CN101624421A - Virus-like particle recombinant protein of virus variation strain VP2 gene of infectious bursal disease - Google Patents
Virus-like particle recombinant protein of virus variation strain VP2 gene of infectious bursal disease Download PDFInfo
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Abstract
The invention relates to virus-like particle recombinant protein of a virus variation strain VP2 gene of an infectious bursal disease, belonging to the field of biologic pharmacy. The IBDV variation strain (AH1) VP2 gene is cloned, converted and transfected to obtain a recombinant baculovirus vBac-VP2; an infected Sf9 insect cell has specific fluorescence, and the antigen valence of the infected Sf9 insect cell is above 1.6*10<3>; the molecular weight of a recombinant VP2 protein is 53kDa, and the recombinant VP2 protein is in the state of virus-like particles; an indirect ELISA detection method established for an envelope antigen by the purified recombinant VP2 protein has good specificity and sensibility; an immune chicken can resist IBDV virulent attack, and the protection ratio achieves 100 percent. The novel virus-like particle recombinant VP2 protein prepared by the IBDV variation strain VP2 gene has high pertinence on the immune prevention of a current prevalent IBDV virulent strain and good practical value and popularization prospects.
Description
One, technical field
The present invention relates to the expression and the application of infectious bursal disease virus (IBDV) variant (AH1) VP2 gene viruses sample particle recombinant protein, belong to field of biological pharmacy.
Two, background technology
Infectious bursal disease (Infectious Bursal Disease, IBD) be one of main transmissible disease of causing China and the serious financial loss of world's aviculture, be that what to be caused by infectious bursal disease virus (IBDV) is the transmissible disease of principal character with infringement chick Lymphoid tissue, particularly central immune organ-fabricius bursa.The harm of this disease comprises two aspects: the one, cause the chicken morbidity and the death in 3～8 ages in week, and the appearance of highly virulent strain (vvIBDV) especially in recent years increases the chick mortality ratio greatly; The 2nd, this disease of chick early infection then causes immunosuppression, causes the chicken group that the susceptibility of other eqpidemic disease is increased and the immunne response ability of vaccine is reduced or failure, causes huge indirect economic loss.
Vaccine inoculation is the effective means of this disease of prevention, and in the middle and later periods of the eighties in last century, worldwide having broken out at antigenicity and aspect such as pathogenic and classical strains has the very IBDV variation strain of big-difference, and poultry husbandry is inflicted heavy losses on.The appearance of variant and highly virulent strain (vvIBDV) strengthens this sick prevention and control difficulty, IBD immunoprophylaxis failure become major issue in the poultry diease prevention and control [Wang Yongshan etc. cause the characterization of molecules of the infectivity bursa of Fabricius virus VP 2 gene of immuning failure in the recent period. Chinese veterinary science, 2008,38 (12): 1013-1019].At present, the conventional attenuated vaccine and the inactivated vaccine that use, still there are deficiency in its security and manufacturing process: for the prevention variation strain adopt in the strong virus force living vaccine exist bigger Biosafety problem, because potential gene resortment may between different I BDV strain, bring hidden danger for these sick prevention and control, inactivated vaccine exists the difficulty of antigen prepd.Therefore, in current I BD prevention and control real work, need that immune efficacy is strong, biological safety is high badly and at the appearance of the novel I BD recombinant vaccine of current popular strain.
IBDV belongs to birnavirus section birnavirus and belongs to, and genome comprises two fragments of (A) little (B) greatly, and five kinds of viral protein: VP1, VP2, VP3, VP4 and VP5 are arranged.VP2 accounts for 51% of viral protein, is the primary structure albumen of IBDV, is again the main protection antigen of virus, with the variation of the inducing of virucidin, antigen and virulence and apoptotic induce etc. relevant.The hypervariable region of VP2 be viral neutrality monoclonal antibody in conjunction with required area, the point mutation in this district is the variation of IBDV antigenic drift, virulence and and then causes the major cause of classical vaccine strain immuning failure.So far, the research of IBD recombinant vaccine is target gene with VP2 all, and the expression system of employing comprises: the polyepitope vaccines of intestinal bacteria, yeast, Fowlpox virus vector, rhabdovirus expression vector, nucleic acid vaccine and recent report etc.Subject matter in the IBD recombinant vaccine research at present: or the proteic expression amount of reorganization IBDV is not high enough, or reorganization IBDV protein renaturation difficulty is big, or the recombinant protein that comes from a kind of strain is not suitable for other variation strain.Therefore, be very necessary further exploring new technological line aspect VP2 gene Selection, expression system and the expression strategy.
