人前列腺干细胞抗原的克隆及表达

Cloning and expression of human prostate stem cell antigen

目的克隆人前列腺干细胞抗原(PSCA)编码基因,并在大肠杆菌中表达PSCA蛋白.方法从培养的人前列腺癌细胞株中提取总RNA,反转录成cDNA,通过聚合酶链反应(PCR)方法扩增PSCA编码基因,克隆至pMD18-T载体,经DNA测序证实后亚克隆至谷胱甘肽巯基转移酶(GST)融合表达载体pGEX-5X-1,重组质粒转化大肠杆菌,异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达PSCA/GST融合蛋白.结果从培养的人前列腺癌细胞株中扩增出编码PSCA的cDNA(241 bp),序列测定证实与Genebank上登录的序列一致;成功构建了GST融合表达载体pGEX-5X-1/PSCA;表达了PSCA/GST融合蛋白(34.5×103).结论成功克隆了人PSCA基因,构建了GST融合表达载体并表达了PSCA/GST蛋白.

Objective To clone the cDNA of human prostate stem cell antigen (PSCA) and ex- press the corresponding protein. Methods Total RNA was isolated from cultured human prostate cancer cell lines and mRNA was reversly transcribed into cDNA. Then PCR was used to amplify the PSCA cod- ing region. The PCR product was cloned into pMD18-T plasmid and sequenced, then subcloned into vec- tor pGEX-5X-1. The PSCA protein was expressed in E. coli of JM109 as fusion protein with glutathione S-transferase (GST) induced by IPTG. Results PSCA coding region (241 bp) was cloned into pMD18- T and the sequence was conformed; Fusion expressing vector of pGEX-5X-1/PSCA was successfully con- structed and correctly expressed PSCA/GST fusion protein (34. 5 ×10~3 ). Conclusion Human prostate stem cell antigen gene is successfully cloned and PSCA/GST fusion expressing vector is constructed. PSCA/GST fusion protein is correctly expressed in E. coli.