CN104878043A - Orf virus virulence gene VIR-deleted (interferon-resistance gene deleted) mutant and its preparing method and application - Google Patents
Orf virus virulence gene VIR-deleted (interferon-resistance gene deleted) mutant and its preparing method and application Download PDFInfo
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Abstract
The invention relates to an orf virus virulence gene VIR-deleted (interferon-resistance gene deleted) mutant and its preparing method and application. The preparing method includes: cloning a gene segment containing an ORFV-SHZ1 VIR virulence gene and a virulence gene by means of PCR (polymerase chain reaction), deleting the VIR virulence gene by means of enzyme digestion, inserting an expression cassette carrying vaccinia virus late promoter P11 and a LacZ reporter gene into a deletion site, and building a recombinant shuttle vector using the LacZ gene as a selection marker; transfecting the recombinant shuttle vector to ORFV infected Vero cells to allow homologous recombination, and acquiring the VIR virulence gene deleted recombinant virus ORFV-DeltaVIR-LacZ by means of plaque screening and purifying. The mutant has the advantages that the ORFV virulence is lowered quickly by means of gene engineering, technical support is provided for the research and development of ORF vaccines, the preparing method features short production cycle, avoidance of domestication, operational convenience and quickness and the like, and the preparing method is widely applicable.
Description
Technical field
The present invention relates to genetically engineered for technical field of veterinary biology, be specifically related to a kind of sheep of virus attenuated vaccine and preparation method thereof.
Background technology
Sore mouth virus is also known as sheep infective warts (Contagious Ecthyma, CE), contagiousness impetiginous dermatitis (Contagious pustular dermatitis, CPD), sheep infective running sore (Ecthyma Contagiosum) etc., by sheep of virus (Orf virus, the contagious disease of a kind of sheep and goat ORFV), forms the thick scab of erythema, papule, tubercle, blister, warts, ulcer and excipuliform for feature with the skins such as lip, tongue, nose, breast and mucous membrane clinically.This disease is by immediate contagion, with the most susceptible of the lamb at 3 ~ 6 monthly ages, normal popular in mass-sending property; Deer, camel, musk-ox, people also can infect.The countries and regions that this disease is being concentrated sheep raising are widely current, and cause more serious financial loss to sheep husbandry.
Vaccine inoculation is the Main Means of this disease of prevention and control, conventional ORF deactivation vaccine and Attenuate vaccine play a role in the generation and epidemiological process of this disease of prevention, it can reduce the infection rate of disease to a certain extent, but existing conventional vaccine also exists certain defect.ORF deactivation vaccine uses the method for physics or chemistry by ORFV deactivation, make its feeling of loss metachromia and fecundity, although this vaccine security is high, stability is strong, but due to certain change that the antigenicity of virus in inactivation process occurs, duration of immunity is shorter, immune effect is not ideal enough, and the humoral immunoresponse(HI) of main induction body, can not the cellular immunization of inducing specific.
Attenuated vaccine ORFV virulent strain is carried out domestication by cell continuous passage obtain attenuation strain, and its advantage is that immunogenicity is strong, and body can be induced to produce strong humoral and cellular immune response, and immunizing power is comparatively lasting.As CN201110452835.7 discloses a kind of ORFV virulence weakening method, utilize bovine testicle cell system, cultivated by the continuous passage of sheep of virus, sheep of virus virulence is caused to cause weak, prepare to produce for sheep of virus attenuated vaccine and provide reliable and stable method, in order to obtain a large amount of stable vaccines.But its shortcoming is that the R&D cycle is long, domestication difficulty is high, is difficult to obtain the desirable attenuation strain of immunogenicity in the short period of time.In recent years, along with the appearance of ORFV virus variant, existing ORF vaccine makes it not provide to protect completely, thus causes frequently breaking out of sheep infective pustule.Therefore the method reducing ORFV virulence by the technique means research with outstanding advantage is fast necessary, for the research and development of ORF vaccine provide technical support.
Summary of the invention
The object of the invention is for shortcomings such as the existing domestication sheep of virus low virulent strain cycle are long, immunogenicity easily changes, gene engineering method is adopted to pass through sheep of virus virulence gene VIR (interferonresistance gene, the interferon-resistant factor) carry out lacking to realize its virulence and reduce fast, provide a kind of virulence of preparing significantly reduce but still keep good immunogenic attenuated vaccine preparation method.
