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Method for using CRISPR/Cas9 for preparing recombinant pseudorabies virus

A porcine pseudorabies virus and virus technology, applied in the field of genetic engineering, can solve the problems of blindness in the process of recombinant virus screening and purification, complex thinking and operation, and heavy workload, and achieve the goals of shortening the experimental period, reducing workload, and improving efficiency Effect

Inactive Publication Date: 2019-02-12
WUHAN KEQIAN BIOLOGY CO LTD
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Problems solved by technology

[0004] In order to solve the problems existing in the prior art, overcome the shortcomings of blindness, tediousness, and heavy workload in the process of screening and purification of recombinant viruses in the prior art, and use two sets of gene editing techniques, the thinking and operation are relatively complicated, and the workload is heavy. Big disadvantage, the purpose of the present invention is to provide a gene editing technology using only CRISPR / Cas9, by inserting fluorescent marker sequences and irrelevant sequences in the gE coding region of PRV successively, so as to achieve the purpose of easy operation and improved screening efficiency

Method used

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  • Method for using CRISPR/Cas9 for preparing recombinant pseudorabies virus
  • Method for using CRISPR/Cas9 for preparing recombinant pseudorabies virus
  • Method for using CRISPR/Cas9 for preparing recombinant pseudorabies virus

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Embodiment 1

[0032] Experimental materials: ST cells are porcine testis cell lines (ATCC ID: CRL-1746); homologous recombination template vector FLAG-HA-pcDNA3.1 (Addgene ID 52535 hereinafter referred to as pcDNA3.1), PCR premix system primestar purchased from Takara (R045); T4 ligase and buffer were purchased from Quanshijin Biological Company (FL101); nucleic acid molecular weight MarkerDM1000 was purchased from Kangwei Century (CW0634); plasmid mini-extraction kit was purchased from OMEGA (D6943-02); DNA gel Recovery kit OMEGA (D2500-02); Lipofectamine 2000, a liposome transfection reagent, was purchased from Invitrogen (1803716); all endonucleases were purchased from Dalian Baobio.

[0033] The homologous recombination template uses FLAG-HA-pcDNA3.1 as the vector and consists of the following three parts: the upstream homology arm gE1, the EGFP with CMV enhancer and promoter, and the downstream homology arm gE2.

[0034] The upstream homology arm gE1 is shown in SEQ ID NO.2, the sequen...

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Abstract

The invention belongs to the technical field of gene engineering, and particularly discloses a method for using CRISPR / Cas9 for preparing recombinant pseudorabies virus. According to the method, the gene editing technology CRISPR / Cas9 is used, a coded sequence of fluorescent protein is introduced into a gE editing area of PRV, and after positive recombinant virus is screened out successfully, thesame technology is used for replacing a fluorescent protein gene with an irrelevant sequence (LysC sequence with 500 bp) to form insertion mutation, so that gE protein expression deletion of the PRV is achieved. According to the method for using the CRISPR / Cas9 for preparing the recombinant pseudorabies virus, visible screening of the fluorescent protein from nothing to something and then from something to nothing is used, a screening procedure is simplified, the recombinant virus construction efficiency is improved, and an experiment cycle is shortened; moreover, by introducing insertion mutation into the gE editing area of the pseudorabies virus (PRV), the prepared recombinant virus can be used for researching and developing PRV attenuated vaccine, and meanwhile, the irrelevant sequencecan be used for identifying and judging immunotoxicity and natural toxicity.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a method for preparing recombinant porcine pseudorabies virus by using CRISPR / Cas9. Background technique [0002] The Chinese patent application whose publication number is CN106929485A discloses a pseudorabies virus genetically engineered gB recombinant attenuated vaccine strain and its application. The plaque screening technology used in this technical solution is blind screening, and the screening of recombinant viruses is a purely probabilistic event. After screening, the virus is amplified and cultured to extract the genome for PCR detection, and the workload is relatively large. [0003] The Chinese patent application with the publication number CN104894075A discloses the method and application of CRISPR / Cas9 and Cre / lox system editing pseudorabies virus genome to prepare vaccines. This technical solution uses two gene editing technologies, and the ...

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Application Information

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IPC IPC(8): C12N15/113C12N15/90C12N15/65C12N7/01
CPCC07K14/005C12N7/00C12N15/113C12N15/65C12N15/902C12N2310/10C12N2710/16721C12N2710/16722C12N2310/20
Inventor 徐高原孙芳周明光张华伟郝根喜
Owner WUHAN KEQIAN BIOLOGY CO LTD
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