摘要
目的:克隆人膀胱癌UROC28基因并在原核细胞 中表达.方法:用RT PCR从人膀胱癌患者组织中扩增 UROC28基因cDNA,并克隆到载体pUC 19中.经测序证实 后,用HindⅢ/EcoRI双酶切,亚克隆到原核表达载体pGEX 4T 1,并转化E.coliDH5α菌株.取工程菌,用IPTG诱导表 达,对表达产物进行SDS PAGE鉴定.结果:①经RT PCR、测 序和酶切鉴定,成功地克隆了人膀胱癌UROC28基因;②经 IPTG诱导的重组质粒pGEX 4T UROC28表达出Mr约为 42000的融合蛋白,与预期的结果相符.结论:成功克隆到人 膀胱癌UROC28基因,并在E.coliDH5α中表达出GST UROC28融合蛋白.
AIM: To clone UROC28 gene of human bladder cancer and to express it in E. coli DH5α. METHODS: The fragment of UROC28 gene was amplified by RT-PCR from human bladder cancer tissue and cloned to pUC19. The DNA fragment from pUC19-UROC28 digested with HindⅢ and EcoRⅠ was ligated to XhoⅠ/EcoRⅠ-digested prokaryotic expression vector pGEX-4T-1. The expression of fusion protein was induced with IPTG and the expressed product was identified by SDS-PAGE. RESULTS: ① The sequencing and endonucleases digestion analysis showed that the fragment of UROC28 gene cDNA was inserted into the vectors pUC19 and pGEX-4T-1; ② SDS-PAGE showed that UROC28 gene was expressed in E. coli DH5α. CONCLUSION: We have successfully constructed the recombinant plasmid of the UROC28 gene and successfully expressed the UROC28 gene, which will help further researches on the function of UROC28 gene and the preparation of antibodies to UROC28 protein.
出处
《第四军医大学学报》
北大核心
2005年第3期206-209,共4页
Journal of the Fourth Military Medical University
关键词
膀胱癌
UROC28基因
克隆
原核表达
bladder neoplasms
UROC28 gene
cloning
prokaryotic expression