The object of the invention is to implement by following technical solution:
A preparation method for sheep of virus virulence gene VIR deletion mutantion strain, concrete steps are as follows:
A, employing PCR method clone comprise the gene fragment of ORFV-SHZ1 strain VIR virulence gene and flanking sequence, VIR virulence gene is fallen by enzymatic cleavage methods disappearance, the expression cassette with vaccinia virus late promoter P11 and LacZ reporter gene is inserted, the recombinant shuttle vector that to build with LacZ gene be selection markers at deletion segment;
B, recombinant shuttle vector is transfected in Vero cell that ORFV infects and carries out homologous recombination, obtain the recombinant virus ORFV-Δ VIR-LacZ of VIR virulence gene disappearance through plaque screening purifying.
The sequence of described ORFV-SHZ1 strain VIR virulence gene is as shown in SED ID NO.1.
The gene fragment comprising ORFV-SHZ1 strain VIR virulence gene and flanking sequence described in steps A, when producing, preferred method Viral extraction test kit extracts ORFV-SHZ1 pnca gene group DNA, the sequence containing VIR virulence gene and upper underarm is gone out with P1-P2 primer amplification, reclaim pcr amplification product, it is connected with pMD18-T-simple carrier, obtain pVIR recombinant plasmid, wherein:
The sequence of P1 is: 5 '-ATCGTGAACACCAAGACCT-3 ',
The sequence of P2 is: 5 '-TTGTGCACGCTGCGCGC-3 '.
Described primer P1 sequence as shown in SED ID NO.2, primer P2 sequence is as shown in SED ID NO.2.
The gene order of the upper and lower homology arm of described VIR gene is as shown in SED ID NO.4.
In described steps A, enzymatic cleavage methods disappearance is fallen the preferred operations of VIR virulence gene and is: with Sph I and BglII (629-827bp) double digestion pVIR recombinant plasmid, carry out deletion mutantion to VIR gene.
Restriction enzyme XbaI is adopted to cut the gene fragment containing vaccinia virus late promoter P11 and reporter gene lacZ from pSC-11 shuttle vectors with the expression cassette of vaccinia virus late promoter P11 and LacZ reporter gene in described steps A.
Plaque screening purifying in step B adopts DMEM stoste to the dilution of homologous recombination poison, inoculation Vero cell, day by day transfectional cell is observed, after cytopathy to appear (CPE) and locus coeruleus, after preferred obvious cytopathy to be found and locus coeruleus, asepticly take locus coeruleus, multigelation, renewed vaccination Vero cell, after cytopathy to appear (CPE) and locus coeruleus, vaccinization Vero cell, obtains the recombinant virus ORFV-Δ VIR-LacZ of purifying.
Multigelation generally carries out being advisable for 2 ~ 6 times.
A kind of sheep of virus virulence gene VIR deletion mutantion strain, its TCID
50be 1 × 10
-3.5/ mL.
The present invention adopts gene engineering method to reduce fast by lacking to realize its virulence to sheep infective pustule virus VIR virulence gene, for preparation virulence significantly reduces but still keeps good immunogenic attenuated vaccine to provide a kind of fast and reliable preparation method.And with short production cycle, do not need domestication can obtain the desirable attenuation strain of immunogenicity.Even if ORFV virus variation, also can weaken virulence rapidly, cure sheep infective pustule.
Accompanying drawing explanation
To be that blue plaque that restructuring ORFV-Δ VIR-LacZ produces is aseptic dig out purification result figure to Fig. 1;
Fig. 2 is the recombinant virus CPE that produces on Vero cell of poison and blue plaque;
Fig. 3 is the growth curve of ORFV-Δ VIR-LacZ and ORFV-SHZ1 strain.
Embodiment
The invention provides a kind of construction process preparing sheep of virus VIR virulence gene gene-deleted strain, significantly reduce for the preparation of virulence but still keep good immunogenic attenuated vaccine Candidate Strain, comprising the following aspects content:
1, the cell cultures of ORFV
With Vero cell cultures ORFV SHZ1 epidemic strain, the DMEM liquid of growth media containing 8% calf serum, the DMEM liquid of maintenance medium containing 2% calf serum.Connect poison when Growth of Cells becomes individual layer, when typical CPE appears in 80% cell, receive poison.Multigelation three times, the centrifugal 10min of 5000rpm, gets supernatant as the material extracting STb gene.
