Studies On Recombinant Human Anti-rabies Antibody

Rabies is an acute rabies viral infectious disease of the central nervous system affecting almost all mammals, including humans. And it is considered to be universally fatal. The most effective postexposure prophylaxis should always include a combination of active and passive immunization, advocated by the World Health Organization. Administration of anti-rabies virus serum in conjuction with vaccine is recommended. But the shortage in supply, side effects as heterologous protein and the risk of transferring blood-borne diseases limit the application of anti-rabies virus serum as postexposure therapy. With the development of gene engineering, recombinant antibody technology has provided new ideas. The fragment antigen binding (Fab) and the single-chain Fv (scFv) of humanized antibody to rabies virus are stably produce in E. coli. But they can not function the same way as intact immunoglobulin for they lack the integrated actions including antibody dependent cellular cytotoxicity and antibody mediated cell disruption. For further study the production of intact immunoglobulin to rabies virus is essential.In the 20th century, numerous recombinant antibodies have resulted from the development of engineering antibody and the clarity of gene construct. As the rising of phage display library, the screen of high-quality and high-affinity antibodies have been made as possible. But it is difficult to obtain high-affinity antibodies because the modifying of recombinant antibodies is absent in phage display library. So, we want to mend a expression system of antibodies library for the cloning and expression of natural human scFv-Fc antibody. And then, study has been done on the large-scale fermentation process of RS3 which is a scFv-Fc antibody against rabies antigen in the New Brunswick Scientific bioflow 5000 fermentor, and we create a method to purify RS3 by SP Sepharose XL and Phenyl Sepharose 6 Fast Flow. The general expression vectors are intact human antibody heavy chain expression vector pPICZαCH and light chain expression vector pPICZαCκwhich were prepared by our laboratory previously. And the template is the specific monoclonal antibody RS3 that are from human anti-rabies virus scFv-Fc small molecule antibody library.1.Construction of yeast expression and screen system library of scFv-Fc(1) Construction of human micromolecule antibody scFv-Fc expression vector pPICZα/Fc: 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 human antibody expression vector pPICZα/Fc-scFv: The heavy chain antibody expression vector pPICZαIgH and light chain antibody expression vector pPICZαIgκare obtained from the lymphocytes that immuned with the antigens of rabies virus. 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 antibody expression vector pPICZα/Fc-scFv.(3) Identification of human antibody pPICZα/Fc-scFv expression system: Cloned the VH and VκcDNA of human anti- rabies virus antibody into the improved full human antibody expression vector pPICZα/Fc-scFv, established the expression system of full human anti- rabies virus antibody in Pichia pastoris X-33. Analyzed the recombinant protein by using ELISA,SDS-PAGE and Western blot after transformation. The results showed : the recombinant micromolecule antibody of 55 KD molecular weight appeared. So human anti- rabies virus micromolecule antibody with high affinity can be secreted in the expression system.2.Studies on pilot-scale fermentation and purification process of scFv-Fc(1) Studies on large-scale fermentation process of scFv-Fc:Pichia pastoris has many advantages as a kind of expression host, and it's very suitable for large-scale expression of the extraneous proteins. So after identifying the bioactivities of the scFv-Fc that is recombinant RS3 can bind to rabies virus antigen, we explored the large-scale fermentation process of scFv-Fc and found that the best pH is pH5.4±0.2, DO between 20%～30% and the supply speed of methanol is 9～9.5ml/h/L initial fermentation volume. The concentration of scFv-Fc in the broth can reached 50～70mg·L-1.(2). The method to purify RS3 at large-scale:We found that 40% saturation of (NH4)2SO4 (pH6.0) was suitable to purify RS3 with Phenyl Sepharose 6 Fast Flow. Then the eluate could be purified by SP Sepharose XL on pH4.0. This method overcame the shortcomings of the affinity chromatography and anion chromatography, and the yield coefficient was higher than 60%. 3.Expression of the recombinant full human anti-rabies virus antibody(1) expression of recombinant full heavy chainBy using recombinant technology, cDNA of the heavy chain and light chain of anti-rabies virus micromolecule antibody were integrated to the vector pPICZαCH and pPICZαCκpreviously prepared. The recombinant vector was transformed to Pichia pastoris induced by methanol. The supernatant was analysied by using ELISA,SDS-PAGE and Western-blot. The result showed that the highest expression appeared at 72 hours.(2) expression of the recombinant full human anti-rabies virus antibodyThe positive plasmid was picked and transformed to Pichia pastoris which were cultured under different concentration of Zeocin condition. Pichia pastoris was induced by methanol and the supernatant was analysed by using ELISA and SDS-PAGE and immuno-blotting. The result showed that the highest expression reached at 96 hours. Then the purified products were ananysed by using Western-blot. The result showed 55kD and 25kD specialized bands under reduction condition and 150kD band under nonreduction condition. The purified rabies virus antigen was dealt with the same way and there appeared 57kD band. All those proved the expression of secreted full immunoglobin.