【摘要】 目的:利用大肠杆菌系统表达牛蛙核糖核酸酶(RC-RNase),观察其对人肝癌细胞的体外杀伤作用。方法:构建PET32-RC-RNase重组原核表达载体,转化入大肠杆菌BL21(DE3)p lyS中并利用IPTG诱导表达。工程菌经超声碎菌、离心后,表达产物在变性条件下经N i-NTA-agarose亲合层析法纯化及复性,细胞毒试验检测其对体外培养的人肝癌细胞的杀伤活性。结果:经IPTG诱导后,大肠杆菌中表达的RC-RNase融合蛋白相对分子量(M r)约为31 KD,与预期大小基本相一致,主要以包涵体的形式存在。经纯化及复性后,获得纯度达到95%以上且对体外培养的人肝癌细胞具有明确杀伤活性的RC-RNase融合蛋白。结论:在大肠杆菌中表达的RC-RNase融合蛋白对体外培养的肝癌细胞具有一定的杀伤作用。更多还原

【Abstract】 objective: To express the Rana catesbiana ribonuclease(RC-RNase) gene in E.coli and observe its killing effect on human hepatocellular carcinoma cell in vitro.Methods: The prokaryotic expression vector PET32-RC-RNase was contructed and transformed into E.coli BL21(DE3)plyS which was induced to express by IPTG.The E.coli was lysed by ultrasonication and centrifugated.After purified by Ni-NTA-agarose affinity chromatography and refolded,the killing effect of the expressed products on human hepatocellular carcinoma cell in vitro was detected by cytotoxicity test.Results: The RC-RNase fusion protein was successfully induced to express in the form of inclusion body in E.coli by IPTG.Its molecular weight was about 31KD,which was consistent with that anticipated.After purified and refolded,the RC-RNase fusion protein with purity more than 95% was(obtained.)The results of cytotoxic test showed that the RC-RNase fusion protein had obvious killing effect on(human) hepatocellular carcinoma cell in vitro.Conclusion: The RC-RNase fusion protein expressed in E.coli has killing effect to some extent on human hepatocellular carcinoma cell in vitro,which lays the foundation for further studying its activity and function.更多还原