SARS冠状病毒PUP5蛋白的表达、纯化及鉴定

Expression,purification and identification of SARS coronavirus putative uncharacterized protein 5

经RT-PCR扩增了SARS冠状病毒(SARS-CoV)BJ01株的PU5基因(putativeuncharacterizedgene5)的cDNA,将其克隆到原核表达载体pGEX-6P-1中,命名为pGEX-PU5,测序正确后将阳性质粒转化到大肠杆菌BL21(DE3)中,IPTG诱导表达,表达的融合蛋白命名为pGEX-PUP5,并用GlutathioneSwpharoseTM4B亲和层析柱对表达产物进行纯化,然后用PreScissionTMProtease酶对融合蛋白进行解离,获得PUP5蛋白,用抗SARS-CoV卵黄抗体IgY和GST-tag单克隆抗体分别对表达的目的蛋白做Westernblot鉴定,证明所表达的蛋白具有免疫学活性。本研究应用原核表达系统表达了PUP5蛋白,为进一步解析该蛋白功能奠定了基础。

The complementary DNA of putative uncharacterized gene 5 （PU5） was prepared fi＇om SARS coronavirus （SARS-CoV） gene RNA by RT-PCR. The amplified fi＇agment was cloned into the plasmid vector pGEX-6P-1. The recombinant vector was designated pGEX-PUS. The putative uncharacterized protein 5 （PUP5） of SARS-CoV was expressed in E. coli as fusion protein with GST protein at N-terminus. The recombinant protein was identified by Western blot. The soluble parts of the cell extract were then purified by Glutathione SwpharoseTM 4B and purified putative uncharacterized protein 5 was harvested by PreScissionTM Protease witch cleaved the purified protein.