Three, summary of the invention
Technical problem
The present invention is directed to the subject matter that exists in the research of popular present situation of IBD and recombinant vaccine; adopt new technological line; on goal gene is selected; adopt the IBDV variant (AH1) that causes immuning failure in the recent period; this variant has the VP2 gene of the strong malicious characterization of molecules of IBDV completely; the VP2 gene order of the vaccine strain (B87 strain) that this VP2 gene and current prevention are used has than big-difference; on expression system; utilize baculovirus expression system to have and the similar posttranslational modification of high cell system; can make the exogenous gene expression product have the advantage that natural activity form and expression product can be self-assembled into virus-like particle (VLP); preparation virus-like particle recombinant VP 2 albumen, development is at the new and effective IBD virus-like particle recombinant vaccine and the detection reagent of current I BDV epidemic isolates.
Technical scheme
Infectious bursal disease virus variant (AH1) VP2 gene viruses sample particle recombinant protein is that the recombinant baculovirus vBac-VP2 expression that obtains with the pBac-VP2 transfection Sf 9 insect cell obtains by structure recombinant baculovirus expression plasmid pBac-VP2.
Its recombinant baculovirus expression plasmid pBac-VP2 and recombinant baculovirus vBac-VP2, its construction process is: express reading frame design VP2 gene amplification primer according to VP2 gene order and donor plasmid pFastBacHTA:
Forward VP2F (36): 5 '-CAGATCTGAATTCATGGCGAACCTGCAAGATCAAAC-3 '
Reverse VP2R (34): 5 '-CTACTACCTCCTTATGGCCCGGATTATGTCTTTG-3 '
Recombinant baculovirus expression plasmid primers designed M13F (17):
Upstream sequence: 5 '-GTTTTCCCAGTCACGAC-3 '
Downstream sequence: 5 '-CAGGAAACAGCTATGAC-3 '
Cloned plasmids pUC-VP2 (AH1) and the donor plasmid pFastBacHTA that contains 6 Histidine His are used EcoR I and Hind III double digestion respectively, VP2 gene with IBDV variant AH1, directed cloning is gone among the donor plasmid pFastBacHTA of baculovirus expression system, make up reorganization donor plasmid pFastBacHTA-VP2, conversion contains the E.coli DH10Bac competence of Bacmid and Helper plasmid, identify Bacmid DNA with VP2 gene forward amplimer VP2F (36) and M13R (17) primer downstream sequence PCR, the band that can to get a size be 2100bp, acquisition contains the recombinant baculovirus expression plasmid pBac-VP2 of VP2 gene, use the pBac-VP2 transfection Sf 9 insect cell, obtain recombinant baculovirus vBac-VP2.
Described recombinant baculovirus expression plasmid pBac-VP2 and recombinant baculovirus vBac-VP2 all contain 6 Histidine His labels and marmor erodens TEV proteolytic enzyme recognition site, are convenient to the proteic purifying of recombinant VP 2.
Above-mentioned infectious bursal disease virus variant (AH1) VP2 gene viruses sample particle recombinant protein can be applied in IBDV detection method and preparation immunological reagent.Its purification process is: infect the Sf9 insect cell with the 3rd generation recombinant baculovirus vBac-VP2, treat after the complete pathology of cell, the collecting cell culture, the centrifugal 30min collecting cell of 3000rpm, resuspended with HisTrap HP binding buffer liquid, and add 20 μ l 100mmol/L PMSF, place 0 ℃ of ice-water bath, ultrasonic power 500～600W, each ultrasonic time 10min, repeat 2～4 times ultrasonic, limpid to cell suspension, 4 ℃, the centrifugal 30min of 10000rpm, remove cell debris, get supernatant, use HisTrap HP chromatography column purification of Recombinant VP2 albumen then to specifications with 0.45 μ m aperture membrane filtration.
Its IBDV serum antibody ELISA detection method is: uses recombinant VP 2 albumen to the 5 μ g/ml of the carbonate buffer solution dilution purifying of pH9.6, wraps by elisa plate, and 100 μ l/ holes, 4 ℃ are spent the night; With PBST washing 3 times, each 2 minutes, pat dry; Every hole adds the PBST confining liquid 300 μ l that contain volume ratio 10%FBS, 37 ℃, 2h; With PBST washing 3 times, each 2 minutes, pat dry; Add serum sample to be checked, 37 ℃, 1h; With PBST washing 3 times, each 2 minutes, pat dry; The anti-chicken IgG100 of 1: the 2000 rabbit μ l that adds the HRP-mark, 37 ℃, 1h; With PBST washing 4 times, each 2 minutes, pat dry; Add each one of developer A liquid (tetramethyl biphenyl amine aqueous solution), B liquid (hydrogen peroxide urea solution), the magazine colour developing; 2M H
2SO
4Stop, survey A
450Value.
