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
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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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