CN104878043B - Sheep of virus virulence gene VIR deletion mutation strains and its preparation method and application - Google Patents
Sheep of virus virulence gene VIR deletion mutation strains and its preparation method and application Download PDFInfo
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Abstract
The present invention relates to a kind of sheep of virus virulence gene VIR deletion mutation strains and its preparation method and application, preparation method, using genetic fragment of the PCR method clone comprising ORFV SHZ1 strain VIR virulence genes and flanking sequence, VIR virulence genes are missed out with enzymatic cleavage methods, in expression cassette of the deletion segment insertion with vaccinia virus late promoter P11 and LacZ reporter gene, the recombinant shuttle vector using LacZ genes as selection markers is built；Recombinant shuttle vector is transfected into the Vero cells of ORFV infection and carries out homologous recombination, the recombinant virus ORFV Δ VIR LacZ for obtaining VIR virulence genes missing are purified through plaque screening.The present invention ORFV virulence is quickly reduced by genetic engineering means, the research and development for ORF vaccines provide technical support, have the characteristics that it is with short production cycle, need not tame, be easy to operate, can be widely used.
Description
Technical field
The present invention relates to genetic engineering to be used for technical field of veterinary biology, and in particular 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., be by
The contagious disease of a kind of sheep and goat caused by sheep of virus (Orf virus, ORFV), clinically with mouth
The skins such as lip, tongue, nose, breast and mucous membrane form erythema, papule, tubercle, blister, warts, ulcer and excipuliform thickness scab and are characterized.Should
Disease is by immediate contagion, and most susceptible with the lamb at 3~6 monthly ages, normal is in that mass-sending property is popular；Deer, camel, musk-ox, people also may be used
Infection.The disease is widely current in the countries and regions that sheep raising is concentrated, and more serious economic loss is caused to sheep husbandry.
Vaccine inoculation is that the prevention and control sick Main Means, conventional ORF inactivated vaccines and Attenuate vaccine are preventing the sick generation
Played a role with epidemiological process, it can reduce the infection rate of disease, but existing conventional vaccine to a certain extent
In the presence of it is certain the defects of.ORF inactivated vaccines are to be inactivated ORFV with physics or chemical method, it is lost infectious and breeding
Ability, this vaccine is although safe, stability is strong, but due in inactivation process viral antigenicity occur it is certain
Change, duration of immunity is shorter, and immune effect is not ideal enough, and the humoral immune response of main induction body, it is impossible to inducing specific
Cellular immunity.
Attenuated vaccine is that ORFV velogen strains are carried out into domestication by cell continuous passage to obtain attenuation strain, its advantage
It is that immunogenicity is strong, can induce body and produce strong humoral and cellular immune response, immunity is more lasting.Such as
CN201110452835.7 discloses a kind of ORFV virulence weakening method, using bovine testicle cell system, passes through sheep
The continuous passage culture of blue tongue virus, sheep of virus virulence is caused to cause weak, preparing production for sheep of virus attenuated vaccine carries
Reliable and stable method is supplied, to obtain largely stable vaccine.But its shortcoming is R＆D cycle length, and domestication difficulty is high,
It is difficult to obtaining immunogenicity in the short time is preferably attenuated strain.In recent years, it is existing with the appearance of ORFV virus variants
ORF vaccines prevent it from providing complete protection, so as to cause frequently breaking out for sheep infective pustule.It is therefore desirable to by tool
The method that the technological means research for having outstanding advantage quickly reduces ORFV virulence, the research and development for ORF vaccines provide technical support.
The content of the invention
The purpose of the present invention is easily changed for length of existing domestication sheep of virus low virulent strain cycle, immunogenicity
The shortcomings of change, passed through using gene engineering method to sheep of virus virulence gene VIR (interferon resistance
Gene, interferon resistance factor) lacked to realize that its virulence quickly reduces, there is provided one kind prepare virulence significantly reduce but still
Keep the attenuated vaccine preparation method of good immunogenicity.
The purpose of the present invention is implemented by following technical solution：
A kind of preparation method of sheep of virus virulence gene VIR deletion mutation strains, is comprised the following steps that：
A, using genetic fragment of the PCR method clone comprising ORFV-SHZ1 strain VIR virulence genes and flanking sequence, enzyme is used
Blanking method misses out VIR virulence genes, and vaccinia virus late promoter P11 and LacZ reporter gene is carried in deletion segment insertion
Expression cassette, build the recombinant shuttle vector using LacZ genes as selection markers；
B, recombinant shuttle vector is transfected into the Vero cells of ORFV infection and carries out homologous recombination, purified through plaque screening
Obtain the recombinant virus ORFV- Δs VIR-LacZ of VIR virulence genes missing.
