抗人HAb18G分子单链抗体基因的构建及在大肠杆菌中的分泌表达

Construction and secretory expression in E.coli of anti-HAb18G molecule ScFv gene

目的 :构建抗人HAb18G分子单链抗体基因 ,并在大肠杆菌中进行分泌表达。方法 :通过连接肽 (Gly4Ser) 3基因序列将已成功克隆的抗人肝癌单抗HAb18轻、重链可变区基因拼接成单链抗体 (ScFv)基因 ,并在 5′和 3′端引入相应的酶切位点 ,克隆至改造的分泌性表达载体pCANTAB 5His之中 ,转化到E coliHB2 15 1中进行诱导表达。亲和层析纯化表达蛋白后 ,以SDS PAGE电泳、Westernblot及ELISA等方法分析检测表达产物。结果 :经限制性酶切鉴定及DNA测序分析 ,证实基因构建正确。SDS PAGE电泳和Westernblot分析表明ScFv基因在大肠杆菌中成功表达。表达产物的相对分子质量 (Mr)为 31kD ,与理论预期值相符 ,并且可与相应的抗原特异结合。结论 :成功实现了抗人HAb18G分子单链抗体的原核分泌表达 ,为其在人肝癌诊治中的进一步应用奠定了基础。

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.