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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.
OBJECTIVE: To clone UROC28 gene of human bladder cancer and express in prokaryotic cells.Methods: UROC28 cDNA was amplified from human bladder cancer tissues by RT-PCR and cloned into vector pUC 19. After confirmed by sequencing, Double digested and subcloned into the prokaryotic expression vector pGEX 4T 1 and transformed into E. coli DH5α strain.The recombinant plasmid was induced by IPTG and identified by SDS PAGE.RESULTS: ①Through RT PCR, sequencing and restriction enzyme digestion , Successfully cloned human bladder cancer UROC28 gene; ② IPTG-induced recombinant plasmid pGEX 4T UROC28 expression of Mr about 42000 fusion protein, consistent with the expected results.Conclusion: Human bladder cancer UROC28 gene was cloned successfully, and in the The GST UROC28 fusion protein is expressed in E. coli DH5a.