UROC28基因cDNA的克隆及其原核表达

Cloning and prokaryotic expression of human UROC28 gene cDNA

目的:克隆人UROC28基因cDNA并在原核细胞中表达。方法:用RT-PCR从前列腺癌PC-3细胞系中扩增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UROC28基因cDNA;②经IPTG诱导的重组质粒pGEX-4T-UROC28表达出相对分子质量(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UROC28基因cDNA,并在E.coliDH5α中表达出GST-UROC28融合蛋白。

Objective：To clone the cDNA of human UROC28 gene and express it in E.coli DH5α. Methods： The fragment of UROC28 gene was amplified by RT-PCR from human prostate cancer cell line PC-3 and was cloned into pUC19. The DNA fragment from pUC19-UROC28 digested with HindⅢ and EcoRI was ligated to the 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： The sequencing and endonuclease digestion analysis showed that the fragment of UROC28 gene cDNA has been inserted into vectors pUC19 and pGEX-4T-1. SDS-PAGE showed the UROC28 gene was expressed in E.coli DH5α. Conclusion：The recombinant plasmid of the UROC28 gene can be constructed successfully, and the UROC28 fusion protein can be expressed in E.coli DH5α.