2, the extraction of ORFV genomic dna
Get 200 μ L ORFV SHZ1 strain virus liquid respectively, add in 1.5mL centrifuge tube; Then in centrifuge tube, add the Solution A of 200 μ L, after concuss mixing, room temperature places 5min; Add the Solution B of 75 μ L again, after mixing, the centrifugal 5min of 12000rpm; Supernatant liquor is transferred in new Collection Tube (2mL provides in test kit), add 250 μ L Virahols (1 ﹪ glacial acetic acid), mixing of turning upside down; Be placed in by Spin Column in test kit on another new Collection Tube (2mL provides in test kit), be transferred to by above-mentioned supernatant liquor in Spin Column, the centrifugal 1min of 12000rpm, abandons filtrate; Be added in Spin Column by the Rinse A of 500 μ L, room temperature leaves standstill 1min, and the centrifugal 1min of 12000rpm, abandons filtrate; Be added in Spin Column by the Rinse B of 800 μ L, the centrifugal 1min of 12000rpm, abandons filtrate; Again carry out the centrifugal 1min of 12000rpm; Be placed in by Spin Column on the centrifuge tube of new 1.5mL, add sterile purified water or the Elution Buffer of 50 μ L in Spin Column film centre, room temperature leaves standstill 1min; 12000rpm centrifugal 1min wash-out viral DNA, the DNA collected is for next step operation or-20 DEG C of preservations.
3, the pcr amplification of VIR virulence gene and upper underarm and clone
According to ORFVSA00 pnca gene group sequence (AY386264) announced in GenBank, primer P1 (5 '-ATCGTGAACACCAAGACCT-3 ') and primer P2 (5 '-TTGTGCACGCTGCGCGC-3 '), with the pcr amplification product amplified containing VIR virulence gene and upper underarm, synthesized by Beijing Hua Da genome company.The primer of synthesis is carried out low temperature brief centrifugation, adds sterilizing distilled water and be made into 100pmol/uL.
Extract ORFV-SHZ1 pnca gene group DNA with Viral extraction test kit, carry out pcr amplification reaction as template.PCR reaction conditions is as follows: reaction system is: 10 × buffer (containing MgCl2) 2.0 μ L, 2.5mmol/LdNTP 0.6 μ L, the each 0.5 μ L of 20mmol/L upstream and downstream primer, DNA profiling 1 μ L, TaqDNA (2.5U/mL) polysaccharase 0.5 μ L, supplies volume to 20 μ L with sterile purified water.Reaction conditions is: 95 DEG C of denaturation 5min, 94 DEG C of sex change 50s, 61 DEG C/62 DEG C/60 DEG C/59 DEG C/54 DEG C annealing 40s, and 72 DEG C extend 120s, totally 35 circulations, and last 72 DEG C extend 10min.Obtaining clip size is the amplified production of 1550bp, conforms to the DNA fragmentation length of expection.Reclaim pcr amplification product, be cloned into pMD18-T-simple carrier and obtain pVIR recombinant plasmid.PVIR is checked order, confirms that successful clone obtains VIR virulence gene and upper and lower arm pieces section.
4, the structure of shuttle vectors p Δ VIR-LacZ and qualification
4.1p11-LacZ the acquisition of gene fragment
Cut gene fragment containing vaccinia virus late promoter P11 and reporter gene lacZ with restriction enzyme XbaI from pSC11 shuttle vectors, carry out gel electrophoresis and reclaim object fragment (3845bp), and two ends cohesive terminus is filled.
The structure of 4.2 shuttle vectors p Δ VIR-LacZ
With Sph I and BglII (629-827bp) double digestion pVIR recombinant plasmid, deletion mutantion is carried out to VIR gene, reclaim large fragment, and carry out two ends cohesive terminus and fill.Be connected containing p11 promotor with pVIR large fragment with the gene fragment of LacZ, obtain recombinant shuttle vector p Δ VIR-LacZ.Verified by PCR and order-checking p Δ VIR-LacZ.
