Type I interferon receptor gene knockout bovine kidney cell line as well as construction method and application thereof

A technology of gene knockout and construction method, which can be applied in application, genetic engineering, plant genetic improvement and other directions, can solve the problems of slow proliferation and low virus titer, and achieve the effect of high mutation efficiency, low cost, and simple and fast construction method.

Pending Publication Date: 2022-08-09
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The purpose of the present invention is to solve the problem of slow proliferation and low virus titer of BVDV in MDBK cells, to provide a gene knockout bovine kidney cell line, to improve the p

Method used

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  • Type I interferon receptor gene knockout bovine kidney cell line as well as construction method and application thereof
  • Type I interferon receptor gene knockout bovine kidney cell line as well as construction method and application thereof
  • Type I interferon receptor gene knockout bovine kidney cell line as well as construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031]Example 1: Construction of MDBK cells containing Cas9 protein

[0032] The CRISPR / Cas9 gene knockout system is a new generation tool for gene knockout because of its convenient construction and flexible design. The system consists of Cas9 protein with DNA cleavage activity and guide RNA that recognizes specific targets. Since the target recognition can be achieved only by editing the guide RNA in the system, the efficiency of constructing gene knockout vectors is greatly improved. . Among them, the construction of a stable cell line that stably expresses Cas9 protein is a prerequisite for gene knockout. Currently, the commonly used method for constructing a stable cell line of Cas9 protein is as follows: packaging lentivirus expressing Cas9 protein, Cas9 lentivirus infecting target cells, Screening and functional verification of stably transfected cell lines.

[0033] In this experiment, a lentiviral expression vector for Cas9 protein was constructed. Taking advantage ...

Embodiment 2

[0053] Example 2: Construction of IFNAR1 knockout MDBK monoclonal cells

[0054] 1. Resuscitate and subculture the MDBK cells containing Cas9 protein selected in the laboratory in the early stage

[0055] First, the MDBK cells containing Cas9 protein (MDBK-Cas9-I) previously preserved were taken out from liquid nitrogen, and were quickly placed in a 37°C water bath with constant shaking to thaw the cells. Put the thawed cells into a sterile operating table, and use a pipette to suck up 5 mL of cell culture medium (DMEM containing 5% FBS and 2% double antibody), then suck up the cell suspension, pipet repeatedly for several times, and pour it into a centrifuge tube. 1000r / min, centrifuge for 5 minutes, discard the supernatant. Add 2 mL of cell culture medium to a centrifuge tube, pipetting evenly, and then transfer the cells to a six-well plate, set at 37°C, 5% CO 2 Cultured in a constant temperature incubator.

[0056] Subculture was performed when the cells were about 95% ...

Embodiment 3

[0092] Example 3: Validation of monoclonal cell phenotype of IFNAR1 knockout MDBK cells

[0093] Recover wild-type MDBK, and two IFNAR1 knockout MDBK cell lines numbered sgRNA2-10 and sgRNA4-L in T25 cell flasks, and count the cells after they are full, and take 3.5 × 10 cells respectively. 6 3 cells were plated on T25 cell flasks, and the cells were counted after 24 hours of adherence, and then inoculated with BVDV at 0.1 MOI. After the cytopathic effect was complete, the cells were collected, and the wild-type MDBK, sgRNA2-10 and sgRNA4-L cells were measured respectively. Obtained TCID of BVDV 50 , observe the phenotypic differences.

[0094] Repeat the above process, respectively connect IBRV and BPIV-3 to verify whether there is a difference in their phenotypes. The result is as Image 6 It was shown that the viral titers of the IFNAR1-knockout MDBK cell lines numbered sgRNA4-L were significantly increased, regardless of whether it was BVDV, IBRV, or BPIV-3.

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Abstract

The invention discloses an I-type interferon receptor (IFNAR) gene knockout bovine kidney cell line, which is obtained by carrying out gene editing on bovine kidney cells by utilizing a CRISPR/Cas9 gene editing technology and carrying out monoclonal selection. Phenotype verification shows that after the IFNAR1 knockout bovine kidney cell line does not express IFNAR1 and is infected with a bovine viral diarrhea virus (BVDV), an infectious bovine rhinotracheitis virus (IBRV) and a 3-type bovine parainfluenza virus (BPIV-3), the virus titer can be remarkably improved, so that the IFNAR1 knockout bovine kidney cell line can be used for separating and identifying bovine-derived virus clinical strains and improving the virus proliferation efficiency.

Description

technical field [0001] The invention belongs to the field of biology, and relates to a bovine kidney cell line knocked out of type I interferon alphareceptor (IFNAR1), and also relates to the application of the cell line in the isolation, identification, replication and proliferation of bovine-derived viruses and the same. build method. Background technique [0002] Interferon (IFN) is a cytokine with antiviral, antitumor and immune regulation functions. Generally divided into three categories: type I interferon represented by IFN-α, IFN-β, type II interferon represented by IFN-γ and type III interferon represented by IFN-λ. Studies have shown that IFN-I can inhibit type I interferon signaling and promote the control of persistent viral infections. The antiviral effect of interferon is indirectly dependent on the autocrine or paracrine pathway. After the virus infects cells, the target cell will release interferon, and the released interferon will bind to IFN receptors on ...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/867C12N15/113C12N15/12C12N7/00
CPCC12N5/0686C12N15/86C12N15/1138C07K14/7156C12N7/00C12N2310/20C12N2510/00C12N2800/107C12N2740/15043C12N2770/24351C12N2760/18651C12N2710/16051Y02A50/30
Inventor 郭爱珍耿元晨姜传文杨浩项志杰陈颖钰胡长敏陈曦陈建国徐肖文覃城皇
Owner HUAZHONG AGRI UNIV
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