Anti-hepatitis B virus surface antigen completely humanized human antibody and use thereof

A fully human antibody and surface antigen technology, applied in antiviral agents, antiviral immunoglobulins, antibodies, etc., can solve the problems of uncertain risk factors, limited production methods, and complex components of hepatitis B immunoglobulins. The effect of low immunogenicity

Inactive Publication Date: 2015-11-18
SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] However, there are many unsafe factors in the current HBIG as a source of therapeutic antibodies
At the same time, the composition of hepatitis B immunoglobulin is complex, the risk factors are relatively uncertain, and the production method is limited. These factors restrict the clinical use of hepatitis B therapeutic antibodies [3]

Method used

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  • Anti-hepatitis B virus surface antigen completely humanized human antibody and use thereof
  • Anti-hepatitis B virus surface antigen completely humanized human antibody and use thereof
  • Anti-hepatitis B virus surface antigen completely humanized human antibody and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1: Anti-HBV surface antigen fully human antibody A 3 D. 5 preparation of

[0030] 1. Cloning of antibody light chain constant region and heavy chain constant region genes and construction of their vectors

[0031] Isolate healthy human lymphocytes with lymphocyte separation medium, extract total RNA with Trizol reagent, design primers according to the sequences reported in literature [4] and literature [5], and use QIAGEN OneStep RT-PCRKit to amplify the heavy chain and light chain constant regions of human antibodies Gene. The heavy chain and light chain constant region genes of the human antibody were respectively connected to the expression vector AbVec plasmid, and the antibody heavy chain constant region nucleotide vector IgG-AbVec plasmid and the antibody light chain constant region nucleotide vector Igκ-AbVec were respectively constructed Plasmid, after sequencing verification, it was confirmed that the correct clone was obtained. Wherein, the nucleotid...

Embodiment 2

[0040] Example 2: Anti-HBV surface antigen fully human antibody A 3 D. 5 specificity

[0041] Detection of fully human monoclonal antibody A by Elisa method 3 D. 5 Specifically binds to HBsAg protein.

[0042] The recombinant HBsAg protein and the control protein cIg were coated on the ELISA plate, and after being blocked by the blocking solution, the blank group, different concentrations of the control protein cIg, and different concentrations of expressed and purified A 3 D. 5 Antibody incubation, adding Goatanti-humankappa-HRP after washing the plate, TMB chromogenic solution for color development, microplate reader reads at 450nm wavelength.

[0043] Draw according to antibody concentration and corresponding OD value figure 1 ,Depend on figure 1 The analysis shows that with the increase of the antibody concentration, the corresponding reading value of the HBsAg protein group is also correspondingly increased. However, the reading value of the coated control protein...

Embodiment 3

[0044] Example 3: Anti-HBV surface antigen fully human antibody A 3 D. 5 Binding epitope identification

[0045] Combining the antigenic epitope region of hepatitis B surface antigen (HBsAg) with neutralizing activity reported in previous literature, a biotin-labeled short peptide of hepatitis B surface antigen was synthesized to measure the binding of fully human hepatitis B surface protein monoclonal antibody on HBsAg area. Such as figure 2 As shown in the HBsAg pattern diagram, the synthetic four biotin-labeled HBsAg short peptide P 1 (aa:104-120), P 2 (aa:121-137), P 3 (aa:139-148), P 4 (aa:149-163), the amino acid sequences of P1-P4 are shown in SEQ ID NO.9-SEQ ID NO.12. Among them, the antigenic short peptide P 1 and P 4 is a linear structure, P 2 and P 3 ring structure [6] .

[0046] A was analyzed by ELISA 3 D. 5 The binding epitope of the monoclonal antibody, composed of image 3 It can be seen that A 3 D. 5 Antibody binds to P 3 Location.

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Abstract

The invention provides an anti-hepatitis B virus surface antigen completely humanized human antibody A3D5 and a gene for encoding the antibody. Experiments show that the antibody A3D5 can specifically bind to an HBsAg protein, and has good HBV neutralization activity in order to possibly prevent HBV infection related hepatitis, hepatic cirrhosis and liver cancer. The antibody is a completely humanized human antibody obtained through cloning HBsAg specific memory B cells in the peripheral blood of hepatitis B vaccine inoculated volunteer, has lower immunogenicity than murine, chimeric and humanized antibodies, and can be used to prepare hepatitis B virus related hepatopathy prevention or treatment drugs or diagnose reagents.

Description

technical field [0001] The invention belongs to the field of biotechnology. More specifically, the invention discloses the preparation technology of anti-hepatitis B surface protein antibody and its application in preventing HBV infection and treating HBV-related hepatocellular carcinoma. Background technique [0002] Hepatitis B virus (HBV) is a double-stranded DNA virus, which can easily cause chronic hepatitis B after infection in the human body, and then lead to liver cirrhosis and (or) liver cancer. According to statistics, in recent years, there are about 350 million HBV-infected people in the world, and about 1 million to 1.5 million people die each year from acute or chronic HBV infection-induced liver failure, liver cirrhosis and / or liver cancer. Therefore, preventing HBV infection has become a worldwide public health problems [1] . Hepatocellular carcinoma (HCC, referred to as liver cancer) is closely related to viral hepatitis, is one of the most important cause...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/08C12N15/13A61K39/42A61P31/20G01N33/569
Inventor 李博华孟艳春王华菁杨扬于晓杰
Owner SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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