The preparation method of its virus-like particle recombinant vaccine is: get the Sf9 insect cell culture that recombinant baculovirus vBac-VP2 infects; freeze thawing three times; detect with the IBDV antibody sandwich ELISA; ELISA tires 〉=and 1600; add isopyknic white oil immunological adjuvant; mixing and emulsifying is IBD virus-like particle recombinant vaccine.
The application method of its virus-like particle recombinant vaccine is through the chest muscle injection, 0.4ml//time, carries out the 2nd immunity behind the 1st the immune 14d.
Beneficial effect characteristics of the present invention and advantage are as follows:
IBDV belongs to birnavirus section birnavirus and belongs to, and genome comprises two fragments of (A) little (B) greatly, and five kinds of viral protein: VP1, VP2, VP3, VP4 and VP5 are arranged.VP2 accounts for 51% of viral protein, is the primary structure albumen of IBDV, is again the main protection antigen of virus, with the variation of the inducing of virucidin, antigen and virulence and apoptotic induce etc. relevant.The hypervariable region of VP2 be viral neutrality monoclonal antibody in conjunction with required area, the point mutation in this district is the variation of IBDV antigenic drift, virulence and and then causes the major cause of classical vaccine strain immuning failure.So far, the research of IBD recombinant vaccine all is target gene with VP2, subject matter in the IBD recombinant vaccine research at present: or the proteic expression amount of reorganization IBDV is not high enough, or reorganization IBDV protein renaturation difficulty is big, or the recombinant protein that comes from a kind of strain is not suitable for other variation strain.Therefore, be very necessary further exploring new technological line aspect VP2 gene Selection, expression system and the expression strategy.
Virus sample particle vaccines has the advantage of the following aspects as preventative vaccine: (1) does not contain viral DNA, does not have infectious; (2) immunogenicity is strong, can stimulate body to produce specific humoral immunity and cellular immunization; (3) good stability is difficult for inactivation.Particle vaccines has not only overcome the shortcoming of traditional attenuated vaccine and inactivated vaccine as a kind of novel vaccine, has also remedied the deficiency of gene vaccine.
The present invention is directed to the subject matter that exists in the research of popular present situation of IBD and recombinant vaccine, adopt new technological line, on goal gene is selected, adopt the IBDV variant (AH1) that causes immuning failure in the recent period, this variant has the VP2 gene of the strong malicious characterization of molecules of IBDV completely, the VP2 gene order of the vaccine strain (B87 strain) that this VP2 gene and current prevention are used has than big-difference, and therefore the immunoprophylaxis to current I BDV has more specific aim; On expression system, utilize baculovirus expression system to have and the similar posttranslational modification of high cell system, can make the exogenous gene expression product have the advantage that natural activity form and expression product can be self-assembled into virus-like particle (VLP), IBDV variant (AH1) VP2 gene has been realized efficiently expressing in insect cell; Cell culture to the VP2 gene recombination baculovirus infects detects with sandwich ELISA method, and recombinant VP 2 is proteic tires 1.6 * 10
3More than, recombinant VP 2 albumen can be self-assembled into virus-like particle, and has found " inclusion body sample " structure first in the insect cell that infects.In addition, before the VP2 gene, increase by 6 Histidine (His) labels and marmor erodens (TEV) proteolytic enzyme recognition site, be convenient to the proteic purifying of recombinant VP 2.The IBDV antibody indirect ELISA detection method of setting up as envelope antigen with the recombinant VP 2 albumen of purifying has good specificity and susceptibility; Sf9 insect cell lysate immunity chicken with recombinate shape virus infection; excitating organism produces immunoprotective antibody effectively, opposing IBDV strong virus attack.
The recombinant VP 2 albumen of the present invention's preparation is higher to the immunoprophylaxis specific aim of current I BDV epidemic isolates, has good practical value and promotion prospect.
Four, description of drawings
Fig. 1 contains the structure of the reorganization donor plasmid pFastBacHTA-VP2 of VP2 gene
Fig. 2 infects Sf9 cell SDS-PAGE and the Western-blotting analytical results of vBac-VP2
Fig. 3 infects the Sf9 cell electron microscopic examination result of vBac-VP2
Five, embodiment
IBDV variant (AH1) VP2 gene line causes clone the IBDV variant (AH1) of immuning failure with the RT-PCR method in Anhui from December, 2007, this variant has the whole significant amino-acid residue of IBDV virulent strain, VP2 gene seven peptide motifs are SWSASGS, 222,253,256,279,284, amino-acid residue on 294 and 299 is respectively A, Q, I, D, A, I and S, with the VP2 gene order of existing commercially available vaccine strain B87 have than big-difference [Wang Yongshan etc. cause the characterization of molecules of the infectivity bursa of Fabricius virus VP 2 gene of immuning failure in the recent period. Chinese veterinary science, 2008,38 (12): 1013-1019].Therefore, the prevention and control to current I BDV have more specific aim and practical significance.
