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HCBP 6 (Hepatitis C Virus Core-Binding Protein 6) gene knockout cell line and construction method thereof

A gene knockout and cell line technology, applied in the field of molecular biology, can solve problems such as incompleteness and instability of the HCBP6 method

Active Publication Date: 2018-06-12
THE FIFTH MEDICAL CENT OF CHINESE PLA GENERAL HOSPITAL
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0007] The purpose of the present invention is to provide a method for stably knocking out the HCBP6 gene from the genome against the instability and incompleteness of the current transient knockout HCBP6 method. In addition to being used for HCBP6 function research, the method can also be used to establish HCBP6 expression deficient cell line

Method used

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  • HCBP 6 (Hepatitis C Virus Core-Binding Protein 6) gene knockout cell line and construction method thereof
  • HCBP 6 (Hepatitis C Virus Core-Binding Protein 6) gene knockout cell line and construction method thereof
  • HCBP 6 (Hepatitis C Virus Core-Binding Protein 6) gene knockout cell line and construction method thereof

Examples

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Embodiment 1

[0051] Example 1. Knockout of the HCBP6 gene in liver cancer cells HepG2 based on CRISPR-Cas9

[0052] 1. Human HCPB6 gene knockout sgRNA design

[0053] According to the National Center for Biotechnology Information (NCBI), the human HCBP6 gene is located on chromosome X of the human genome, with a full length of 30128 bp, and its protein coding sequence is 151..283 (SEQ ID No.1), 6615. .6765 (SEQ ID No.2), 19721..19796 (SEQ ID No.3), 24882..25013 (SEQ ID No.4), 27807..27884 (SEQ ID No.5), encoding the above 5 segments Enter the sequences into the sgRNA online design website: http: / / crispr.mit.edu / , select unique genomic region (23-500nt) in the sequence type column (sequence type), find out the series of sgRNA, and add the lentiCRISPRV2 vector ( figure 1 ) on the reverse complementary sequence to design the sgRNA of the gene to be knocked out.

[0054] This polyoligonucleotide should be complementary to the 20bp base in front of the NGG PAM (proto-spacer adjacentmotif) at ...

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Abstract

The invention discloses an HCBP 6 (Hepatitis C Virus Core-Binding Protein 6) gene knockout cell line and a construction method thereof. The construction method for the HCBP 6 gene knockout cell line is characterized in that a CRISPER / Cas9 (CRISPR associated protein 9) technology is adopted to construct the HCBP 6 gene knockout cell line, a fragment which conforms to a 5'-N[X]-NGG-3' or 5'-CNN-N[X]-3' sequence arrangement rule in the coding sequence of HCPB6 protein is taken as a target sequence, wherein N presents any one of A, G, C and T, X is greater than or equal to 14 or less than or equalto 30, in addition, X is an integer, and N[X] presents X continuous deoxyribonucleotide. The construction method lays a foundation for HCV (hepatitis C virus) infection mechanism research. The HCBP 6gene knockout constructed by the invention forms frameshift mutation on a genome level, the gene can be transferred to the next generation along with the division and the proliferation of cells, anda stable HCBP 6 gene knockout cell line is formed.

Description

technical field [0001] The invention belongs to the field of molecular biology, and relates to a HCBP6 gene knockout cell line and a construction method thereof. Background technique [0002] Hepatitis C virus (Hepatitis C Virus) is one of the main pathogens causing chronic hepatitis, liver cirrhosis and liver cancer. At present, the main treatment method is interferon plus ribavirin, but the effect is not very satisfactory, especially for patients with decompensated liver cirrhosis. Therefore, it is of great significance to further study the pathogenesis of HCV and develop new antiviral drugs. [0003] Hepatitis C Virus Core-Binding Protein 6 (Hepatitis C Virus Core-Binding Protein 6) is a protein that can bind to the hepatitis C virus core protein. Studies have found that HCBP6 protein can up-regulate and down-regulate the expression levels of a series of different genes, and these differentially expressed genes are closely related to cell signal transduction, proliferat...

Claims

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Application Information

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IPC IPC(8): C12N15/867C12N15/90C12N5/10
CPCC07K14/4702C12N5/0693C12N9/22C12N15/86C12N15/907C12N2510/00C12N2740/15043C12N2800/107C12N2810/10
Inventor 钟彦伟范华昊张敏史策
Owner THE FIFTH MEDICAL CENT OF CHINESE PLA GENERAL HOSPITAL
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