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Separating method of hepatitis B virus

A technology of hepatitis B virus and separation method, applied in the field of hepatitis B virus separation, can solve the problems of inability to effectively block HBV replication and reinfection, lagging research progress, affecting new treatment methods and drug research and development, etc.

Inactive Publication Date: 2013-03-27
冉兵
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AI Technical Summary

Problems solved by technology

Chronic hepatitis B still lacks effective means of control and a complete solution. Antiviral drugs commonly used in clinical practice have limited efficacy and cannot effectively block the replication and reinfection of HBV in liver tissue
The relative lag in the research progress of the molecular mechanism of the interaction between HBV and liver cells is one of the important reasons that seriously affect the development of new treatments and drugs

Method used

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Embodiment Construction

[0006] 1. Different gene fragments of PreS1 were amplified by PCR method, cloned into eukaryotic secretory expression vector pCMV-CFT with Flag tag, and the Flag-PreS1 fusion protein was further isolated and purified.

[0007] 2. Take the normal liver tissue (about 40 g) of the patient undergoing partial hepatic lobectomy, separate hepatocytes according to the two-step collagenase perfusion method reported by Protzer U et al., culture and identify them.

[0008] 3. Fully incubate the purified and expressed Flag-PreS1 fusion protein with primary human hepatocyte (PHH) lysate, the fusion protein and the binding protein complex on the liver cell membrane can pass through the FITC-labeled anti-Flag monoclonal antibody (Sigma) specific binding, the cell-binding activity of the HBV PreS1 fragment expressed in fusion to PHH was detected by flow cytometry.

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Abstract

The invention relates to a separating method of hepatitis B virus. The hepatocyte surface HBV PreS1 binding protein is searched by an immuno-precipitation technology, the receptor gene is separated and identified by a protein time flight mass spectrum and a bioinformatics method, a candidate receptor gene is further cloned, the essence of the virus receptor is probed from the molecular level, and the relationship between the virus and the host cell receptor is disclosed, so that the separation and the identification can be realized.

Description

【Technical field】 [0001] The invention relates to a method for isolating hepatitis B virus, especially HBV has strict host susceptibility and hepatotropism, and the specific peptides of HBV surface antigen in the early stage of infection recognize and bind to specific receptors on liver cells to mediate virus entry cells to initiate the HBV replication cycle. Therefore, the receptor of the virus is the portal for HBV to enter the liver cells, and blocking the combination of HBV and its receptor is one of the important ways to treat hepatitis B. 【Background technique】 [0002] Hepatitis B virus (Hepatitis B Virus, HBV) infection is one of the serious global health problems at present, about 350 million people in the world are infected with HBV, accounting for 6% of the total population. Among them, a considerable number of infected persons develop chronic hepatitis, and a few also develop liver cirrhosis. Chronic hepatitis B still lacks effective means of control and a comp...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/705C07K1/14G01N33/68G01N27/62
Inventor 冉兵
Owner 冉兵
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