CRISPR/Cas9 targeted knockout human hepatitis B virus P gene and specific gRNA thereof

A hepatitis B virus, specific technology, applied in the field of genetic engineering, can solve problems such as difficult to achieve therapeutic effect, easy mutation of HBV

Active Publication Date: 2017-05-24
重庆高圣生物医药有限责任公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, there are two types of anti-HBV drugs in clinical application: interferon and nucleic acid analogues. Since HBV is prone to mutations, drug-resistant mutants are constantly emerging, making it difficult for existing anti-HBV drugs to achieve the desired therapeutic effect.

Method used

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  • CRISPR/Cas9 targeted knockout human hepatitis B virus P gene and specific gRNA thereof
  • CRISPR/Cas9 targeted knockout human hepatitis B virus P gene and specific gRNA thereof
  • CRISPR/Cas9 targeted knockout human hepatitis B virus P gene and specific gRNA thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0014] Example 1 Synthesis of gRNA targeting human hepatitis B virus P gene and construction of CRISPR / Cas9 system vector

[0015] 1. Selection and design of gRNA targeting human hepatitis B virus P gene

[0016] Find the sequence of human hepatitis B virus P gene in Genebank, and design potential target sites in human hepatitis B virus P gene.

[0017] Through the online design tool (http: / / crispr.mit.edu / ) and the design principles of gRNA, the target sites with higher scores on the human hepatitis B virus P gene sequence were evaluated to design gRNA.

[0018] 2. Synthesis of gRNA oligonucleotide sequence targeting human hepatitis B virus P gene and construction of eukaryotic expression vector

[0019] Digest the pSpCas9(BB)-2A-GFP (PX458) plasmid (Addgene plasmid ID: 48138, hereinafter referred to as pSpCas9(BB)) with BbSI, and after 1 hour in a water bath at 37°C, electrophoresis on 1% agarose to recover the digested product (TAKARA gel recovery kit).

[0020] The enzy...

Embodiment 2

[0033] Example 2 Transfection of HEPG2.2.15 cells

[0034] Resuscitate human liver cancer cells (HEPG2.2.15 cells, Shanghai Cell Bank, Chinese Academy of Sciences), put the cells into a culture flask with 10% FBS+DMEM, and store them at 37°C, 5% CO 2 Cells were cultured in an incubator, and the day before transfection, the cells were subcultured.

[0035] Aspirate the culture medium in the HEPG2.2.15 T75 bottle, add 2 mL of 0.25% trypsin taken out of the refrigerator at 4°C, make it evenly cover the bottom of the bottle, place it in the incubator at 37°C for 3-5 min, take it out, and shake it. When the cells are found to be detached from the bottom, shake them all down, add 3 mL of 10% DMEM preheated in a 37°C water bath, and blow and beat with a 10 mL pipette for 6 to 8 times without leaving any dead ends. The pipette can be aligned with the mouth of the bottle by blowing, and the medium can be pushed out with a small force to cover the cells close to the mouth of the bottle...

Embodiment 3

[0047] Example 3 ELISA detection of HBsAg and HBeAg levels in the supernatant of HepG2.2.15 cells

[0048] Cultivate HEPG2.2.15 cells and place the cells on a 6-well plate, and transfect the 8 high-efficiency targeted knockout hepatitis B virus vectors obtained in Example 2, respectively, into the cells on the 6-well plate, and make 3 copies of each vector Repeat the well, use the same amount of PBS to replace the carrier (positive control), and refer to Example 2 for the specific transfection method.

[0049] Change the medium 24 hours after transfection, take the cell supernatant, centrifuge to remove the cell bodies or debris in the supernatant, take the cell supernatant 72 hours after transfection, and centrifuge at 5000 rpm to remove the cell bodies or debris in the supernatant, according to Hepatitis B virus surface antigen (HBsAg) and e antigen diagnostic kit (HBsAg) (enzyme-linked immunoassay, Aikang Biotechnology Co., Ltd.) operating instructions, the supernatant stoc...

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Abstract

The invention relates to the technical field of genetic engineering, and particularly relates to a guide RNA (gRNA) sequence based on a CRISPR/Cas9 system and combinations thereof, as well as gRNA for a specific targeted knockout hepatitis B virus cccDNA P gene. In the invention, 30 gRNAs are designed according to design principles of CRISPR/Cas9, have a sequence table as shown in SEQ ID NO.1-30, and are constructed on a PX458 vector, to screen out four more efficient gRNAs. The CRISPR/Cas9 system guided by the four gRNAs and combinations thereof is utilized in a human hepatocellular carcinoma cell line (HepG2.2.15) to effectively knock out the human hepatitis B virus cccDNA P gene. The gRNA of the specific targeted hepatitis B virus cccDNA prepared in the invention can accurately target the hepatitis B virus cccDNA and realize gene knockout. The preparation method is simple in operation; the gRNA has a good targeting property; the CRISPR/Cas9 system is high in knockout efficiency.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a CRISPR / Cas9 method for specifically knocking out the P gene of human hepatitis B virus and a gRNA for specifically targeting the P gene of human hepatitis B virus. Background technique [0002] Hepatitis B virus (HBV) is a DNA virus belonging to the family Hepadnaviridae. HBV can cause chronic hepatitis, acute hepatitis, liver cirrhosis, liver cancer and other diseases. HBV is a worldwide epidemic disease. About 350 to 400 million people are infected with chronic HBV in the world. In my country, the infection rate of HBV is as high as 60% to 70%, and 93 million people are carrying hepatitis B virus. Among them, patients with chronic hepatitis B About 20 million cases. The purpose of hepatitis B treatment is to reduce liver lesions, prevent or delay the development of liver cirrhosis and liver cancer, and the ultimate goal is to completely eliminate the v...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/85
CPCC07K14/005C12N15/1131C12N15/85C12N2310/10C12N2730/10122C12N2810/10
Inventor 周勇申友锋
Owner 重庆高圣生物医药有限责任公司
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