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Preparation method and application of completely humanized monoclonal antibody aiming at hepatitis B virus (HBV) surface protein

A monoclonal antibody, surface protein technology, applied in the direction of antibodies, antiviral immunoglobulins, applications, etc., can solve the problems of uncertain risk factors, complex components of hepatitis B immunoglobulin, limited production methods, etc., and achieve low immunogenicity. sexual effect

Inactive Publication Date: 2015-11-04
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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  • Preparation method and application of completely humanized monoclonal antibody aiming at hepatitis B virus (HBV) surface protein
  • Preparation method and application of completely humanized monoclonal antibody aiming at hepatitis B virus (HBV) surface protein
  • Preparation method and application of completely humanized monoclonal antibody aiming at hepatitis B virus (HBV) surface protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1: Fully Human Antibody B against Hepatitis B Surface Antigen 5 h 6 preparation of

[0029] 1. Cloning of antibody light chain constant region and heavy chain constant region genes and construction of their vectors

[0030]Lymphocytes from healthy people were isolated with lymphocyte separation medium, total RNA was extracted with Trizol reagent, primers were designed according to the sequences reported in literature [4] and literature [5], and the heavy chain and light chain of human antibodies were amplified using the QIAGEN OneStep RT-PCR Kit Chain constant region 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...

Embodiment 2

[0040] Example 2, Fully Human Antibody B against Hepatitis B Surface Protein 5 h 6 specificity

[0041] Detection of fully human monoclonal antibody B by Elisa method 5 h 6 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 B 5 h 6 Antibody incubation, adding Goat anti-human kappa-HRP after washing the plate, TMB chromogenic solution for color development, and microplate reader reading at a wavelength of 450nm.

[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 co...

Embodiment 3

[0044] Example 3: Fully Human Antibody B against Hepatitis B Surface Protein 5 h 6 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

[0046] (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] .

[0047] B was analyzed by ELISA 5 h 6 The binding epitope of the monoclonal antibody, composed of image 3 It can be seen that B 5 h 6 Antibody binds to P 2 ...

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Abstract

The invention provides a completely humanized monoclonal antibody B5H6 aiming at hepatitis B virus (HBV) surface protein and a gene for encoding the antibody. An experiment shows that the antibody B5H6 can be specifically combined with HBsAg protein, has relatively good HBV neutralization activity and then can resist the progresses of HBV infection related hepatitis, liver cirrhosis and liver cancers. Meanwhile, because the antibody is a completely humanized monoclonal antibody cloned from memory B cells with specific HBsAg in peripheral blood of a volunteer vaccinated with a hepatitis B vaccine, the antibody has immunogenicity lower than that of murine, chimeric and humanized antibodies, and can be used for preparing medicaments or diagnostic reagents for preventing or treating hepatitis B virus related hepatic diseases.

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 causes ...

Claims

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Application Information

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IPC IPC(8): C07K16/08C12N15/13C12N15/85C12N5/10A61K39/395A61P31/20G01N33/577
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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