小鼠Nanog基因的克隆及其在大肠杆菌中的表达

按照nanog基因编码序列设计合成引物,利用RT-PCB从小鼠的囊胚期胚胎中扩增得到该 基因,并将该基因克隆到pET-28b(+)载体上,获得pET-28b(+)-nanog原核表达重组质粒,限制 性酶分析和DNA序列测定均证实该克隆插入片段为nanog基因编码序列。重组质粒转化大肠杆 菌BL21(DE3),经IPTG诱导表达,在大肠杆菌表达系统中获得了高效表达,western杂交证实该 蛋白具有6-His抗原活性,从而证实目的蛋白为Nanog蛋白。

Cloning of Mouse Nanog Gene and Its Expression in E . coli

The primers specific for the mouse nanog coding sequence was designed and synthesized. The nanog coding sequence was amplified from mouse blastosphere by using RT-PCR and inserted into vector pET-28b( ) to obtain the recombinant expression plasmid pET28b( )-nanog. The restriction enzymes analysis and DNA sequence detection confirmed that the inserted fragment of the recombinant plasmid was the mouse nanog coding sequence. The recombinant DNA was transformed into the host cells BL21(DE3). The E. coli BL21(DE3) transformed with pET-28b( )-nanog was induced with IPTG. We obtained the effective expression. The result of western blot showed that this fusion protein reacted specifically with the antibodies to 6His and proved that the aimed protein was Nanog.