The Molecular Identification of 4.3 shuttle vectors p Δ VIR-LacZ
The shuttle vectors p Δ VIR-LacZ single endonuclease digestion built can obtain the fragment of 7835bp, completes conform to expection size.The product of 2400bp can be amplified with LacZ primer, show that shuttle vectors p Δ VIR-LacZ builds correct.
5, shuttle vectors p Δ VIR-LacZ transfection
5.1Vero monolayer cell culture
Vero cell passes in six orifice plates, every hole 2mL, for subsequent use when cell 70%-80% is laid at the bottom of plate.
5.2Vero cell inoculation vaccinia virus
By ORFV virus liquid: ORFV is inoculated 6 orifice plate Vero cells by cell culture fluid=1:10 volume ratio, every hole 200 μ L.37 DEG C of absorption 2h, then inhale and abandon virus liquid, add containing 2% calf serum DMEM nutrient solution, 37 DEG C, 5%CO
24-6h cultivated by incubator.Use the liposome transfection Vero cell of Invitrogen company again.
5.3 liposome-mediated Transfected Recombinant Plasmid Vero cells
Preparation A liquid and B liquid.A liquid is that DMEM stoste adds 4 μ L liposomes, and B liquid is that DMEM stoste adds 1.6 μ g recombinant plasmids.A liquid, B liquid respectively prepare 130 μ L, and A liquid is dropwise added B liquid, mixes gently, make transfection liquid, totally 260 μ L after placing 5min clock by room temperature, and room temperature places more than 30min.Transfection liquid dropwise inoculating cell, every hole meets 250 μ L, connects 5 holes altogether, and negative control is done in the 6th hole.Then at 37 DEG C, 5%CO
2cultivate 4-6h in incubator, inhale afterwards and abandon supernatant, add containing 2% calf serum DMEM nutrient solution, at 37 DEG C, 5%CO
2cultivate in incubator, observe transfectional cell day by day, after cytopathy to appear (CPE) and backboard, multigelation 3 times, collects culture, centrifugally removes cell debris, preserve the stoste of supernatant as vaccinia virus recombinant, for screening purification of Recombinant bovine vaccine poison.Day by day the change of every hole is observed.The blue plaque of picking.After locus coeruleus occurs,
6, the plaque purification of recombinant virus and qualification
The purifying of 6.1 recombinant virus ORFV-Δ VIR-LacZ
Do 10 times of doubling dilutions by DMEM stoste to restructuring poison, general dilution gradient is 10
-1to 10
-6, each extent of dilution connects holes.Vero cell is inoculated the restructuring poison of different gradient, and every hole adds 200 μ L viral dilution liquid respectively, and control group cell culture fluid replaces, and then puts 37 DEG C of 5%CO
2absorption 1h is cultivated in incubator.With Vero cell in PBS wash buffer plate, wash the virus of not adsorbing off.2 × DMEM the nutrient solution got containing 16% calf serum adds in equivalent 3% agar (keeping 37 DEG C), mixing.Every hole adds this mixture of 2mL, and horizontalization platform, after agar cooled and solidified, puts 37 DEG C of 5%CO
2cultivate 2 days in incubator.2 × DMEM the nutrient solution got containing 16% calf serum adds in equivalent 3% agar (keeping 37-40 DEG C), then lucifuge adds X-gal solution, 20 μ L/mL.After above admixture mixing, every hole adds 1mL, sets level platform after agar cooled and solidified, at 37 DEG C of 5%CO
2cultivate in incubator, observe the change of every hole day by day.After locus coeruleus occurs, choose obviously the most concentrated several locus coeruleus pickings (Fig. 1), the locus coeruleus of picking is received in preprepared Vero Tissue Culture Flask and breed, for next purifying is prepared.Take turns plaque purification through 5, filter out purer recombinant virus.
The PCR qualification of 6.2 recombinant virus ORFV-Δ VIR-LacZ
Collect the 5th generation purifying recombinant virus ORFV-Δ VIR-LacZ and the culture of ORFV-SHZ1 strain, extract viral DNA respectively, carry out PCR qualification with primer P1-P2.ORFV-SHZ1 amplified band size is 1550bp, and recombinant virus ORFV-Δ VIR-LacZ amplifies object stripe size is 5145bp.
