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Monoclonal antibody of hepatitis B virus X protein and use thereof

A monoclonal antibody and protein technology, applied in antiviral immunoglobulins, antibodies, antiviral agents, etc., can solve the problems of low efficiency of monoclonal antibodies and weak immunogenicity of HBx

Inactive Publication Date: 2011-04-27
王虹
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the preparation methods of HBx monoclonal antibodies mostly use genetic engineering to express and isolate HBx full-length protein as the antigen for immunizing animals and screening monoclonal antibodies, which has the problem of weak autoimmunogenicity of HBx and low efficiency of obtaining monoclonal antibodies

Method used

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  • Monoclonal antibody of hepatitis B virus X protein and use thereof
  • Monoclonal antibody of hepatitis B virus X protein and use thereof
  • Monoclonal antibody of hepatitis B virus X protein and use thereof

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0033] Example 1, Screening of HBx Monoclonal Antibody

[0034] 1. Synthesis of HBx N-terminal and C-terminal antigenic epitope polypeptides: Antigenic Peptides antigenic epitope analysis online software was used to analyze the antigenic epitope of the amino acid sequence of HBx protein. The results showed that the HBx protein N-terminal 11-20 amino acid sequence PARDVLCLRP and the HBx protein C-terminal 127-150 amino acid sequence IRLKVFVLGGCRHKLVCSPAPCNF were segments with higher epitope index. The HBx N-terminal and C-terminal antigenic epitope polypeptides shown in polypeptide sequence 1 (SEQ 1) and polypeptide sequence 2 (SEQ 2) were synthesized by artificial or chemical synthesis method respectively.

[0035] 2. Cross-linking of HBx N-terminal or C-terminal antigenic epitope polypeptide and carrier protein keyhole limpet hemocyanin (KLH): dissolving maleimide-activated keyhole limpet hemocyanin (KLH) maKLH in water (Themo Scientific company product), so that the concent...

example 2

[0039] Example 2, Preparation, purification and identification of HBx monoclonal antibody

[0040]1. Preparation and purification of HBx monoclonal antibody: For large-scale preparation of monoclonal antibody, hybridoma cells were inoculated into the peritoneal cavity of mice, and the ascites of mice was collected to obtain antibodies. Antibody purification can be carried out by methods such as salting out, gel filtration, and affinity chromatography. We prefer the ammonium sulfate-octanoate precipitation method for purification. (1) Preparation of ascites: first intraperitoneally inject 0.5ml Pristane (pristane) or liquid paraffin into BaLb / c mice, and then intraperitoneally inject 1×10 6 Ascites can be produced 7 to 10 days after the inoculation of cells. Closely observe the health status of the animals and signs of ascites. When the ascites is as much as possible, and the mice die frequently, the mice are killed and the ascites is collected. (2) Purification of monoclonal...

example 3

[0046] Example three, HBx monoclonal antibody is used to prepare HBx protein enhanced chemiluminescence immunoassay kit

[0047] 1. HBx N-terminal and C-terminal epitope-specific monoclonal antibody pairing experiment: HBx N-terminal epitope-specific monoclonal antibodies N1E3, N2F2, N2G4 and HBx C-terminal epitope-specific monoclonal antibodies C3B2, C5E8 Perform reciprocal pairing experiments. Experiment 1: Use 50 μl of 20 μg / ml N-terminal epitope-specific monoclonal antibodies N1E3, N2F2 and N2G4 as capture antibodies to coat microwell plates, and the coating buffer is carbonate buffer (PH9.5). HRP-labeled HBx C-terminal epitope-specific monoclonal antibodies C3B2 and C5E8 were used as detection antibodies, adding serially diluted recombinant HBx protein and HBx positive quality control serum, washing with washing solution 3 times to wash off non-specific binding antigens, adding HRP-labeled 1E34 and 3B23 monoclonal antibodies were used for paired comparison experiments, a...

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Abstract

The invention relates to a monoclonal antibody of hepatitis B virus X protein (HBx), which is characterized by having specificity reaction to N-end epitope or C-end epitope of HBx, but having no reaction to keyhole limpet hemocyanin (KLH) of carrier protein and other expressed proteins of hepatitis B virus (HBV). The preparation method of the monoclonal antibody comprises the steps of: fusing N-end and C-end antigen epitope polypeptides of HBx respectively with mice immunized with KLH in crosslinking way, and myeloma cells and screening to obtain the monoclonal antibody. The monoclonal antibody can be used for various immunodetection reagents and directed therapeutic drugs of HBx protein or HBx antibody and applied to diagnosis and treatment of diseases such as hepatitis b virus (HBV) infection, hepatocellular carcinoma (HCC) and the like.

Description

technical field [0001] The present invention relates to a monoclonal antibody, specifically a monoclonal antibody specific to hepatitis B virus X protein (HBx), and the use of the monoclonal antibody in the preparation of HBx immunoassay reagents and drugs for treating liver diseases . Background technique [0002] my country is an area with a high incidence of hepatitis B virus (HBV) infection, and more than 10% of people carry hepatitis B virus. HBV infection is the main factor causing liver diseases such as liver fibrosis, liver cirrhosis and liver cancer. In my country, currently about 80% of patients with hepatocellular carcinoma (HCC) are infected with hepatitis B virus (HBV), and the risk of developing HCC in HBV-infected patients is more than 200 times that of those without infection. [0003] The latest research shows that HBV X protein (HBx) has a wide range of gene transcription regulation, which is closely related to the development of HCC in chronic HBV infect...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/08G01N33/577A61K51/10A61K47/48A61K45/00A61K39/42A61P31/20A61P1/16A61P35/00A61K47/64
Inventor 王虹
Owner 王虹
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