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Construction and Expression in E. coli of scFv against Human Lung Adenocarcinoma
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Abstract:
Abstract Objectives: To construct a single-chain antibody (scFv) against lung adenocarcinoma and research its expressing conditions for establishing a clinical base. Methods: The variable genes of McAb anti-human lung adenocarcinoma, which were cloned from hybridoma cell WLA-2C4, were inserted into phagemid pFUW80 and expressed scFv in XL1-Blue. Also the scFv genes were inserted into another two vectors pTH and pTF. The corresponding host strains were Top10 and GI724/GI698, the inducing agents were IPTG and tryptophan, respectively. SDS-PAGE and ELISA methods were used to assay the expression products-phage antibodies in XL1-Blue and inclusion bodies in Top10 and GI724/GI698. Results: The scFv had the same specificity of the original monoclonal antibody against the antigen of A549 cell. The inclusion bodies were obtained at high yield 18.6% of the total E.coli proteins. Conclusion: A scFv against lung adenocarcinoma was successfully constructed and expressed in E. coli.
Keywords:
human lung adenocarcinoma ; single chain Fv antibody (scFv) ; phage antibody ; ELISA ; inclusion body ; renaturation