6.3 the pcr amplification product sequence verification of recombinant virus ORFV-Δ VIR-LacZ
Sequencing result shows, and the sequence of primer P5, P6 amplification contains P11 promoter sequence and the part LacZ gene order of homology arm gene order and insertion on part VIR virulence gene; The sequence of primer P7, P8 amplification to contain under VIR virulence gene homology arm gene order and inserts gene order.
7, recombinant virus ORFV-Δ VIR-LacZ virulence determination
7.1 recombinant virus ORFV-Δ VIR-LacZ and parent's strain TCID
50measure
ORFV-Δ VIR-LacZ and ORFV SHZ1 strain are inoculated Vero cell respectively, occurs that cytopathy measures TCID after receiving poison
50.Result shows, the TCID of parental virus ORFV-SHZ1
50be 1 × 10
-5.5the TCID of/mL, ORFV-Δ VIR-LacZ gene-deleted strain
50be 1 × 10
-3.5/ mL.Compared with parent ORFV, ORFV-Δ VIR-LacZ virus titer obviously reduces (P<0.05), has significant impact after VIR virulence gene disappearance is described on the propagation of ORFV on cell.
7.2 recombinant virus ORFV-Δ VIR-LacZ are in Vero cell growth characteristics
ORFV-Δ VIR-LacZ and ORFV-SHZ1 strain are inoculated Vero cell respectively, measures TCID every 6h results virus
50, draw growth curve.Result ORFV-Δ VIR-LacZ is similar with the growth curve of ORFV-SHZ1 strain, but virus titer reduces about 2 orders of magnitude (as shown in Figure 3), and this shows that the disappearance of VIR virulence gene significantly affects the propagation of ORFV.
The pathogenic detection of 7.3 recombinant virus ORFV-Δ VIR-LacZ
Select healthy 2 the monthly age lamb 9, be divided into I ~ III group, often organize 3.With recombinant virus ORFV-Δ VIR-LacZ and the ORFV-SHZ1 strain of Vero cell cultures, then use virus liquid the lamb of lip scratch inoculation I group and II group respectively, often only inoculate 0.5mL (1 × 10
-5.5tCID
50/ 0.1mL), control group (III group) inoculates aseptic PBS 0.5mL in the same way simultaneously, isolated rearing respectively, carries out 10d observation after inoculation to inoculation site morbidity.Result occurs only having occurred erythema at its position such as bicker, lip at recombinant virus ORFV-Δ VIR-LacZ after inoculation for 1 week, and papule, warts have appearred in ORFV-SHZ1 strain after inoculation lamb lip; Control group has no any pathology simultaneously.
8, recombinant virus ORFV-Δ VIR-LacZ genetic stability and the immunogenicity of expressing LacZ gene detect
The genetic stability of 8.1 recombinant virus ORFV-Δ VIR-LacZ
By recombinant virus ORFV-Δ VIR-LacZ continuous passage on Vero cell correct for qualification, get F1, F5, F10, F15 generation virus LacZ primer respectively and carry out PCR qualification, all can amplify object band, show that this recombinant virus has good genetic stability; The malicious valency in recombinant virus F1, F5, F10, F15 generation is respectively 1 × 10
-5.5/ mL, 1 × 10
-5.3/ mL, 1 × 10
-5.6/ mL and 1 × 10
-5.5/ mL.
8.2 recombinant virus ORFV-Δ VIR-LacZ immunogenicities detect
Select healthy 2 the monthly age lamb 24, be divided into I ~ III group, often organize 8.With Vero cell cultures recombinant virus ORFV-Δ VIR-LacZ, by recombinant virus respectively with 1 × 10
-5.5tCID
50/ 0.1mL and 5 × 10
-4.5tCID
50the dosage of/0.1mL, the lamb of lip scratch inoculation I group and II group, often only inoculates 0.5mL; Establish Normal group (III group), control group inoculates aseptic PBS 0.5mL in the same way simultaneously, and isolated rearing respectively, detects lamb cellular immune level, humoral immunity level after inoculation.
