鸡Oct-1 POU结构域基因的克隆、表达及纯化

根据GenBank已发表的鸡Oct-1基因序列(M29972)设计一对引物,利用RT-PCR技术从SPF鸡肝脏中扩增出编码Oct-1POU功能域的cD-NA序列,序列比较表明,与GenBank中发表的鸡Oct-1基因的同源性为99.6%,推导的氨基酸的同源性为99%。构建了原核表达载体pET-POU,利用IPTG在大肠杆菌中诱导表达,并对表达产物进行了纯化,结果表明,Oct-1POU功能域在大肠杆菌中获得高效可溶性表达,融合蛋白的分子量约为37kD,表达产物经His.Bind亲和层析得到纯化的蛋白。

Gene cloning, expression and purification of chicken Oct-1 POU domain

A pair of primers were designed according to the published sequence of chicken oct-1 gene, and the chicken oct-1 pou domain gene was amplified from SPF chicken liver mRNA by RT-PCR. Sequence comparison with the published oct-1 pou domain showed that the homology of nucleotide acids was 99.6%, and the homology of amino acids was 99%. The amplified cDNA fragment was cloned into the pET-32a and got a recombinant plasmid pET-pou, and then pET-pou was transformed into E.coli BL21. A recombinant protein of 37 Ku was highly expressed after induced by IPTG and purified by Ni-nitriloniacetic affinity chromatography.