Porcine pseudorabies attenuated strain prepared from CRISPR/Cas9 and application thereof
A technology of porcine pseudorabies virus and porcine pseudorabies, applied in the direction of viruses, virus peptides, antiviral agents, etc., can solve the problems of difficult to effectively control porcine pseudorabies, economic losses, and inability to provide complete immune protection for PRV mutant strains, etc. Achieve the effects of low knockout efficiency, high safety, and improved gene editing efficiency
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0041] Example 1 Construction of gRNA and Cas9 protein expression recombinant plasmid and US7 / US8 / US9 / US2 gene deletion homology arm plasmid
[0042] 1.1 Construction of gRNA and Cas9 protein expression recombinant plasmids
[0043] a) Use BbsⅠ to digest the Cas9 protein expression plasmid pX335-cas9n, and perform gel recovery; b) According to the sequence of the deleted genes (US7, US8, US9 and US2), use the online design tool (http: / / crispr.mit.edu / ) , designing a single guideRNA (gRNA) sequence targeting the missing fragment; c) synthesizing a single-stranded DNA primer, and annealing the primer; d) ligation, constructing the annealed primer into the linearized Cas9 protein expression plasmid pX335-Cas9n by ligation and transformation, wherein Table 1 is the primer sequence used to amplify the gRNA, figure 1 Flowchart for constructing gRNA and Cas9 protein recombinant plasmids, figure 2 Sequencing validation results for gRNA construction. In the figure, A is pX335-Cas9n...
Embodiment 2
[0048] Example 2 Obtaining of Porcine Pseudorabies Virus Gene Deletion HB-1 Strain (△US7 / US8 / US9 / US2)
[0049] In this example, the recombinant plasmids pX335-Cas9n-US7gRNA and pX335-Cas9n-US2gRNA prepared in Example 1 are used to knock out the gene fragments of US7, US8, US9 and US2 of the porcine pseudorabies virus variant strain PRV HB strain, the specific process as follows:
[0050] After the HB strain of porcine pseudorabies virus mutant strain was isolated, the constructed pSK-Cas9n-US7gRNA and pSK-Cas9n-US2gRNA were co-transferred into ST cells by liposome transfection. The result is as Figure 5 shown), collect the cell culture supernatant, inoculate the transfection product into ST cells, harvest the cell fluid producing viral plaques, purify the plaques with low-melting point agarose, pick 10 plaques randomly after lesion, and use detection primers (As shown in Table 2) Perform PCR amplification on US7 / US8 / US9 / US2 to identify recombinant strains and wild strains. ...
Embodiment 3
[0064] Example 3 The preparation method of porcine pseudorabies virus gene deletion HB-1 strain attenuated vaccine
[0065] 1. Cell Preparation
[0066] 1.1 Cell recovery and proliferation: Take the suspended ST seed cells out of the liquid nitrogen tank, immediately place them in a 37°C water bath to dissolve rapidly, centrifuge at 1000r / min for 5min at room temperature, and discard the supernatant. Resuspend the cells with ST cell medium to make the initial cell density 0.5-0.7×10 6 pieces / ml. At 37°C, 120r / min with 5% CO 2 After being cultured in a horizontal shaker for 48 hours, the cell density reached 4.0-6.0×10 6 pieces / ml.
[0067] 1.2 Primary reactor culture: when the cell density in the shake flask reaches 4.0-5.0×10 6 cells / ml, transfer to the primary bioreactor, add ST cell culture medium to make the initial cell density 0.5-0.7×106 cells / ml. Bioreactor cultivation parameters: temperature 37°C, pH value 7.0, rotational speed 50r / min, dissolved oxygen (DO) 40%...
PUM
Login to View More Abstract
Description
Claims
Application Information
Login to View More