Pseudorabies virus gE/gI deletion mutant strain of double expression gC gene and construction and application thereof
A technology of pseudorabies virus and porcine pseudorabies virus, which is applied in the field of animal virology and genetic engineering, can solve the problems of ineffective resistance to infection by virulent mutant strains, and achieve good immune protection effect, good immune effect, and neutralizing antibodies high level effect
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Embodiment 1
[0063] Example 1 Construction of homologous recombination vector (pMD-LA-RA; pMD-LA-RA-EGFP; pMD-LA-gC-RA)
[0064] 1. According to the gene sequence of the PRVZJ01 strain registered in GenBank (accession number: KM061380.1), using the PRV-AH strain of the pseudorabies virus variant as a template, design 2 pairs of primers LA-F / LA-R and RA-F / RA- R (Table 1) is used to amplify the homologous recombination arm fragments located on both sides of the gI gene and the gE gene (including part of the gI and gE genes), wherein the left arm fragment is referred to as LA, and the right arm fragment is referred to as RA.
[0065] 2. Connect the LA fragment and the pMD18-T vector by TA cloning, and then transform and screen to obtain the recombinant plasmid pMD-LA; the recombinant plasmid pMD-LA and RA fragments are double-digested with restriction endonucleases HindШ and PstI respectively , the digested product was ligated and transformed, and the recombinant plasmid pMD-LA-RA was obtaine...
Embodiment 2
[0073] Construction and purification of embodiment 2 recombinant virus
[0074] 1. Recombinant virus rPRV-AH-gI - / gE - / EGFP + the acquisition
[0075] (1) Inoculate monolayer BHK-21 cells with 0.1 MOI of pseudorabies virus mutant strain PRV-AH, and when 70% to 80% of the cells have lesions, it is repeatedly frozen and thawed 3 times, and the PRV-AH virus liquid is harvested;
[0076] (2) Transfection was performed on a 24-well cell culture plate, and the transfection was performed when the BHK-21 cells grew to 80%. refer to 2000Reagent transfection reagent instructions, such as figure 1 As shown in the construction strategy, the recombinant plasmid pMD-LA-RA-EGFP was transfected into BHK-21 cells. After 4 hours of transfection, 0.1 MOI of PRV-AH virus solution was inserted to allow homologous recombination of the plasmid and the viral genome in the cells. , and set up a transfection control group containing only the recombinant plasmid pMD-LA-RA-EGFP at the same time;...
Embodiment 3
[0084] The drawing of embodiment 3 recombinant virus one-step growth curves
[0085] 1. One-step virus growth curve drawing
[0086] rPRV-AH-gI - / gE - / gC + , rPRV-AH-gI - / gE - and PRV-AH strain were inoculated in the culture plate with BHK-21 cells at 1MOI respectively, at 37°C, 5% CO 2 After incubating in a cell culture box for 1 hour, discard the solution, wash twice with PBS, discard the solution, and add DMEM cell maintenance solution containing 2% fetal bovine serum. Collect the virus for the first time 1 hour after inoculation. The virus was harvested every 4 hours between 4 and 72 hours, and the collected virus was stored at -80°C. The virus titers were measured after repeated freezing and thawing of the viruses collected in all time periods three times, and the growth curves of the three viruses within 72 hours were drawn.
[0087] 2. Conclusion
[0088] Such as Figure 8 Shown, PRV-AH, rPRV-AH-gI - / gE - , rPRV-AH-gI - / gE - / gC + The three viruses h...
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