The sequence of the ORFV-SHZ1 strains VIR virulence genes is as shown in SED ID NO.1.
The genetic fragment comprising ORFV-SHZ1 strain VIR virulence genes and flanking sequence described in step A, it is preferred when producing
Method with Viral extraction kit extract ORFV-SHZ1 pnca gene group DNA, with P1-P2 primers amplify virulence gene containing VIR and
The sequence of upper underarm, pcr amplification product is reclaimed, it is connected with pMD18-T-simple carriers, obtain pVIR recombinant plasmids, its
In：
P1 sequence is：5’-ATCGTGAACACCAAGACCT-3’,
P2 sequence is：5’-TTGTGCACGCTGCGCGC-3’.
The primer P1 sequences are as shown in SED ID NO.2, primer P2 sequences are as shown in SED ID NO.2.
The gene order of homology arm is as shown in SED ID NO.4 above and below the VIR genes.
The preferred operations that enzymatic cleavage methods miss out VIR virulence genes in the step A are：With Sph I and BglII (629-
827bp) double digestion pVIR recombinant plasmids, deletion mutation is carried out to VIR genes.
The expression cassette with vaccinia virus late promoter P11 and LacZ reporter gene is using restricted in the step A
Restriction endonuclease XbaI cuts the gene containing vaccinia virus late promoter P11 and reporter gene lacZ from pSC-11 shuttle vectors
Fragment.
Plaque screening purifying in step B is diluted using DMEM stostes to homologous recombination poison, is inoculated with Vero cells, is seen day by day
Examine transfectional cell, it is sterile after obvious cytopathy preferably to be found and locus coeruleus after cytopathy (CPE) to appear and locus coeruleus
Take locus coeruleus, multigelation, renewed vaccination Vero cells, after cytopathy (CPE) to appear and locus coeruleus, vaccinization Vero is thin
Born of the same parents, the recombinant virus ORFV- Δs VIR-LacZ purified.
Multigelation typically be advisable for 2~6 times.
A kind of sheep of virus virulence gene VIR deletion mutation strains, its TCID50For 1 × 10-3.5/mL。
The present invention uses gene engineering method by being lacked sheep infective pustule virus VIR virulence genes to realize
Its virulence quickly reduces, and is significantly reduced for preparation virulence but still keeps the attenuated vaccine of good immunogenicity to provide one kind quickly
Reliable preparation method.And it is with short production cycle, need not tame and can obtain immunogenicity and be preferably attenuated strain.Even if
ORFV virus variations, virulence can also be weakened rapidly, cure sheep infective pustule.
Brief description of the drawings
Fig. 1 digs out purification result figure for blue plaque is sterile caused by restructuring ORFV- Δs VIR-LacZ；
Fig. 2 is recombinant virus poison caused CPE and blue plaque on Vero cells；
Fig. 3 is the growth curve of ORFV- Δ VIR-LacZ and ORFV-SHZ1 strains.
Embodiment
The present invention provides a kind of construction method for preparing sheep of virus VIR virulence gene gene-deleted strains, for preparing virulence
Significantly reduce but still keep the attenuated vaccine Candidate Strain of good immunogenicity, including the following aspects content：
1st, ORFV cell culture
With Vero cell culture ORFV SHZ1 epidemic strains, DMEM liquid of the growth-promoting media containing 8% calf serum, maintaining liquid is used
DMEM liquid containing 2% calf serum.Poison is connect when cell growth is into individual layer, poison is received when typical CPE occurs in 80% cell.Instead
Multiple freeze thawing three times, 5000rpm centrifugation 10min, takes material of the supernatant as extraction STb gene.
2nd, the extraction of ORFV genomic DNAs
200 μ L ORFV SHZ1 strain virus liquid are taken respectively, are added in 1.5mL centrifuge tubes；Then 200 are added into centrifuge tube
μ L Solution A, after acutely concussion mixes, room temperature places 5min；75 μ L Solution B are added, after being well mixed,
12000rpm centrifuges 5min；Supernatant is transferred in new Collection Tube (2mL, kit in provide), adds 250 μ
L isopropanols (1 ﹪ glacial acetic acids), mixing of turning upside down；Spin Column in kit are placed in another new
On Collection Tube (2mL, kit in provide), above-mentioned supernatant is transferred in Spin Column, 12000rpm
1min is centrifuged, abandons filtrate；500 μ L Rinse A are added into Spin Column, are stored at room temperature 1min, 12000rpm centrifugations
1min, abandon filtrate；800 μ L Rinse B are added into Spin Column, 12000rpm centrifugation 1min, abandon filtrate；Again
Carry out 12000rpm centrifugations 1min；Spin Column are placed on new 1.5mL centrifuge tube, in Spin Column films
50 μ L sterile purified water or Elution Buffer is added at centre, is stored at room temperature 1min；12000rpm centrifugation 1min elution diseases
Malicious DNA, the DNA being collected into are used to operate in next step or -20 DEG C of preservations.
