Cloning And Expression Of Full Human Anti-HBs Antibody

Numerous new application of antibody for basic research as well as clinical use, have resulted from the development of engineering antibody. However, compared with nature human antibody, the Fab and ScFv fragment can't restore the avidity, effect functions due to the absence of Fc fragment; and the humanized mouse monoclonal antibody may induce HAMA because of its heterology. So it is important to establish a expression system for the cloning and expression of nature full human antibody.We created a full human antibody expression vector pPICZαIgG based on the vector pPICZα, and modified it by inserting of synthesized oligos and PCR mediated site-specific mutations, established the expression system of full human antibody in Pichia pastoris. Constructed the human heavy chain and light chain antibody expression library which derived from the HBsAg immunized lymphocyte, and screened the cDNA of human anti-HBs antibody from it by Western blot and ELISA. Then expressed anti-HBs antibody in the expression system. Identify the recombinant antibody, evaluate the advantage of Pichia pastoris expression system to express full human antibody.1. Construction of full human antibody expression system.(1)Construction of the heavy chain antibody expression vector pPICZαIgH and light chain antibody expression vector pPICZαIgκ: For many applications, it is useful to restore Fc mediated antibody functions such as avidity, effect functions and a prolonged serum half-life. So designed the primer to amplify human CH and CκcDNA, and cloned it into yeast expression vector pPICZα. Modified the cloning site for antibody variable region DNA. The results showed: The vector contained human constant region cDNA was constructed.(2)Construction of full human antibody expression vector pPICZαIgG: Assembled the expression cassette contained CH cDNA and the one contained CκcDNA into one vector, constructed the co-expression vector for convenient ,rapid expression of full human IgG.(3)Modification of the full human antibody expression vector pPICZαIgG: ①Using PCR mediated site-specific mutations, introduced new restriction site ClaI at 3' end of the expression cassette for the ligation of expression cassette with head-to-tail orientation. The results showed: The two(or more) expression cassette could assemble in correct orientation. ②The plasmid pPICZαIgG contained multimers by ligating two expression cassette, so it cannot be linearized because any enzyme that cuts in the 5' AOXI region will cut in all of the 5' AOXI regions present in the multimer. Through site-directed mutagenesis against PmeI site in the expression cassette contained CκcDNA, we created a mutant form of pPICZαIgG, which could be linearized. The results showed :Integration efficiency of the improved vector was increased significantly. So besides full human antibody, the improved vector could also express other multimers.(4)Identification of the full human antibody expression system: Cloned the human VH and VκcDNA into expression vector pPICZαIgH, pPICZαIgκ and pPICZαIgG. Analyzed the recombinant protein by Western blot after transformation. The results showed : the recombinant heavy, light chain and full human antibody of 55KD, 23KD and 140 KD molecular weight appeared. So the heavy chain, light chain and full human antibody can be secreted in the expression system. 2. Screening of anti-HBs antibody . (1)Construction of antibody expression libraries: Extracted total RNA from the HBsAg immunized lymphocyte, amplified all human VH and VκcDNA. Then inserted the VH and VκcDNA into yeast expression vector pPICZαIgH and pPICZαIgκ, prepared human heavy chain and light chain antibody expression library. DNA analysis indicated good diversity of the libraries.(2).Screening of anti-HBs antibody: Identified the culture supernatants by ELISA, obtained positive clone of anti-HBs heavy chain (light chain) antibody from 100 clones. Isolated total DNA from positive Pichia pastoris cells, amplified the VH and VκDNA. Compare ou...