1 experiment material
1.1 plasmid, bacterial classification and cell
The cloned plasmids pUC-VP2 (AH1) that contains the VP2 gene is [public, Wang Yongshan etc. cause the characterization of molecules of the infectivity bursa of Fabricius virus VP 2 gene of immuning failure in the recent period. Chinese veterinary science, 2008,38 (12): 1013-1019] be, with gene constructed the forming of VP2 of RT-PCR method from December, 2007 amplification the sick chicken bursa tissue of the domestic IBD immunoprophylaxis failure that takes place in Anhui.IBD attenuated vaccine (the B87 strain is available from Nanjing Tianbang Bio-industry Co., Ltd.).Contain the donor plasmid pFastBacHTA of 6 * His label, the E.coli DH10Bac that contains Bacmid and Helper plasmid, Sf9 insect cell, E.coli TOP10 all available from Invitrogen company.
1.2 main agents
The anti-IBDV hyper-immune serum of chicken system obtains with the immune SPF chicken of IBD attenuated vaccine (B87) (5 times), with DEAE Mierocrystalline cellulose (DE32) purifying (Liu Yubin, Gou Shijin chief editor. the animal immunology experimental technique. Changchun: Jilin science tech publishing house, 1989,8-15);
The anti-chicken igg antibody reference literature preparation of the rabbit of anti-chicken igg antibody of rabbit and HRP mark (Liu Yubin, Gou Shijin chief editor. the animal immunology experimental technique. Changchun: Jilin science tech publishing house, 1989,150-151; Wang Yongshan, Hao Chong, Hou Shikuan, Liu Zi, Ma Conglin, Yin Zhen. anti-chicken IgG studies on Monoclonal Antibody. animal doctor's college journal, 1989,9 (2): 109-114).
MiniBEST plasmid purification test kit, dna gel reclaim test kit, X-gal, IPTG all available from TaKaRa company; BAC/PAC DNA Isolation kit is available from OMEGA company; The Lipofectamine2000 transfection reagent is available from Invitrogen company; Foetal calf serum, Grace ' s insect cell substratum are available from GIBCO company.The goat anti-rabbit igg antibody of FITC mark, DAB colour developing liquid are available from Wuhan Boster Biological Technology Co., Ltd..
2 experimental techniques
2.1 primer design is with synthetic
Express reading frame design VP2 gene amplification primer according to VP2 gene order and donor plasmid pFastBacHTA:
Forward VP2F (36): 5 '-CAGATCTGAATTCATGGCGAACCTGCAAGATCAAAC-3 '
Reverse VP2R (34): 5 '-CTACTACCTCCTTATGGCCCGGATTATGTCTTTG-3 '
Recombinant baculovirus expression plasmid primers designed M13F (17):
Upstream sequence: 5 '-GTTTTCCCAGTCACGAC-3 '
Downstream sequence: 5 '-CAGGAAACAGCTATGAC-3 '
Above primer is synthetic by Invitrogen company.
2.2VP2 the structure of gene recombination donor plasmid
Cloned plasmids pUC-VP2 (AH1) and donor plasmid pFastBacHTA (wherein containing 6 Histidine (His) labels and marmor erodens (TEV) proteolytic enzyme recognition site) are used EcoR I and Hind III double digestion respectively, through 1% agarose gel electrophoresis, reclaim VP2 and carrier DNA fragment respectively, DNA glue reclaims the test kit purifying, T4DNA Ligase connects, Transformed E .coli TOP10 competence bacterium, be coated on on 1.5% agar plate of LB preparation (containing penbritin 100 μ g/ml), cultivate 14h for 37 ℃.Choose single colony inoculation and go into LB (containing penbritin 100 μ g/ml), 12h is cultivated in jolting, extract plasmid with MiniBEST plasmid purification test kit, EcoR I and Hind III double digestion are identified, visible two bands of 1% agarose gel electrophoresis, be about 1.4Kb and 5.0Kb respectively, obtain to contain the reorganization donor plasmid pFastBacHTA-VP2 (Fig. 1) of VP2 gene.
