在大肠杆菌中高效表达具有抗病毒活性的可溶性犬α7干扰素

摘要

为了在大肠杆菌表达系统中高效表达具有抗病毒活性的犬0干扰素(CaIFN-α),本研究从犬肝脏中PCR获得CaIFN-α第7亚型(CaIFN-α7)基因,通过重叠延伸PCR方法在CaIFN-α7的N端融合连接内含肽SspDnaB intein (SDI)基因,并将该融合基因连接至pMAL-c2X载体中,构建含有内含肽和麦芽糖结合蛋白基因的CaIFN-α重组质粒.重组质粒在大肠杆菌Rosetta中经IPTG诱导高效表达可溶性CaIFN-α蛋白,经直链淀粉树脂亲和层析纯化后可以得到纯度较高的目的蛋白.重组蛋白经MDCK-CDV检测系统证明具有生物学活性,为该干扰素的大量生产及临床应用奠定了基础.%To achieve high-level expression of soluble recombinant CaIFN-α7 in E.coli, a fusion gene of Ssp DnaB intein (SDI) coding sequence on the upstream of the CaEFN-α7 cDNA was generated by overlap extension (SOE)-PCR, and inserted into pMAL-c2X to construct the recombinant plasmid of pMAL-SDI-CalFN-α for expressing in E.coli. The SDS-PAGE analysis indicated that the expressed fusion protein was about 80 ku and mainly expressed in soluble form which was reacted positively with rabbit anti-CaIFN-α7 serum identified by western blot. Moreover, the purified fusion protein by amylose resin column possessed the antiviral activity as detecting in MDCK/CDV system.