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Human anti-HBV surface antigen genetic engineering antibody, and preparation method and application thereof

A technology of genetically engineered antibodies and surface antigens, applied in genetic engineering, antiviral agents, antiviral immunoglobulins, etc., can solve problems such as mass production limitations, limited applications, and infections

Active Publication Date: 2014-02-19
中国疾病预防控制中心病毒病预防控制所
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  • Abstract
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AI Technical Summary

Problems solved by technology

However, most McAbs are of mouse origin, and the heterogeneous reaction of mouse McAbs greatly limits the application of McAbs as therapeutic agents in humans.
As an antibody component, immunoglobulin (Vaccinia immune globulin, VIG) mainly comes from the immune serum of donors (patients in convalescent stage). It takes a lot of manpower and financial resources to obtain positive serum and pass safety testing, which makes its mass production difficult. At the same time, because it comes from serum, it is prone to infection of blood-borne diseases

Method used

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  • Human anti-HBV surface antigen genetic engineering antibody, and preparation method and application thereof
  • Human anti-HBV surface antigen genetic engineering antibody, and preparation method and application thereof
  • Human anti-HBV surface antigen genetic engineering antibody, and preparation method and application thereof

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Experimental program
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Embodiment 1

[0032] Embodiment 1 Materials and methods

[0033] 1. Viruses, cells, and carriers: Hepatitis B virus HBV was provided by the Hepatitis Department of the Institute of Viral Disease Prevention and Control, Chinese Center for Disease Control and Prevention. Human liver cancer cell line HepG2 (ATCC); insect cells Sf9 were from American Cell Culture Center (ATCC). The strain is XLI-Blue (Stratagene, USA), and the vector is pComb3H (40kb), donated by Scripps Research Institute, USA. Baculovirus expression vector is pAC-K-Fc (German PROGEN PR3003) (Liang, M.F., Stefan, D., Li, D.X., Queitsch, I., Li, W., and Bautz, E.F. Baculovirus expression cassette vectors for rapid production of complete human IgG from phage displayselected antibody fragments. Journal of Immunological Methods. 247:119-130). Hepatitis B surface antigen (CHO-HBsAg) was purchased from Beijing Wantai Biological Pharmaceutical Company; mouse anti-hepatitis B surface antigen monoclonal antibody 3E7 (Mouse Anti-Hepat...

Embodiment 2

[0060] Embodiment 2 result and analysis

[0061] 1. Screening of human anti-HBsAg antibody library

[0062] Successfully built 4 Kappa libraries and 4 Lambda libraries, the library capacity is 1×10 7 Above, the light and heavy chain insertion rate is above 95%. The Kappa sub-library and the Lambda sub-library were packaged separately. After three rounds of screening with purified hepatitis B surface antigen, it was found that the Kappa sub-library was enriched in three rounds of screening, and the capacity of the eluted library gradually increased, while the Lambda sub-library did not appear. Pick 100 Kappa sub-bank clones and 100 lambda sub-bank clones, ELISA results showed that there were 30 HBsAg-positive clones, all from the Kappa sub-bank, and 10 antibody clones with different sequences were found by sequencing, as shown in Table 2:

[0063] Table 2 Output of antibody library packaging and enrichment screening

[0064]

[0065] 2. Sequence analysis of human anti-HBs...

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Abstract

The invention discloses a human anti-HBV surface antigen genetic engineering antibody, and a preparation method and application thereof. According to the invention, the phage surface display technology is adopted, peripheral blood lymphocyte of person having high titer surface-antibody after immunization of hepatitis B vaccine is collected is obtained, a human anti-HBV surface antigen genetic engineering antibody library is established through a genetic engineering means, and a Fab section of the specific anti-HBV surface antigen genetic engineering antibody is obtained through screening. The obtained Fab section of the antibody is named to be HBFab21. The amino acid sequence of a light chain variable region of the HBFab21 is shown in SEQ ID No.1, and the amino acid sequence of a heavy chain variable region of the HBFab21 is shown in SEQ ID No.2. According to the invention, a foundation for hepatitis B virus infection prevention and researches on treatment to hepatitis B virus is laid; through the foundation, an antibody product having an effect of neutralizing hepatitis B virus infection is obtained, and can be applied to preparation of a clinical drug or diagnostic reagent for liver diseases relating to hepatitis B virus.

Description

technical field [0001] The invention relates to genetic engineering antibody technology, in particular to a human-sourced anti-hepatitis B virus surface antigen genetic engineering antibody; the invention also relates to the application of the antibody in the preparation of drugs for preventing or treating hepatitis B virus-related liver diseases. Background technique [0002] Hepatitis B virus infection is a major public health problem worldwide. After acute hepatitis B, 5%-10% of persistent HBV infection develops into chronic liver diseases including chronic active hepatitis, liver cirrhosis and primary hepatocellular carcinoma. It is estimated that 500,000 to 1,000,000 people worldwide die each year from various liver diseases related to HBV. my country is a big country with hepatitis, and the carrier rate of hepatitis B virus is 9.75% of the population. There are 420 million hepatitis B virus infected people in the world, and about 130 million people in my country are c...

Claims

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Application Information

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IPC IPC(8): C07K16/08C12N15/13C12N15/866C12N5/10A61K39/42A61K48/00A61P31/20A61P1/16G01N33/569
Inventor 梁米芳毕胜利孙丽娜郭瑜刘洋张福顺李川李德新
Owner 中国疾病预防控制中心病毒病预防控制所
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