Immunity lamb cellular immune level detects (spleen lymphocyte proliferation test): 10d after immunity, and often group gets 2 lambs, asepticly after putting to death gets its spleen and is separated splenic lymphocyte, carries out ion vitro immunization and tests.The concrete operation step that splenic lymphocyte is separated is as follows: (1) takes a blood sample, and separation of serum, puts-20 DEG C of preservations; (2) put to death after lamb, asepticly get its spleen, be placed in the RPMI-1640 of 3mL containing 2% foetal calf serum, with the inner core grinding spleen of sterilizing syringe, use 100 order sterile nylon net filtrations afterwards, collect splenic lymphocyte suspension; (3) adjusting cell concns with 1640 perfect mediums containing 10% foetal calf serum is 1 × 10
7individual/mL; (4) seed cells in 96 porocyte culture plates by every hole 100 μ L, and set ConA and stimulate hole (final concentration of ConA is 5 μ g/mL), do not add the control wells of stimulator, every hole is established 3 to repeat and is set 3 nutrient solutions not adding cell as blank well.(5), after the mixing of each hole mixture, 37 DEG C of CO are positioned over
2cultivate 48h in cell culture incubator, before cultivation terminates, 4h adds 10 μ L MTT (5g/L), and then puts back to continuation cultivation 4h in incubator; (6) after cultivation terminates, supernatant discarded, every hole adds the DMSO of 100 μ L, and after vibration mixing, room temperature places 10min; (7) microplate reader measures the OD value in each hole, 570nm place.Result is as shown in table 1, and recombinant virus immune group (the Ith and II group) stimulates specific antigens ORFV and shows obvious T lymphopoiesis ability, OD
570nmvalue is between 0.41 ~ 0.51; To heterogenetic antigen ConA stimulate index of Response between 0.45 ~ 0.58, stimulate higher than ORFV; And control group (III group) does not show obvious lymphproliferation response, its OD to ORFV stimulation
570nmvalue is only 0.10 ~ 0.14.
Immunity lamb humoral immunity level detects: measured lamb serum neutralizing antibody by the method for fixed virus dilute serum: the mensuration of (1) viral median tissue cell infection amount: calculate viral TCID according to Reed-MuenchShi method
50value, then becomes 200 TCID by viral dilution
50, after mixing with equivalent serum, each dosage of inoculation contains 100 TCID
50.(2) process of serum to be checked: serum to be checked is put deactivation 30min in 56 DEG C of water-baths, to destroy complement and other heat labile non-specific molecules that kills the virus, then carries out continuous 2 times of dilutions with DMEM nutrient solution by serum to be checked.(3) sense is done: get quantitative ORFV and isopyknic serum to be checked, is placed in 2mL EP and manages interior fully mixing, 37 DEG C, feel work 1 ~ 2h in 5%CO2 incubator.(4) inoculate: the virus inoculation that sense is done to terminate is in the 96 porocyte culture plates being paved with 80%Vero, and each extent of dilution does 3 repetitions, and each hole inoculum size is 10 ~ 20 μ L.Be placed in 37 DEG C, 5%CO
2after hatching 1 ~ 2h in incubator, with containing 5% calf serum with after changing liquid containing 1% dual anti-DMEM, be again placed in 37 DEG C, 5%CO
2cultivate in incubator.(5) according to Reed-MuenchShi method calculation result.Using the NAT of most high dilution as serum to be checked that the cell of 50% can be protected not produce cytopathy reaction.The NAT of recombinant virus ORFV-Δ VIR-LacZ immune group lamb serum will apparently higher than control group as can be seen from Table 2.
Table 1 recombinant virus ORFV-Δ VIR-LacZ immunity lamb splenic lymphocyte is through the post-stimulatory OD of ORFV and ConA
570nmvalue (X ± SD)
Table 2 recombinant virus ORFV-Δ VIR-LacZ immunity lamb serum ORFV specificity neutralizing antibody detects
| After immunity | I group | II group | III group |
| 2nd week | 1:32 | 1:64 | 1:2 |
| 4th week | 1:64 | 1:128 | 1:4 |
| 6th week | 1:64 | 1:128 | 1:4 |
| 8th week | 1:32 | 1:64 | 1:4 |
Claims (7)
1. a preparation method for sheep of virus virulence gene VIR deletion mutantion strain, concrete steps are as follows:
A, employing PCR method clone comprise the gene fragment of ORFV-SHZ1 strain VIR virulence gene and flanking sequence, VIR virulence gene is fallen by enzymatic cleavage methods disappearance, the expression cassette with vaccinia virus late promoter P11 and LacZ reporter gene is inserted, the recombinant shuttle vector that to build with LacZ gene be selection markers at deletion segment;
B, recombinant shuttle vector is transfected in Vero cell that ORFV infects and carries out homologous recombination, obtain the recombinant virus ORFV-Δ VIR-LacZ of VIR virulence gene disappearance through plaque screening purifying.