2.3VP2 the structure of gene recombination baculovirus expression plasmid
With reorganization donor plasmid pFastBacHTA-VP2 Transformed E .coli DH10Bac competent cell (containing BacmidDNA and Helper plasmid), in the LB substratum 37 ℃, 220rpm, 4h does a series of dilutions (10 with LB
-1, 10
-2, 10
-3), coat on 1.5% agar plate of LB preparation and (contain 50 μ g/ml kantlex, 7 μ g/ml gentamicins, 10 μ g/ml tsiklomitsins and 100 μ g/ml X-gal and 40 μ g/ml IPTG), cultivate 16h for 37 ℃, choose white single bacterium colony, be inoculated in and contain three kinds of microbiotic (50 μ g/ml kantlex, 7 μ g/ml gentamicins, 10 μ g/ml tsiklomitsins) among the LB, 16h is cultivated in 37 ℃ of joltings, extract reorganization BacmidDNA with BAC/PAC DNA Isolation kit, carry out pcr amplification (93 ℃ of 3min with VP2 gene forward amplimer VP2F (36) and M13R (17) primer downstream sequence, 94 ℃ of 45s, 55 ℃ of 45s, 72 ℃ of 5min, 30 circulations, last 72 ℃ of 7min), the band that is about 2100bp with the visible size of 1% agarose gel electrophoresis, 2092bp conforms to theoretical value, illustrate that the successful swivel base of VP2 gene goes among the Bacmid DNA, obtain to contain the recombinant baculovirus expression plasmid pBac-VP2 (and wherein containing 6 Histidine (His) labels and marmor erodens (TEV) proteolytic enzyme recognition site) of VP2 gene.
2.4VP2 the acquisition of gene recombination baculovirus
In transfection the day before yesterday, in 6 porocyte culture plates, prepare monolayer cell with the Sf9 insect cell of logarithmic phase.To identify that good pBac-VP2 presses Lipofectamine2000 working instructions transfection Sf9 cell, during transfection, discard old substratum, and with Grace ' the s liquid washed cell of serum-free antibiotic-free, then with the pBac-VP2-Lipofectamine2000 mixture tiling Sf9 cell monolayer of hatching, 27 ℃, 5h discards the pBac-VP2-Lipofectamine2000 mixture, and every hole adds and contains 10% serum and antibiotic Grace ' s complete culture solution, putting 27 ℃ continues to cultivate, observe pathology every day, after 5 days, it is big that transfection hole visible part cell rounding becomes, cytopathy reached more than 90% in the 6th day, collect sick cell and supernatant, the centrifugal 10min of 1500rpm, supernatant liquor are the recombinant baculovirus vBac-VP2 stoste (P1 generation) that contains the VP2 gene, continue to go down to posterity 2 times, obtain P3 for recombinant baculovirus.It is contrast with transfection reagent transfection Sf9 cell that experiment is set up with Bacmid plasmid that does not insert exogenous genetic fragment and list synchronously.
2.5VP2 the evaluation of gene recombination baculovirus
2.5.1PCR identify
Get the Sf9 insect cell culture 500 μ l of the recombinant baculovirus vBac-VP2 infection that contains the VP2 gene, freeze thawing 3 times, add 5ul Proteinase K (20mg/ml) and 100ul 10%SDS, 42 ℃ of water-bath 1h, add isopyknic phenol chloroform extracting 1 time, 4 ℃, the centrifugal 5min of 12000rpm gets supernatant liquor, adds the 3mol/L NaAc of 1/10 volume and the dehydrated alcohol of 2.5 times of volumes in supernatant liquor, put-20 ℃, 6h, 4 ℃, the centrifugal 10min of 12000rpm abandons supernatant liquor, washes precipitation 1 time with 70% ethanol, dry, with 30ul pH8.0TE dissolving, carry out pcr amplification (93 ℃ of 3min, 94 ℃ of 45s with VP2 gene forward amplimer VP2F (36) and M13R (17) primer downstream sequence, 55 ℃ of 45s, 72 ℃ of 5min, 30 circulations, last 72 ℃ of 7min), be about the band of 2100bp with the visible size of 1% agarose gel electrophoresis, show and contain the VP2 gene among the recombinant baculovirus vBac-VP2.
2.5.2 indirect immunofluorescence assay (IFA) detects
Experiment is carried out in 24 porocyte culture plates.Recombinant baculovirus (P3 generation) is inoculated into the Sf9 cell, behind the cultivation 72h, inhales and abandon cell culture fluid, wash 2 times with the nutrient solution of serum-free, in the cell cultures hole, add the dehydrated alcohol 1ml/ hole of-20 ℃ of precoolings then, 4 ℃ of fixing 30min, wash 3 times with PBS, pat dry; Add the anti-IBDV hyper-immune serum of chicken of 40 times of dilutions, hatched 2 hours for 37 ℃ in 200 μ l/ holes, and PBS washing 5 times pats dry; Add the anti-chicken igg antibody of rabbit, 1h is hatched for 37 ℃ in 200 μ l/ holes, and PBS washing 5 times pats dry; Add the goat anti-rabbit igg antibody working fluid of the FITC mark of 50 times of dilutions, 1h is hatched for 37 ℃ in 200 μ l/ holes, and PBS washing 5 times places under the fluorescent microscope and observes.Set up baculovirus (Baculovirus) the infection Sf9 cell and the normal Sf9 cell of no VP2 gene to be contrast synchronously.Under fluorescent microscope, can see that infection P3 presents stronger fluorescence for the Sf9 insect cell of vBac-VP2 virus, the cell that infects wild-type baculovirus does not then have fluorescence with normal cell.Show that the VP2 gene is in expressed in insect cells.