3rd, the PCR of VIR virulence genes and upper underarm amplifications and clone
According to the ORFVSA00 pnca gene group sequences (AY386264) announced in GenBank, primer P1 (5 '-
ATCGTGAACACCAAGACCT-3 ') and primer P2 (5 '-TTGTGCACGCTGCGCGC-3 '), with amplifying the base of virulence containing VIR
The pcr amplification product of cause and upper underarm, is synthesized by Beijing Huada gene company.The primer of synthesis is subjected to low temperature brief centrifugation,
Add sterilizing distilled water and be made into 100pmol/uL.
ORFV-SHZ1 pnca gene group DNA are extracted with Viral extraction kit, pcr amplification reaction is carried out as template.
PCR reaction conditions are as follows：Reaction system is:The μ L of 10 × buffer (containing MgCl2) 2.0 μ L, 2.5mmol/L dNTP 0.6,
Each 0.5 μ L of 20mmol/L upstream and downstream primers, the μ L of DNA profiling 1 μ L, TaqDNA (2.5U/mL) polymerase 0.5, use sterile purified water
Volume is supplied to 20 μ L.Reaction condition is:95 DEG C of pre-degeneration 5min, 94 DEG C of denaturation 50s, 61 DEG C/62 DEG C/60 DEG C/59 DEG C/54 DEG C
Anneal 40s, 72 DEG C of extension 120s, totally 35 circulations, last 72 DEG C of extensions 10min.Obtain the amplification that clip size is 1550bp
Product, it is consistent with expected DNA fragmentation length.Pcr amplification product is reclaimed, pMD18-T-simple carriers is cloned into and obtained
PVIR recombinant plasmids.PVIR is sequenced, it was demonstrated that successful clone obtains VIR virulence genes and upper and lower arm pieces section.
4th, shuttle vector p Δs VIR-LacZ structure and identification
4.1p11-LacZ the acquisition of genetic fragment
Cut with restriction enzyme XbaI from pSC11 shuttle vectors containing vaccinia virus late promoter P11 and report
Gene lacZ genetic fragment is accused, carries out gel electrophoresis recovery purpose fragment (3845bp), and both ends cohesive terminus,cohesive termini is mended
It is flat.
4.2 shuttle vector p Δs VIR-LacZ structure
With Sph I and BglII (629-827bp) double digestion pVIR recombinant plasmids, deletion mutation is carried out to VIR genes, returned
Large fragment is received, and carries out both ends cohesive terminus,cohesive termini filling-in.Genetic fragment containing p11 promoters and LacZ and pVIR large fragments are connected
Connect, obtain recombinant shuttle vector p Δs VIR-LacZ.Verified by PCR and sequencing p Δs VIR-LacZ.
4.3 shuttle vector p Δs VIR-LacZ Molecular Identification
The shuttle vector p Δ VIR-LacZ single endonuclease digestions built can obtain 7835bp fragment, and phase is completed with expected size
Symbol.2400bp product can be amplified with LacZ primers, shows that shuttle vector p Δs VIR-LacZ structures are correct.
5th, shuttle vector p Δs VIR-LacZ is transfected
5.1Vero monolayer cell culture
Vero cells are passed in six orifice plates, standby when cell 70%-80% is laid on plate bottom per hole 2mL.
5.2Vero cells are inoculated with vaccinia virus
By ORFV virus liquids：Cell culture fluid=1:ORFV is inoculated with 6 orifice plate Vero cells by 10 volume ratios, per the μ L of hole 200.
37 DEG C of absorption 2h, then inhale and abandon virus liquid, add and contain 2% calf serum DMEM nutrient solutions, 37 DEG C, 5%CO2Incubator culture 4-
6h.The liposome transfection Vero cells of Invitrogen companies are used again.
