Ago2 gene knockout BHK-21 cell line

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

Active Publication Date: 2021-11-05
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] Aiming at the low titer of the current Seneca virus production cell line, the present invention provides a BHK-21 cell line with Ago2 gene knockout, which uses CRISPR/Cas9 technolog

Method used

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  • Ago2 gene knockout BHK-21 cell line
  • Ago2 gene knockout BHK-21 cell line
  • Ago2 gene knockout BHK-21 cell line

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1 Design and screening of sgRNA targeting Ago2

[0024] 1. Design of sgRNA targeting Ago2

[0025] Using the Syrian hamster Ago2 protein gene (Gene ID: 101843722) as a template, select the front and common exon of all mutants. The sequence length of the exon must not be a multiple of 3. Use the gRNA online design website http: / / crispor .tefor.net / Design and screen out the appropriate sequence and add CACCG sequence at the front end as gRNA-F. At the same time, add AAAC to the front end and C at the end of the reverse complementary sequence of the screened sequence as gRNA-R, which corresponds to The oligo DNA sequence is shown in Table 1:

[0026] Table 1 oligo DNA sequence corresponding to sgRNA

[0027] After the above primers are synthesized, the temperature is gradually lowered from 80°C to 30°C and annealed to complete renaturation to obtain hybrid strands. Digest the pX458 plasmid with BbSI, electrophoresis and recover large fragments. Ligate the hy...

Embodiment 2

[0033] Example 2 Construction of Ago2 gene knockout BHK-21 cell line

[0034] 1. Establishment of Ago2 knockout BHK-21 cell line

[0035] The PX458-sgRNA1 with the highest cleavage efficiency was selected and transfected into BHK-21 cells by lipo3000 liposome transfection method. After 48 hours of culture, the positive cells were obtained by flow cytometry, and placed in a medium containing 1% double antibody, 10% FBS DMEM nutrient solution at 37°C 5% CO 2 The incubator is used for culturing and multiplying. The DNA extracted from the monoclonal cell line was amplified by PCR with corresponding primers. PCR products were sequenced, and cells with homozygous mutant sequences were selected for preservation. The comparison between the preserved homozygous mutant sequence and the wild-type sequence is as follows figure 2 Shown: Insertion of 2 bases (CG) at the target position, this frameshift mutation is expected to cause frameshift, premature termination and loss of function...

Embodiment 3

[0039] Example 3 Seneca virus infection of Ago2 knockout cell line and virus replication rate

[0040] 1. Viral RNA expression

[0041] BHK-21-Ago2 Δ- When the cells and BHK-21 cells are cultured to about 80% confluence, suck off the medium, inoculate the cells with 0.01 MOI Seneca virus, incubate in the incubator for 1 hour, suck off the virus solution, add PBS to wash the cells twice Add DMEM medium containing 2% FBS. Supernatant and cell samples were collected at 4h, 8h, 12h, 16h, 20h, and 24h of infection, respectively. The kit was used to extract the total RNA and carry out SYBR Green qPCR after reverse transcription. The primers are as follows:

[0042] SVA-QRTU: 5'-AGAATTTGGAAGCCATGCTCT-3';

[0043] SVA-QRTL: 5'-GAGCCA ACATAGAAACAGATTGC-3';

[0044] The result is as Figure 4 As shown, after Seneca virus infection, the Ago2 knockout cell line (Ago2 Δ- ) was significantly higher than the control cell line (BHK-WT) in the replication rate of Seneca virus.

[0045]...

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Abstract

The invention belongs to the field of molecular biology and cytobiology, and provides an Ago2 gene knockout BHK-21 cell line. The preservation number of the cell line is CCTCC NO:C2021206. The Ago2 gene knockout BHK-21 cell line can be used for Seneca proliferation and production of Seneca vaccines. The Ago2 gene knockout BHK-21 cell line is successfully constructed by using a CRISPR/Cas9 technology; the replication rate of Seneca virus in the cell line is increased, the virus titer is remarkably increased, and the cell growth rate is not remarkably influenced, so that the cell line can be used for commercial production of the Seneca vaccines.

Description

technical field [0001] The invention belongs to the fields of molecular biology and cell biology, and in particular relates to a BHK-21 cell line with Ago2 gene knockout. Background technique [0002] Senecavirus A (SVA), formerly known as Seneca Valley virus (Seneca Valleyvirus), was renamed Senecavinus A by the International Committee on Taxonomy of Viruses (ICTV) in 2016. The only member of the Cavirus genus. Porcine primary vesicular disease broke out in Canada in 2007. The pathogen detection of vesicular disease confirmed that the pathogens such as foot-and-mouth disease and swine vesicular disease were negative, but SVA was positive. Therefore, SVA is the pathogen that causes porcine primary vesicular disease. Initially, the virus was occasionally detected in pig herds, but there have been no reports of the virus causing disease. Therefore, Seneca is considered an emerging zoonotic disease. From November 2014 to early 2015, the disease broke out successively in Braz...

Claims

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Application Information

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IPC IPC(8): C12N5/20C12N15/113C12N15/85C12N7/00C12R1/91
CPCC12N5/0686C12N15/113C12N15/85C12N7/00C07K14/47C12N2310/20C12N2510/00C12N2770/32051C12N2770/32034
Inventor 李琛吴晓燕李俊时建立刘畅彭喆徐绍建韩红王硕马英茹
Owner INST OF ANIMAL SCI & VETERINARY MEDICINE SHANDONG ACADEMY OF AGRI SCI
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