2.5.3 sandwich ELISA detects
Dilute anti-IBDV chicken IgG to 5 μ g/ml with coating buffer, coated elisa plate then, 100 μ l/ holes, 4 ℃ are spent the night, PBST washing 5 times, each 5min pats dry; With the PBST sealing hydroful hole sealing that contains volume ratio 10%FBS, 37 ℃, 2h, PBST washing 5 times, each 5min pats dry; Add the good P4 of ultrasonication for cell, 100 μ l/ holes, 37 ℃, 2h, PBST washing 5 times, each 5min pats dry; The anti-IBDV chicken IgG that adds the HRP mark of dilution in 1: 1000,100 μ l/ holes, 37 ℃, 1h, PBST washing 5 times, each 5min pats dry; Colour developing.Feminine gender (normal Sf9 cell) and blank (PBST) contrast are set up in experiment synchronously.P4 is for vBac-VP2 virocyte ultrasonic degradation thing, antigen valence 1.6 * 10
3More than, show that the VP2 gene has obtained efficiently expressing in insect cell.Normal Sf9 cell and PBST control wells are then all negative.
2.5.4 immunoblotting assay (Western blotting)
With the Sf9 cell of P3 for the vBac-VP2 infection, behind twice of the Sf9 cell of the wild baculovirus infection that the Bacmid transfection produces and the normal Sf9 cell usefulness 0.01mol/L pH7.2PBS centrifuge washing, it is resuspended to press original volume 1% adding PBS, after three freeze thawing, add 5 * SDS-Loading Buffer, advance the SDS-PAGE electrophoretic analysis of 12% separation gel and 5% concentrated glue according to a conventional method, be transferred on the nitrocellulose filter, after transfer printing finishes, nitrocellulose filter is spent the night with 4 ℃ of sealings of 5% skim-milk, then with one anti-(the IBDV hyper-immune serum of chicken), two anti-(the anti-chicken igg antibodies of the rabbit of HRP mark) are effect with it respectively, the differential protein band is observed in the colour developing of DAB substrate.P3 is near vBac-VP2 virocyte culture visible differential protein band (3,4,6 swimming lanes among Fig. 2) 53kDa.
2.5.5 electron microscopic observation
Infect the Sf9 insect cell with P3 for vBac-VP2, treat after the complete pathology of cell, collecting cell, freeze thawing three times, 3000rpm, centrifugal 30min gets supernatant and observes with the film-making of negative staining electron microscope technology.With collecting sick cell with quadrat method, not freeze thawing, the centrifugal 30min sedimentation cell of 3000rpm, 2% glutaraldehyde stationary liquid is fixed, ultrathin section(ing), electron microscopic observation sets up normal Sf9 insect cell to do blank simultaneously.Can find virus-like particle at P3 in for the insect cell of vBac-VP2 virus infection, (Fig. 3 a) for the about 60nm of diameter; Sick cell with the vBac-VP2 virus infection prepares ultrathin section(ing), can find " inclusion body sample " structure (Fig. 3 b) that is formed in cell by virus-like particle.
2.6 the proteic purifying of recombinant VP 2
Get the cell culture that 300ml vBac-VP2 infects, centrifugal collecting cell, resuspended with 12ml binding buffer liquid, and add 20 μ l 100mmol/LPMSF, under the condition of ice bath ultrasonication limpid to cell suspension, 4 ℃, the centrifugal 30min of 10000rpm, remove cell debris, get supernatant with 0.45 μ m aperture membrane filtration, then according to HisTrap HP specification sheets purification of Recombinant VP2 albumen.The recombinant VP 2 albumen of purifying is behind SDS-PAGE, and BandScan5.0 analyzes with the gel images analysis software, and the proteic purity of recombinant VP 2 reaches (4 swimming lanes among Fig. 2) more than 90%.
2.7 the proteic application of recombinant VP 2
2.7.1IBDV the foundation of antibody indirect ELISA detection method
With recombinant VP 2 albumen to the 5 μ g/ml of the carbonate buffer solution of pH9.6 dilution purifying, bag is by elisa plate, 100 μ l/ holes, and 4 ℃ are spent the night; With PBST washing 3 times, each 2 minutes, pat dry; Every hole adds the PBST confining liquid 300 μ l that contain volume ratio 10%FBS, 37 ℃, 2h; With PBST washing 3 times, each 2 minutes, pat dry; Add serum sample to be checked, 37 ℃, 1h; With PBST washing 3 times, each 2 minutes, pat dry; The anti-chicken IgG of rabbit (1: 2000) the 100 μ l that add the HRP-mark, 37 ℃, 1h; With PBST washing 4 times, each 2 minutes, pat dry; Add each one of developer A liquid (tetramethyl biphenyl amine aqueous solution), B liquid (hydrogen peroxide urea solution) liquid, the magazine colour developing; 2M H
2SO
4Stop, survey the A450 value, result of determination.Chicken serum (positive serum) behind the immune 20d of IBD vaccine (B87 strain) detects hole A
450Value 〉=0.90, and the A of SPF chicken serum, bovine serum and PBST control wells
450Value is 0.00.
