肿瘤相关抗原Trop-2胞外区片段蛋白的表达及其特性分析

Expression and characterization of Trop-2 extracellular domain protein of tumor relative antigen

目的:从临床乳腺癌组织中扩增肿瘤相关抗原Trop-2胞外肽段基因,在大肠杆菌中表达重组蛋白,并对该蛋白的生物学特性进行鉴定。方法:RT-PCR扩增Trop-2胞外肽段基因并克隆于pMD18-T载体中进行测序分析,用NcoⅠ和EcoRⅠ将含Trop-2胞外区重组基因的质粒双酶切后,克隆到pBAD-gⅢ中。重组质粒经酶切鉴定、核酸序列分析后转化大肠杆菌TOP10,以L-阿拉伯糖诱导表达融合蛋白,表达的蛋白经后His柱亲和层析纯化后,用Western blot、ELISA检测鉴定。结果:重组表达质粒经酶切鉴定为阳性,核酸序列分析正确。SDS-PAGE和Western blot显示,表达的重组蛋白分子质量约为38ku,此抗原能够与商业化抗体特异性结合。结论:成功制备Trop-2胞外区重组基因表达的蛋白,并证明原核表达的Trop-2胞外肽段保持了原有的抗原性。

Objective：To amplifiy of Trop-2 gene from clinic tumor tissue and express Trop-2 protein in E.coli. TOP10,and an alyze its immunological characters with Western blot and ELISA. Methods：The Trop-2 gene was amplifiied by RT-PCR and cloned into pMD18-T. After sequenced,the Trop-2 gene was digested with NcoI and EcoRI and cloned into expression vector pBAD-gⅢ. The constructed vector had been identified by nucleotide sequence analysis,and then transformed into E.coli. TOP10. After induced with L-arabinase,the recombinant protein was purified with HisTrap affinity chromatography,and confirmed by Western blot and ELISA. Results：The Trop-2 was amplicated and sequenced,and the recombinant plasmid was constructed correctly. SDS-PAGE analysis showed that the recombinant protein was about 38 ku. The purified protein could be used as an antigen to detect the specific antibodies with Western blot and ELISA. Conclusion：The recombinant extracellular protein of Trop-2 could be expressed with high performance, and the antigenicity of the purified protein was certificated with the specific antibody.