Dicer gene knockout BHK-21 cell line

A BHK-21, gene knockout technology, applied in the field of molecular biology and cell biology, can solve the problems of low titer and increase, and achieve the effect of increasing virus titer

Pending Publication Date: 2021-12-31
INST OF ANIMAL SCI & VETERINARY MEDICINE SHANDONG ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In view of the low titer of the current Seneca virus production cell line, the present invention provides a Dicer gene knockout BHK-21 cell line, which uses CRISPR / Cas9 technology to edit the BHK-21 cell line, and successfully knocks out Dicer gene, the replication rate of Seneca virus was accelerated in this cell line, and the virus titer was significantly increased

Method used

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  • Dicer gene knockout BHK-21 cell line
  • Dicer gene knockout BHK-21 cell line
  • Dicer gene knockout BHK-21 cell line

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Design and screening of Dicer targeting sgRNA 1 Example

[0023] 1. Design of targeted sgRNA Dicer

[0024] Syrian hamsters Dicer protein gene (Gene ID: 101839371) length of the sequence as a template, choose the front and is a total of exon all mutants, exon 3 must not be a multiple use gRNA online design website http: / / crispor .tefor.net / filter design and adding a suitable sequence CACCG sequence at a front end, a gRNA-F, while the reverse complement of a sequence selected AAAC distal plus, plus C-terminal as gRNA-R, the corresponding oligo DNA sequence as shown in table 1:

[0025] Table oligo DNA corresponding to sequences 1 sgRNA

[0026]

[0027] After the above primers synthesized gradient lowered from 80 ℃ -30 ℃ annealing to obtain complete renaturation hybrids, were named sgRNA1-4. The PX458 BbSI digested plasmid, electrophoresis and recovered large fragment. Hybridizing strands and the recovered plasmid was digested PX458 ligation, transformation Trelief TM ...

Embodiment 2

[0033] Example 2 Dicer knockout cell line BHK-21 construct

[0034] Select the highest cleavage efficiency PX458-sgRNA1 using the lipofection method lipo3000 transfected into BHK-21 cells, cultured for 48 hours, using flow sorted cells obtained positive cells, placed in double antibody containing 1%, 10% FBS-DMEM nutrient solution 37 ℃ 5% CO 2 Incubator cultured propagation. The monoclonal cell line extract the DNA, and PCR amplified with respective primers. PCR products were sequenced, with the selection of homozygous mutant cells were stored sequence. Save homozygous mutant sequence with a wild-type sequence such as figure 2 FIG: 1 base insertion (A) at the target position, which is expected to result in a frameshift mutation protein translated from frameshift, early termination and loss of function. High mutation location near the sequencing peak pattern quality, reliable sequencing results show. The wild-type and homozygous mutant preserved cells were lysed and the supernatant...

Embodiment 3

[0036] Example 3 Seneca virus infection Dicer knockout cytoplasmic and virus replication rate

[0037] Virus RNA expression quantity

[0038] BHK-21-DICER Δ- When the cells and BHK-21 cells were cultured to approximately 80% of the convergence, the medium was sued out of the medium, and cells were inoculated with 0.01 moi Seneca virus. Incubate for 1 h in the incubator, sucking the virus solution, adding PBS to wash twice The DMEM medium containing 2% FBS was added. The supernatant and cell samples were collected in infection 4H, 8H, 12H, 16H, 20H, 24H. After extracting the total RNA reverse transcription with the kit, Sybr Green QPCR is performed, and the primer is as follows:

[0039] SVA-QRTU: 5'-agaattggaagcccatgctct-3 ';

[0040] SVA-QRTL: 5'-gagcca actagaaacagattgc-3 ';

[0041] Such as image 3 As shown, the Seneca virus is 16h after infection, Dicer Knockout Cell Series (DICER) Δ- ) The replication rate of the Senineri virus is significantly higher than that of the control ...

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Abstract

The invention belongs to the field of molecular biology and cell biology, and provides a Dicer gene knockout BHK-21 cell line. The preservation number of the Dicer gene knockout BHK-21 cell line is CCTCC (China Center For Type Culture Collection) NO: C2021232. The Dicer gene knockout BHK-21 cell line can be used for Seneca proliferation and Seneca vaccine production. According to the cell line, the Dicer gene knockout BHK-21 cell line is successfully constructed by using a CRISPR / Cas9 technology; and the Senecavinus A replication rate in the cell line is accelerated, the virus titer is remarkably improved, and the cell line can be used for commercial production of Seneca vaccines.

Description

Technical field [0001] The present invention is in the field of molecular biology and cell biology, particularly to one kind of BHK-21 cell line Dicer knockout. Background technique [0002] Seneca virus (Senecavirus A, SVA), has been called Seneca Valley virus (Seneca Valleyvirus), 2016, the International Committee on Taxonomy of Viruses (ICTV) changed its name to Senecavinus A, is a micro RNA viruses Ke Saini the only members of the virus genus card. 2007 Canadian primary outbreak of swine vesicular disease, foot and mouth disease and swine vesicular determine the disease were negative pathogen by pathogen detection vesicular disease and SVA is positive, therefore, SVA is the primary cause of pathogenic swine vesicular disease. Initially, pigs occasionally can detect the virus, but the virus has not been reported to cause disease. Therefore, Seneca is considered to be an emerging zoonosis. November 2014 to early 2015, in Brazil and the United States have the disease outbreak. S...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/88C12N15/113C12N15/55C12N7/00C12R1/93
CPCC12N5/0686C12N15/88C12N15/1137C12N9/22C12N7/00C12N2510/00C12N2310/20C12N2770/32051
Inventor 吴晓燕李俊李琛时建立刘畅彭喆徐绍建韩红王硕郑理美
Owner INST OF ANIMAL SCI & VETERINARY MEDICINE SHANDONG ACADEMY OF AGRI SCI
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