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Pseudorabies virus TK, gE, gI and gG gene deletion strain as well as preparation method and application thereof

A pseudorabies virus, pseudorabies virus technology, applied in chemical instruments and methods, microorganism-based methods, biochemical equipment and methods, etc., can solve the problems of off-target, low accurate repair rate, etc., to reduce toxicity and improve immune response. , the effect of improving efficiency

Active Publication Date: 2021-01-29
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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  • Pseudorabies virus TK, gE, gI and gG gene deletion strain as well as preparation method and application thereof
  • Pseudorabies virus TK, gE, gI and gG gene deletion strain as well as preparation method and application thereof
  • Pseudorabies virus TK, gE, gI and gG gene deletion strain as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] The construction of embodiment 1sgRNA expression vector

[0038] In this example, the virus genome sequence is input into the sgRNA online design website (http: / / crispr.mit.edu), the sgRNA sequence with PAM (NGG) is searched, and the sgRNA with a low off-target rate is selected, and the construction method of Zhang Feng's laboratory is adopted. It was cloned into the 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 partial sequences of TK, gE / gI and gG genes were amplified using the PRV CH16 strain genome as a template, and then respectively digested and connected to pcDNA3.1(+) or pBluescript II-SK(+) vectors 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) is inserted between the left and right homologous sequences of TK; the rabbit globin terminator (Rabbit globinterminator) sequence (SEQ ID NO: 1) is inserted between the left and right homologous sequences of gG :2). The sequences of the amplification primers are shown in Table 2.

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

[0045]

Embodiment 3

[0046] The extraction of embodiment 3 PRV genome

[0047] In this example, PK-15 cells were subcultured in T25 cell culture flasks. After 24 hours, the virus was inoculated on the cells at 1 MOI (multiplicity of infection, MOI). Cell lysate, shake evenly to lyse all cells, place in ice for 20min, pour into 2mL EP tube. Add 660 μL of 5M NaCl dropwise, mix gently, and place on ice for 5 hours or in a refrigerator at 4°C overnight. Centrifuge at 12000rpm at 4°C for 30min. Pipette the supernatant into a new 2mL EP tube with the tip of the pipette cut off, add an equal volume of DNA extraction solution, gently invert the EP tube for 5 minutes, centrifuge at 12000rpm at 4°C for 10 minutes, and repeatedly use the DNA extraction solution to extract protein 3 times. Change to a 1.5mL EP tube, add pre-cooled 2 times the volume of absolute ethanol to the final supernatant, mix gently, and stand at -20°C for 2 hours or overnight. Centrifuge at 12,000 rpm at 4°C for 10 min, discard the...

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Abstract

The invention relates to a pseudorabies virus TK, gE, gI and gG gene deletion strain as well as a preparation method and an application thereof. A CRISPR / Cas9 mediated homologous recombination technology specifically comprises the steps that CRISPR / Cas9 serves as a medium to break DNA double strands, then a homologous recombination method is used, homologous sequences transferred into cells are supplemented to notches, DNA double strand repairing is conducted, genome editing is completed, accurate fixed-point editing can be conducted according to the will of a researcher, the gene editing efficiency is greatly improved, and the research and development period of vaccine can be shortened. According to the present invention, the partial sequences of the virulence-related genes TK, gE and gIand the immunosuppression-related gene gG of the pseudorabies virus epidemic strain are deleted by using the technology to obtain the TK, gE, gI and gG gene deleted strain, and the vaccine of the genedeleted strain has advantages of high safety, good hereditary stability, good immunoprotection effect and the like.

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 its preparation method and application. Background technique [0002] Pseudorabies (PR) is an acute infectious disease caused by pseudorabies virus (PRV), and its main features are fever, itching, encephalomyelitis, respiratory and nervous system disorders, etc. 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, which has caused huge economic losses to the pig industry. [0003] At present, vaccination is still one of the main means to prevent and eradicate PR. The early PR vaccines were mainly inactivated vaccines and traditional attenuated live vaccines, which played a certain r...

Claims

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Application Information

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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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