5.3 liposome-mediated Transfected Recombinant Plasmid Vero cells
Prepare A liquid and B liquid.A liquid is that DMEM stostes add 4 μ L liposomes, and B liquid is that DMEM stostes add 1.6 μ g recombinant plasmids.A
Liquid, B liquid respectively prepare 130 μ L, and after room temperature places 5min clocks, A liquid is added dropwise into B liquid, are gently mixed uniformly, transfection liquid is made,
Totally 260 μ L, room temperature place more than 30min.Inoculating cell, every hole meet 250 μ L, connect 5 holes altogether, feminine gender is done in the 6th hole transfection liquid dropwise
Control.Then at 37 DEG C, 5%CO24-6h is cultivated in incubator, inhales abandon supernatant afterwards, add and cultivated containing 2% calf serum DMEM
Liquid, at 37 DEG C, 5%CO2Cultivated in incubator, observe transfectional cell day by day, after cytopathy (CPE) to appear and backboard, instead
Freeze thawing 3 times again, culture is collected, cell fragment is removed in centrifugation, stoste of the supernatant as vaccinia virus recombinant is preserved, for screening
Purification of Recombinant bovine vaccine poison.Day by day observe and change per hole.Picking blueness plaque.After locus coeruleus appearance,
6th, the plaque purification of recombinant virus and identification
6.1 recombinant virus ORFV- Δs VIR-LacZ purifying
10 times of doubling dilutions are done to restructuring poison with DMEM stostes, general dilution gradient is 10-1To 10-6, each dilution factor connects
Holes.The restructuring poison of different gradients is inoculated with Vero cells, adds 200 μ L viral dilution liquid, control group cell respectively per hole
Nutrient solution replaces, and then puts 37 DEG C of 5%CO2Culture absorption 1h in incubator.With Vero cells in PBS rinsing plate, wash
Fall unadsorbed virus.Take 2 × DMEM nutrient solutions containing 16% calf serum to add in the agar of equivalent 3% (being kept for 37 DEG C), mix
It is even.Add the 2mL mixtures per hole, horizontalization platform puts 37 DEG C of 5%CO after agar cooled and solidified2Cultivated 2 days in incubator.Take and contain
2 × DMEM nutrient solutions of 16% calf serum are added in the agar of equivalent 3% (being kept for 37-40 DEG C), then lucifuge addition X-gal molten
Liquid, 20 μ L/mL.After above admixture is mixed, add 1mL per hole, platform is laid flat after agar cooled and solidified, in 37 DEG C of 5%CO2Training
Support and cultivated in case, observe change per hole day by day.After locus coeruleus appearance, the most obvious several locus coeruleus pickings (Fig. 1) most concentrated are chosen,
The locus coeruleus of picking is connected in preprepared Vero Tissue Culture Flasks and bred, is prepared for purifying next time.By 5 wheel plaques
Purifying, filters out purer recombinant virus.
6.2 recombinant virus ORFV- Δs VIR-LacZ PCR identifications
The culture of recombinant virus ORFV- Δ VIR-LacZ and the ORFV-SHZ1 strains of the 5th generation purifying is collected, is extracted respectively
Viral DNA, enter performing PCR with primer P1-P2 and identify.ORFV-SHZ1 amplified bands size is 1550bp, and recombinant virus ORFV-
It is 5145bp that Δ VIR-LacZ, which amplifies purpose band size,.
6.3 recombinant virus ORFV- Δs VIR-LacZ pcr amplification product sequence verification
Sequencing result shows, the sequence of primer P5, P6 amplification contain on the VIR virulence genes of part homology arm gene order and
The P11 promoter sequences and part LacZ gene orders of insertion；The sequence of primer P7, P8 amplification contains VIR virulence genes similarly hereinafter
Source arm gene order and insertion gene order.
7th, recombinant virus ORFV- Δs VIR-LacZ virulence determinations
7.1 recombinant virus ORFV- Δs VIR-LacZ and parent's strain TCID50Measure
ORFV- Δ VIR-LacZ and ORFV SHZ1 strains are inoculated with Vero cells respectively, occur determining after cytopathy receives poison
TCID50.As a result show, parental virus ORFV-SHZ1 TCID50For 1 × 10-5.5/ mL, ORFV- Δ VIR-LacZ gene-deleted strains
TCID50For 1 × 10-3.5/mL.Compared with parent ORFV, ORFV- Δ VIR-LacZ virus titers substantially reduce (P<0.05), say
There is significant impact to propagation of the ORFV on cell after bright VIR virulence genes missing.