2.7.2IBD the preparation of virus-like particle recombinant vaccine; its method is: get the Sf9 insect cell culture that recombinant baculovirus vBac-VP2 infects; freeze thawing three times; detect with the IBDV antibody sandwich ELISA; ELISA tires 〉=and 1600; add isopyknic white oil immunological adjuvant, mixing and emulsifying is IBD virus-like particle recombinant vaccine.
2.7.3 the proteic immune challenge test of recombinant VP 2
100 2 week SPF chickens in age (available from Nanjing Tianbang Bio-industry Co., Ltd.) are divided into three groups at random: recombinant VP 2 albumen is with 60 of immunological adjuvant groups, with the Sf9 insect cell culture of recombinate shape virus infection (ELISA tire 〉=1600) and isopyknic white oil immunological adjuvant (available from Nanjing Tianbang Bio-industry Co., Ltd.) mixing and emulsifying, inject through chest muscle, 0.4ml/ only/time, carry out the 2nd immunity behind the 1st the immune 14d; Single immune programme for children is identical with the former with 20 of immunological adjuvant groups, an injecting immune adjuvant, 0.4ml//time; 20 of blank groups are not injected, and just raise under similarity condition with other two experimental group.Whole experimental chickens all when immunization experiment through the wing venous blood collection, and every interval 14d blood sampling once detects antibody after immunity.
For the first time behind the immune 14d, recombinant VP 2 albumen is 800 with the ELISA antibody titer of immunological adjuvant group, and single with immunological adjuvant group and blank group ELISA antibody titer all less than 100.For the second time behind the immune 14d; recombinant VP 2 albumen reaches more than 3200 with immunological adjuvant group ELISA antibody titer; adopt eye droppings this moment; collunarium; wipe (the BC6-85 strain of the strong poison of three kinds of approach artificial challenges of anus IBDV; available from China Veterinary Drugs Supervisory Inst.); attack only (20 times of Xi Shifashi lens capsule tissue suspensions of toxic agent amount 0.2ml/; it is 8000 that sandwich ELISA is tired); attack the poison back and observe 7d; recombinant VP 2 albumen is 100% (60/60) with the immune protective rate of immunological adjuvant group; and list detects antibody titer still less than 100 with immunological adjuvant group and blank group ELISA; it is all dead to attack malicious chicken; mortality ratio is 100% (40/40); the die of illness fabricius bursa tissue of chicken of inspection finds to have typical IBD pathological change: swelling; hemorrhage.
The usage of IBD virus-like particle recombinant vaccine: the chest muscle injection, 0.4ml//time, carry out the 2nd immunity behind the 1st the immune 14d.
Sequence table
＜110〉Jiangsu Province Agriculture Science Institute
＜120〉infectious bursal disease virus variant VP2 gene viruses sample particle recombinant protein
＜130〉specification sheets
<160>4
<170>PatentIn?version?3.1
<210>1
<211>36
<212>DNA
＜213〉synthetic
<220>
＜221〉VP2 gene amplification primer forward VP2F (36)
<222>(1)..(36)
<223>
<400>1
cagatctgaa?ttcatggcga?acctgcaaga?tcaaac 36
<210>2
<211>34
<212>DNA
＜213〉synthetic
<220>
＜221〉the reverse VP2R of VP2 gene amplification primer (34)
<222>(1)..(34)
<223>
<400>2
ctactacctc?cttatggccc?ggattatgtc?tttg 34
<210>3
<211>17
<212>DNA
＜213〉artificial
<220>
＜221〉primers designed M13F (17) downstream sequence
<222>(1)..(17)
<223>
<400>3
gttttcccag?tcacgac 17
<210>4
<211>17
<212>DNA
＜213〉artificial
<220>
＜221〉primers designed M13F (17) downstream sequence
<222>(1)..(17)
<223>
<400>4
caggaaacag?ctatgac 17
Claims (8)
1, infectious bursal disease virus variant AH1 VP2 gene viruses sample particle recombinant protein is that the recombinant baculovirus vBac-VP2 expression that obtains with the pBac-VP2 transfection Sf 9 insect cell obtains by structure recombinant baculovirus expression plasmid pBac-VP2.
