q column purification and be greater than 90%.Then calculate the content of rVP2 albumen in cocoon shell, result shows that the content of rVP2 albumen in cocoon shell is 110 μ g/g.
The Analysis of Immunogenicity of embodiment 8, rVP2 albumen
By the IBDV vaccine (Shanghai biologics) of highly purified rVP2 albumen and business purchase, through belly immunity SPF mouse in 4 week age, immunity finishes rear collection serum and carries out the two expansion tests of agarose, and result as shown in Figure 8.Result shows, rVP2 proteantigen only can form obvious precipitation line with rVP2 immune serum, can not expand positive serum (Shun Bo biotechnology company limited) with the bursal disease virus fine jade that vaccine serum (hole 3) and business are bought and form precipitation line (Fig. 8 A), and IBDV virus agp antigen (Shun Bo biotechnology company limited) can form obvious precipitation line with vaccine serum and positive serum, but can not form precipitation line (Fig. 8 B) with rVP2 immune serum.Again by the serum of collection by volume for 1:4000 carries out stepwise dilution, until be diluted to 1:2048000, usining respectively rVP2 albumen and IBDV virus agp antigen carries out ELISA experiment as substrate, result as shown in Figure 9.From Fig. 9 A, rVP2 immune serum can with rVP2 albumen generation enzyme linked immunoassay, and present dosage effect, further confirmed in the Mice Body of immune rVP2 albumen, to have produced rVP2 protein antibodies, but vaccine serum only can be when high density and rVP2 albumen there is certain enzyme linked immunoassay.From Fig. 9 B, vaccine serum can with IBDV virus antigen generation immune response, and present dosage effect, and rVP2 immune serum can not produce immune response with IBDV virus antigen.The reason that occurs the above results is that vp2 gene order makes a variation at IBDV camber, and has determined virulence and the antigenic property of different strains.And between the vvIBDV strain of the artificial synthetic coding rVP2 protein sequence of the present invention and IBDV strain sequence, there are differences, cause rVP2 immune serum can not with IBDV antigen None-identified.
Finally explanation is, above preferred embodiment is only unrestricted in order to technical scheme of the present invention to be described, although the present invention is described in detail by above preferred embodiment, but those skilled in the art are to be understood that, can to it, make various changes in the form and details, and not depart from the claims in the present invention book limited range.
Claims (9)
1. be suitable for the chicken infectivity bursa of Fabricius virus structural protein VP2 that silkworm middle division of silkgland is expressed, it is characterized in that: the gene order of described chicken infectivity bursa of Fabricius virus structural protein VP2 is if the 7th of SEQ ID NO.1 is to as shown in the of 1329.
2. be suitable for according to claim 1 the chicken infectivity bursa of Fabricius virus structural protein VP2 that silkworm middle division of silkgland is expressed, it is characterized in that: the aminoacid sequence of described chicken infectivity bursa of Fabricius virus structural protein VP2 is as shown in SEQ ID NO.2.
3. in silkworm, express the expression vector of chicken infectivity bursa of Fabricius virus structural protein VP2 described in claim 1 or 2.
4. expression vector according to claim 3, is characterized in that: described expression vector contains the enhanser hr3 being linked in sequence, the terminator of secretor type sericin 1 gene promotor, chicken infectivity bursa of Fabricius virus structural protein VP2 gene and sericin 1 gene.
5. expression vector according to claim 4, it is characterized in that: described expression vector also contains fluorescent screening marker gene expression cassette and piggyBac swivel base arm sequence, described fluorescent screening marker gene expression cassette is positioned at described enhanser hr3 upstream, and described piggyBac swivel base arm sequence is between fluorescent screening marker gene expression cassette and the terminator of sericin 1 gene.
6. according to the expression vector described in claim 3-5 any one, it is characterized in that: described expression vector is connected into same cut through AscI enzyme, dephosphorylized pBac[3 * p3-EGFPaf by the sequence shown in SEQ ID NO.3 after AscI enzyme is cut] transgene expression vector makes.
7. described in claim 3-6 any one, expression vector is expressed the application in chicken infectivity bursa of Fabricius virus structural protein VP2 at silkworm middle division of silkgland.
8. utilize expression vector described in claim 3-6 any one at silkworm middle division of silkgland, to express the method for chicken infectivity bursa of Fabricius virus structural protein VP2, it is characterized in that, comprise the steps: described expression vector microinjection Eggs of Silkworm, with nontoxic glue sealing, be placed on 25 ℃, relative humidity is to hatch in 90% environment, the positive moth circle of screening transgenic, get positive transgenic bombyx mori cocoon shell, in liquid nitrogen, be ground into powder, then be dissolved in the Tris-Cl containing 20mM, 8M Urea, in the solution of pH8.0, the centrifugal collection supernatant of 15000rpm, purified, obtain chicken infectivity bursa of Fabricius virus structural protein VP2.
9. method according to claim 8, is characterized in that: described purifying, for supernatant is crossed to nickel post, is first washed rinsing by concentration lower than the imidazoles of 30mM, and then with the rinsing of 120mM imidazoles, collects elutriant, finally crosses Q post.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108642059A (en) * | 2018-05-04 | 2018-10-12 | 西南大学 | Transformation suitable for silkworm expression, which has, promotes cell proliferation factor gene and its expression vector and application |
| CN108642059B (en) * | 2018-05-04 | 2020-07-28 | 西南大学 | Modified gene with cell proliferation promoting factor suitable for silkworm expression and expression vector and application thereof |
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