7.2 recombinant virus ORFV- Δs VIR-LacZ are in Vero cell growth characteristics
ORFV- Δ VIR-LacZ and ORFV-SHZ1 strains are inoculated with Vero cells respectively, virus measure is harvested every 6h
TCID50, draw growth curve.As a result ORFV- Δs VIR-LacZ is similar with the growth curve of ORFV-SHZ1 strains, but virus titer
About 2 orders of magnitude (as shown in Figure 3) are reduced, this shows that the missing of VIR virulence genes significantly affects ORFV propagation.
The 7.3 pathogenic detections of recombinant virus ORFV- Δs VIR-LacZ
The 2 monthly age lamb 9 of health is selected, is divided into I~III group, every group 3.With the recombinant virus of Vero cell culture
ORFV- Δ VIR-LacZ and ORFV-SHZ1 strains, then respectively with virus liquid I group and II group of lip scratch inoculation lamb, often
Only inoculation 0.5mL (1 × 10-5.5TCID50/ 0.1mL), while control group (III group) is inoculated with sterile PBS in the same way
0.5mL, raising is individually insulated, 10d observations are carried out to inoculation site morbidity after inoculation.As a result in recombinant virus ORFV- Δs VIR-
LacZ occurs erythema only occur after inoculation for 1 week at positions such as its bicker, lips, ORFV-SHZ1 strains lamb lip after inoculation
There is papule, warts in portion；Control group has no any lesion simultaneously.
8th, recombinant virus ORFV- Δ VIR-LacZ genetic stabilities and the immunogenicity detection of LacZ genes are expressed
8.1 recombinant virus ORFV- Δs VIR-LacZ genetic stability
Correct recombinant virus ORFV- Δs VIR-LacZ continuous passages on Vero cells will be identified, take respectively F1, F5,
F10, F15 generation virus LacZ primers, which enter performing PCR, to be identified, it is amplifiable go out purpose band, it is good to show that the recombinant virus has
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
The 2 monthly age lamb 24 of health is selected, is divided into I~III group, every group 8.With Vero cell culture recombinant viruses ORFV-
Δ VIR-LacZ, by recombinant virus respectively with 1 × 10-5.5TCID50/ 0.1mL and 5 × 10-4.5TCID50/ 0.1mL dosage is in lip
The lamb that I group and II group of portion's scratch inoculation, every inoculation 0.5mL；Normal group (III group) is set simultaneously, control group is with same
Method is inoculated with sterile PBS 0.5mL, is individually insulated raising, lamb cellular immune level, humoral immunity level are entered after inoculation
Row detection.
Immune lamb cellular immune level detection (spleen lymphocyte proliferation experiment)：The 10d after immune, every group takes 2 lambs
Sheep, it is sterile after execution to take its spleen and separate splenic lymphocytes, carry out ion vitro immunization experiment.Splenic lymphocytes separate specific
Operating procedure is as follows：(1) take a blood sample, separate serum, put -20 DEG C of preservations；(2) it is sterile to take its spleen after putting to death lamb, it is placed in 3mL
In 1640 culture medium containing 2% hyclone, spleen is ground with the inner core of sterilizing syringe, afterwards with 100 mesh sterile nylon nets
Filtering, collect splenic lymphocytes suspension；(3) with containing 10% hyclone 1640 complete mediums adjustment cell concentration be 1 ×
107Individual/mL；(4) seeded cells into by every μ L of hole 100 in 96 porocyte culture plates, and set ConA to stimulate hole (ConA end
Concentration is 5 μ g/mL), be not added with the control wells of stimulant, 3 repetitions are set per hole and set 3 nutrient solution conducts for being not added with cell
Blank well.(5) after each hole mixture mixes, 37 DEG C of CO are positioned over248h is cultivated in cell culture incubator, 4h adds before culture terminates
Enter 10 μ L MTT (5g/L), then put back to again in incubator and continue to cultivate 4h；(6) after culture terminates, supernatant discarding, added per hole
100 μ L DMSO, room temperature places 10min after vibration mixes；(7) ELIASA determines the OD values in each hole at 570nm.As a result such as table 1
Shown, recombinant virus immune group (the Ith and II group) stimulates specific antigen ORFV and shows obvious T lymphopoiesis energy
Power, OD570nmValue is between 0.41~0.51；To heterogenetic antigen ConA stimulate index of Response between 0.45~0.58,
Stimulated higher than ORFV；And control group (III group) stimulates ORFV and does not show obvious lymphproliferation response, its OD570nmValue
Only 0.10~0.14.