2, infectious bursal disease virus variant VP2 gene viruses sample particle recombinant protein according to claim 1, the construction process of its recombinant baculovirus expression plasmid pBac-VP2 and recombinant baculovirus vBac-VP2 is: express reading frame design VP2 gene amplification primer according to VP2 gene order and donor plasmid pFastBacHTA:
Forward VP2F (36): 5 '-CAGATCTGAATTCATGGCGAACCTGCAAGATCAAAC-3 '
Reverse VP2R (34): 5 '-CTACTACCTCCTTATGGCCCGGATTATGTCTTTG-3 '
Recombinant baculovirus expression plasmid primers designed M13F (17):
Upstream sequence: 5 '-GTTTTCCCAGTCACGAC-3 '
Downstream sequence: 5 '-CAGGAAACAGCTATGAC-3 '
Cloned plasmids pUC-VP2 (AH1) and the donor plasmid pFastBacHTA that contains 6 Histidine His are used EcoR I and Hind III double digestion respectively, VP2 gene with IBDV variant AH1, directed cloning is gone among the donor plasmid pFastBacHTA of baculovirus expression system, make up reorganization donor plasmid pFastBacHTA-VP2, conversion contains the E.coli DH10Bac competence of Bacmid and Helper plasmid, identify Bacmid DNA with VP2 gene forward amplimer VP2F (36) and M13 R (17) primer downstream sequence PCR, the band that can to get a size be 2100bp, acquisition contains the recombinant baculovirus expression plasmid pBac-VP2 of VP2 gene, use the pBac-VP2 transfection Sf 9 insect cell, obtain recombinant baculovirus vBac-VP2.
3, infectious bursal disease virus variant VP2 gene viruses sample particle recombinant protein according to claim 1 and 2, it is characterized in that, described recombinant baculovirus expression plasmid pBac-VP2 and recombinant baculovirus vBac-VP2 contain 6 Histidine His labels and marmor erodens TEV proteolytic enzyme recognition site, are convenient to the proteic purifying of recombinant VP 2.
4, the application of the described infectious bursal disease virus variant of one of claim 1-3 VP2 gene viruses sample particle recombinant protein.
5, the purification process of the described infectious bursal disease virus variant of one of claim 1-3 VP2 gene viruses sample particle recombinant protein, comprise: infect the Sf9 insect cell with the 3rd generation recombinant baculovirus vBac-VP2, treat after the complete pathology of cell, the collecting cell culture, the centrifugal 30min collecting cell of 3000rpm, resuspended with HisTrap HP binding buffer liquid, and add 20 μ l 100mmol/L PMSF, place 0 ℃ of ice-water bath, ultrasonic power 500～600W, each ultrasonic time 10min, repeat 2～4 times ultrasonic, limpid to cell suspension, 4 ℃, the centrifugal 30min of 10000rpm removes cell debris, get supernatant with 0.45 μ m aperture membrane filtration, use HisTrap HP chromatography column purification of Recombinant VP2 albumen then to specifications.
6, detect the ELISA method of IBDV serum antibody with the described infectious bursal disease virus variant VP2 gene viruses sample particle recombinant protein of one of claim 1-3, comprise: recombinant VP 2 albumen to the 5 μ g/ml that dilutes purifying with the carbonate buffer solution of pH9.6, bag is by elisa plate, 100 μ l/ holes, 4 ℃ are spent the night; With PBST washing 3 times, each 2 minutes, pat dry; Every hole adds the PBST confining liquid 300 μ l that contain volume ratio 10%FBS, 37 ℃, 2h; With PBST washing 3 times, each 2 minutes, pat dry; Add serum sample to be checked, 37 ℃, 1h; With PBST washing 3 times, each 2 minutes, pat dry; The anti-chicken IgG100 of 1: the 2000 rabbit μ l that adds the HRP-mark, 37 ℃, 1h; With PBST washing 4 times, each 2 minutes, pat dry; Adding developer A liquid is that tetramethyl biphenyl amine aqueous solution, B liquid are each one of hydrogen peroxide urea solution, the magazine colour developing; 2M H
2SO
4Stop, survey A
450Value.
7, the virus-like particle recombinant vaccine of the described infectious bursal disease virus variant of one of claim 1-3 VP2 gene viruses sample particle recombinant protein; its preparation method is: get the Sf9 insect cell culture that recombinant baculovirus vBac-VP2 infects; freeze thawing three times; detect with the IBDV antibody sandwich ELISA; ELISA tires 〉=and 1600; add isopyknic white oil immunological adjuvant, mixing and emulsifying is IBD virus-like particle recombinant vaccine.
8, the application of the described virus-like particle recombinant vaccine of claim 7.
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| CN101792742A (en) * | 2010-04-07 | 2010-08-04 | 西北农林科技大学 | Recombinant baculovirus expressing IBDV (Infectious Bursal Disease Virus) immunogen gene and preparation method and application thereof |
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