Construction and secretory expression in E.coli of anti-HAb18G molecule ScFv gene / 中国免疫学杂志

ABSTRACT

Objective:To construct a gene of ScFv against human HAb18G molecule and secretively express it in E coli Methods:The V H and V L genes cloned from McAb HAb18 hybridoma cell were ligated with a flexible linker(Gly4Ser) 3 to construct ScFv gene Then corresponding restriction endonuclease digestion site was introduced into 5′and 3′ end of ScFv gene Finally, it was cloned into expression vector pCANTAB 5His and expressed in E coli HB2151 Expression proteins were purified by affinity chromatography and detected by SDS PAGE electrophoresis?Western blot and ELISA Results:Restriction endonuclease digestion and DNA sequencing proved that ScFv gene was correctly cloned into expression vector SDS PAGE electrophoresis and Western blot analysis showed that ScFv was successfully expressed in E coli HB2151 The relative molecular mass (Mr) of the expression products was 31 kD, according with its predicted Mr value And ELISA assured that expression products had antigen specific binding activity Conclusion:The successful expression of anti HAb18G ScFv gene in E coli laid a solid foundation for its further application in diagnosis and therapy of human hepatoma.