利用GST融合蛋白表达系统表达与纯化人N-Ras蛋白

目的:克隆人N-ras蛋白全长编码区基因,获得其原核表达产物,并对融合蛋白进行纯化。方法:采用PCR技术从人乳腺文库中扩增出人N-ras蛋白全长编码区基因,将其克隆到p GEX-KG载体中,在大肠杆菌Rossate中表达后,利用GST-Sepharose 4B亲和珠对原核表达产物进行纯化,SDS-PAGE鉴定表达与纯化产物。结果:从人乳腺文库中扩增获得约600 bp的DNA片段,并克隆至p GEX-KG载体上,经测序与目的序列完全一致;在大肠杆菌Rossate中诱导表达出相对分子质量约47×103的目的蛋白;纯化后,经鉴定获得了纯度较高的重组蛋白GST-N-Ras。结论:获得了重组蛋白GST-N-ras,为后续深入研究Ras基因与其他癌基因、抑癌基因的相互作用奠定了基础。

Construction and packaging of pseudotype retrovirus containing human N-ras cDNA antisense sequence and its biological effects on human hepatoma cells

N-ras is one of the transforming genes in human hepatic cancer cells.It has been found that N-ras was overexpressed at the mRNA and protein level in hepatoma cells.In order to explore the biological roles of N-ras in human hepatic carcinogenesis and the potential application in control of cancer cell growth,a preudotype retrovirus containing antisense sequence of human N-ras was constructed and packaged.A recombinant retrovirus vector containing antisense or sense sequences of N-ras cDNA was constructed by pZIP-NeoSV(X)1.The pseudotype virus was packaged ang rescued by transfection and infection in PA317 and ψ 2 helper cells.It has been demonstrated that the pseudotype retrovirus containing antisense N-ras sequence did inhibit the growth of human PLC/PRF/5 hepatoma cells accompanied with inhibition of p21 expression,while the retrovirus containing sense sequence had none.The pseudotype virus had no effect on human diploid fibroblasts.