Immune lamb humoral immunity level detection：By the method for fixed virus dilute serum to lamb serum neutralizing antibody
It is measured：(1) measure of viral median tissue cell infection amount：Viral TCID is calculated according to Reed-MuenchShi methods50Value,
Then by viral dilution into 200 TCID50, after being mixed with equivalent serum, each dosage of inoculation contains 100 TCID50。(2)
The processing of serum to be checked：Serum to be checked is put in 56 DEG C of water-baths and inactivates 30min, to destroy complement and other heat labile non-spies
The opposite sex is killed the virus molecule, then serum to be checked is carried out into continuous 2 times of dilutions with DMEM nutrient solutions.(3) sense is made：Take quantitative ORFV and
Isometric serum to be checked, is placed in 2mL EP pipes and fully mixes, 37 DEG C, 1~2h of sense work in 5%CO2 incubators.(4) it is inoculated with：
The virus inoculation that sense is made to terminate is to being paved with 80%Vero 96 porocyte culture plates, and each dilution factor does 3 repetitions, each
Hole inoculum concentration is 10~20 μ L.It is placed in 37 DEG C, 5%CO2After being incubated 1~2h in incubator, with containing 5% calf serum and containing 1%
After dual anti-DMEM changes liquid, 37 DEG C, 5%CO are again placed in2Cultivated in incubator.(5) calculated and tied according to Reed-MuenchShi methods
Fruit.The highest dilution that cell can protect 50% does not produce cytopathy reaction is imitated as the neutralizing antibody of serum to be checked
Valency.The neutralizing antibody titers of recombinant virus ORFV- Δs VIR-LacZ immune group lamb serums will be apparently higher than as can be seen from Table 2
Control group.
It is post-stimulatory through ORFV and ConA that lamb splenic lymphocytes are immunized in the recombinant virus ORFV- Δs VIR-LacZ of table 1
OD570nmIt is worth (X ± SD)
The detection of lamb serum ORFV specificity neutralizing antibody is immunized in the recombinant virus ORFV- Δs VIR-LacZ of table 2
| After immune | 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 (1)
1. a kind of preparation method of sheep of virus virulence gene VIR deletion mutation strains, is comprised the following steps that：
A, using genetic fragment of the PCR method clone comprising ORFV-SHZ1 strain VIR virulence genes and flanking sequence, with digestion side
Method misses out VIR virulence genes, in table of the deletion segment insertion with vaccinia virus late promoter P11 and LacZ reporter gene
Up to box, the recombinant shuttle vector using LacZ genes as selection markers is built；
B, recombinant shuttle vector is transfected into the Vero cells of ORFV infection and carries out homologous recombination, purified and obtain through plaque screening
The recombinant virus ORFV- Δs VIR-LacZ of VIR virulence genes missing；
The genetic fragment comprising ORFV-SHZ1 strain VIR virulence genes and flanking sequence described in step A, preparation method virus
Extracts kit extracts ORFV-SHZ1 pnca gene group DNA, and the sequence of virulence gene containing VIR and upper underarm is amplified with P1-P2 primers
Row, pcr amplification product is reclaimed, it is connected with pMD18-T-simple carriers, obtain pVIR recombinant plasmids, wherein：
P1 sequence is：5’-ATCGTGAACACCAAGACCT-3’,
P2 sequence is：5’-TTGTGCACGCTGCGCGC-3’；
The operation that enzymatic cleavage methods miss out VIR virulence genes in the step A is：Recombinated with Sph I and BglII double digestions pVIR
Plasmid, deletion mutation is carried out to VIR genes；
The expression cassette with vaccinia virus late promoter P11 and LacZ reporter gene uses restriction enzyme in the step A
Enzyme XbaI cuts the gene piece containing vaccinia virus late promoter P11 and reporter gene lacZ from pSC-11 shuttle vectors
Section；
Plaque screening purifying in step B is diluted using DMEM stostes to homologous recombination poison, is inoculated with Vero cells, sterile to take indigo plant
Spot, multigelation, renewed vaccination Vero cells, after cytopathy to appear and locus coeruleus, vaccinization Vero cells, purified
Recombinant virus ORFV- Δs VIR-LacZ.
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| Pathogenesis in lambs and sequence analysis of putative virulence genes of Brazilian orf virus isolates;Mathias Martins等;《Veterinary Microbiology》;20141231;第174卷;第69-77页 * |
| 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;杨海波;《中国优秀硕士学位论文全文数据库 农业科技辑》;20150315(第3期);摘要，第三章第39-51页，第二章 * |
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