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A pseudorabies virus tk, ge, gi and gg gene deletion strain and preparation method and application thereof

A technology of pseudorabies virus and pseudorabies virus, which is applied in chemical instruments and methods, methods based on microorganisms, biochemical equipment and methods, etc., can solve the problems of low precision repair rate and off-target, and achieve the reduction of immunogenicity and improvement of Efficiency, Improving the Effect of the Immune Response

Active Publication Date: 2022-07-19
WUHAN KEQIAN BIOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although CRISPR / Cas9 has the advantages of knocking out multiple genes at the same time, or multiple sgRNAs targeting a gene, there are still problems such as off-target, low precision repair rate, and the need for multiple verifications at the protein level in the later stage

Method used

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  • A pseudorabies virus tk, ge, gi and gg gene deletion strain and preparation method and application thereof
  • A pseudorabies virus tk, ge, gi and gg gene deletion strain and preparation method and application thereof
  • A pseudorabies virus tk, ge, gi and gg gene deletion strain and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1 Construction of sgRNA expression vector

[0038] In this example, the viral genome sequence was input into the sgRNA online design website (http: / / crispr.mit.edu), the sgRNA sequence with PAM (NGG) was found, and the sgRNA with a low off-target rate was selected, and the construction method of Zhang Feng's laboratory was adopted. It was cloned into pX335 vector, and the sgRNA sequence is shown in Table 1.

[0039] Table 1 sgRNA sequences used to target virus-related genes

[0040]

[0041]

Embodiment 2

[0042] Example 2 Construction of transfer plasmids pcDNA3.1-ΔTK, pSK-ΔgE / gI and pSK-ΔgG

[0043] In this example, the genome of PRV CH16 strain was used as a template to amplify the partial sequences of TK, gE / gI and gG genes, and then respectively digested and ligated into pcDNA3.1(+) or pBluescript II-SK(+) vector to construct transfer Plasmids pcDNA3.1-ΔTK, pSK-ΔgE / gI and pSK-ΔgG. Among them, the simian vacuolar virus 40 (sv40) poly A sequence (SEQ ID NO: 1) was inserted between the left and right homologous sequences of TK; the rabbit globin terminator (Rabbit globinterminator) sequence (SEQ ID NO: 1) was inserted between the left and right homologous sequences of gG :2). The amplification primer sequences are shown in Table 2.

[0044] Table 2 Primer sequences related to transfer plasmid construction

[0045]

Embodiment 3

[0046] Example 3 Extraction of PRV genome

[0047] In this example, PK-15 cells were passaged into T25 cell culture flasks, and 24 hours later, the virus was inoculated into the cells at 1 MOI (multiplicity of infection, MOI). Cell lysate, shake uniformly to lyse all cells, place on ice for 20min, and pour into a 2mL EP tube. Add 660 μL of 5M NaCl dropwise, mix gently, and place on ice for 5 h or in a 4°C refrigerator overnight. Centrifuge at 12000rpm for 30min at 4°C. Pipette the supernatant into a new 2mL EP tube with a pipette tip cut off, add an equal volume of DNA extraction solution, gently invert the EP tube for 5min, centrifuge at 12000rpm at 4°C for 10min, and repeat the DNA extraction solution to extract protein 3 times. Change the 1.5mL EP tube, add pre-cooled 2-fold volume of absolute ethanol to the final supernatant, mix gently, and let stand at -20°C for 2h or overnight. Centrifuge at 12000 rpm for 10 min at 4°C, discard the supernatant, add pre-cooled 75% ab...

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Abstract

The present invention relates to a pseudorabies virus TK, gE, gI and gG gene deletion strain and its preparation method and application. CRISPR / Cas9-mediated homologous recombination technology specifically uses CRISPR / Cas9 as a medium to break DNA double-strands, and then use the method of homologous recombination to fill the gap with the homologous sequence transferred into the cell to perform DNA double-strand repair. , Complete genome editing, this technology can perform precise site-specific editing according to the wishes of researchers, greatly improve the efficiency of gene editing, and shorten the development cycle of vaccines. The present invention adopts this technology to delete the partial sequences of the virulence-related genes TK, gE, gI and the immunosuppression-related gene gG of the popular strains of pseudorabies virus to obtain TK, gE, gI and gG gene deletion strains. Vaccines have the advantages of high safety, genetic stability and good immune protection.

Description

technical field [0001] The invention relates to the technical field of veterinary biological products, in particular to a pseudorabies virus TK, gE, gI and gG gene deletion strain and a preparation method and application thereof. Background technique [0002] Pseudorabies (PR) is an acute infectious disease caused by pseudorabies virus (PRV), which is mainly characterized by fever, itching, encephalomyelitis, respiratory and nervous system disorders. Pigs are the natural host and main source of infection of PRV. PRV can be transmitted horizontally through the respiratory tract and digestive tract, and can also be transmitted vertically through semen, mating or placenta. Pigs of all ages can be infected with PRV, causing huge economic losses to the pig industry. [0003] At present, vaccination is still one of the main means of preventing and eradicating PR. The early PR vaccines were mainly inactivated vaccines and traditional live attenuated vaccines, which played a certa...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N7/01C12N15/113A61K39/245A61P31/22C12R1/93
CPCC12N7/00C12N15/113C07K14/005A61K39/12A61P31/22C12N2710/16721C12N2710/16722C12N2710/16734C12N2310/20A61K2039/552
Inventor 徐高原张华伟周明光郝根喜汤细彪宋文博金建云邵伟
Owner WUHAN KEQIAN BIOLOGY CO LTD
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