2. the preparation method of sheep of virus virulence gene VIR deletion mutantion strain according to claim 1, it is characterized in that, the gene fragment comprising ORFV-SHZ1 strain VIR virulence gene and flanking sequence described in steps A, preparation method Viral extraction test kit extracts ORFV-SHZ1 pnca gene group DNA, go out the sequence containing VIR virulence gene and upper underarm with P1-P2 primer amplification, reclaim pcr amplification product, it is connected with pMD18-T-simple carrier, obtain pVIR recombinant plasmid, wherein:
The sequence of P1 is: 5 '-ATCGTGAACACCAAGACCT-3 ',
The sequence of P2 is: 5 '-TTGTGCACGCTGCGCGC-3 '
3. the preparation method of sheep of virus virulence gene VIR deletion mutantion strain according to claim 1 and 2, it is characterized in that, in described steps A, enzymatic cleavage methods disappearance is fallen the preferred operations of VIR virulence gene and is: with Sph I and BglII double digestion pVIR recombinant plasmid, carry out deletion mutantion to VIR gene.
4. the preparation method of the sheep of virus virulence gene VIR deletion mutantion strain according to any one of claim 1-3, it is characterized in that, in described steps A, adopt restriction enzyme XbaI to cut the gene fragment containing vaccinia virus late promoter P11 and reporter gene lacZ from pSC-11 shuttle vectors with the expression cassette of vaccinia virus late promoter P11 and LacZ reporter gene.
5. the preparation method of the sheep of virus virulence gene VIR deletion mutantion strain according to any one of claim 1-4, it is characterized in that, plaque screening purifying in step B adopts DMEM stoste to the dilution of homologous recombination poison, inoculation Vero cell, asepticly takes locus coeruleus, multigelation, renewed vaccination Vero cell, after cytopathy to appear and locus coeruleus, vaccinization Vero cell, obtains the recombinant virus ORFV-Δ VIR-LacZ of purifying.
6. a sheep of virus virulence gene VIR deletion mutantion strain, is characterized in that, its TCID
50be 1 × 10
-3.5/ mL.
7. an application for sheep of virus virulence gene VIR deletion mutantion strain, is characterized in that sheep of virus virulence gene VIR deletion mutantion strain according to claim 6 to be applied in prepare in attenuated vaccine.
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| CN112522215A (en) * | 2019-09-18 | 2021-03-19 | 重庆市畜牧科学院 | 008 gene-deleted recombinant orf virus and construction method thereof |
| WO2022033573A1 (en) | 2020-08-13 | 2022-02-17 | 苏州般若生物科技有限公司 | Mutant ovis spp. infectious pustular dermatitis virus and use thereof |
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| G. DELHON等: "Genomes of the Parapoxviruses Orf Virus and Bovine Papular Stomatitis Virus", 《JOURNAL OF VIROLOGY》 * |
| MATHIAS MARTINS等: "Pathogenesis in lambs and sequence analysis of putative virulence genes of Brazilian orf virus isolates", 《VETERINARY MICROBIOLOGY》 * |
| 李慧霞等: "羊口疮病的研究进展", 《中国预防兽医学报》 * |
| 李杰等: "羊口疮病毒B2L和VIR基因原核表达及抗原性鉴定", 《动物医学进展》 * |
| 杨海波: "新疆石河子地区羊传染性服拖病毒的分离鉴定及其119 基因缺失株研究", 《中国优秀硕士学位论文全文数据库 农业科技辑》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112522215A (en) * | 2019-09-18 | 2021-03-19 | 重庆市畜牧科学院 | 008 gene-deleted recombinant orf virus and construction method thereof |
| CN112522215B (en) * | 2019-09-18 | 2023-06-02 | 重庆市畜牧科学院 | Recombinant orf virus lacking 008 gene and construction method thereof |
| WO2022033573A1 (en) | 2020-08-13 | 2022-02-17 | 苏州般若生物科技有限公司 | Mutant ovis spp. infectious pustular dermatitis virus and use thereof |
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| CN104878043B (en) | 2017-12-05 |
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