Human anti-hepatitis B virus surface antibody as well as preparation method and application thereof

A surface antibody and anti-hepatitis B technology, applied in the field of genetic engineering, can solve the problems of incomplete representation of antibody specificity and affinity, errors, and high requirements for simulation calculation accuracy, so as to ensure operability, reliability, and high affinity active, overcoming a large number of cumbersome effects

Inactive Publication Date: 2015-04-22
JILIN UNIV
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Problems solved by technology

However, since the accumulation of existing protein spatial structure data is much smaller than that of the primary sequence data, and there are only slight differences between different antibodies, the accuracy of simulation calculations is high, so the antibodies obtained by purely calculation methods still have errors and cannot be obtained. Fully representative of antibody specificity and affinity

Method used

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  • Human anti-hepatitis B virus surface antibody as well as preparation method and application thereof
  • Human anti-hepatitis B virus surface antibody as well as preparation method and application thereof
  • Human anti-hepatitis B virus surface antibody as well as preparation method and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0060] Adopt the following steps to carry out the preparation of human source anti-HBV surface antibody:

[0061] (1) Construction of an antibody screening expression platform: based on the Pichia pastoris expression vector pPICZα, PCR-mediated positional mutagenesis technology was used to introduce restriction endonuclease sites into the primers and recombine them into the expression vector; clone the heavy chain and Light chain antibody constant region gene, and design the restriction endonuclease site (ApaI and CpoI) that can be connected with variable region, construct the single-chain antibody expression vector with constant region; Insert artificially synthesized oligonucleotide sequence, Design endonuclease sites (SalI and SpeI) for connecting the variable region with the yeast natural signal peptide, and construct a general expression vector that can secrete and express various heavy chain and light chain antibodies. The light chain expression vector is such as Figure ...

Embodiment 2

[0078] Detection of Antibody Affinity Activity by ELISA

[0079] According to the change of free energy before and after the mutation in Example 1, the selected amino acid was mutated, and the antibody mutant was constructed and linearized. Antibody light and heavy chains were sequentially transferred into Pichia pastoris in different combinations, and the expression was induced by methanol. The expression supernatant was taken, and the antibody affinity activity was detected by ELISA.

[0080] The ELISA plate was coated with recombinant human HBsAg vaccine as antigen, and blocked with 1% BSA. Add the culture supernatant of different clones on the 5th day, that is, the expressed HBsAb, and add the enzyme-labeled secondary antibody 1 hour later, and the chromogenic solution will develop the color. Finally, the absorbance value (A490) at 490nm was detected by a microplate reader to judge the antigen-antibody binding activity.

[0081] When the light chain amino acids of the a...

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Abstract

The invention relates to a human anti-hepatitis B virus surface antibody as well as a preparation method and application thereof. A specific hepatitis B antibody with relatively high affinity activity on hepatitis B virus antigens can be obtained by virtue of antigen-antibody binding activity screening, wherein an amino acid sequence of a light chain variable region of the antibody is shown in SEQ ID No. 1 or SEQ ID No. 2, and an amino acid sequence of a heavy chain variable region of the antibody is shown in SEQ ID No. 3 or SEQ ID No. 4. According to the human anti-hepatitis B virus surface antibody disclosed by the invention, antibody mutation hot spots are analyzed by virtue of a protein molecule modeling technology and are subjected to saturation mutation by taking an amino acid as a unit so as to construct a mutant library and perform biological verification, and thus optimization of the antibody can be achieved. The human anti-hepatitis B virus surface antibody disclosed by the invention can be used for laying a foundation for preventing hepatitis B virus infection and performing treatment research at the same time, so that the human anti-hepatitis B virus surface antibody can be applied to the prevention and treatment of liver diseases caused by hepatitis B virus infection clinically.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a human anti-hepatitis B virus surface antibody and its preparation method and application. Background technique [0002] Acute and chronic hepatitis B caused by hepatitis B virus (HBV) infection is one of the serious infectious diseases that seriously endanger human health and may induce liver cancer. So far, there is no specific drug that can be used to completely control its infection and epidemic break out. Anti-HBV antiserum has been proven to have anti-viral infection effects, especially in blocking mother-to-child transmission and preventing HBV re-infection after liver transplantation, but blood-derived antiserum has the potential to spread other diseases. There are also shortcomings such as low specificity and poor neutralization effect, so the research on anti-HBV recombinant antibody has important social application value. [0003] Existing genetically en...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/08C12N15/13C12N15/81C12N1/19A61K39/42A61P31/20A61P1/16G01N33/576G01N33/569C12R1/84
Inventor 宿晓云
Owner JILIN UNIV
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