大鼠γ-干扰素诱导蛋白-10在大肠杆菌中的表达和鉴定

Expression and identification of rat IFN-γ inducible protein-10 by Escherichia coli expression system

目的原核表达大鼠γ干扰素诱导蛋白10,并对其进行鉴定。方法从大鼠脾细胞中提取RNA,用RTPCR方法得到IP10cDNA,双酶切将其插入pET32a(+)载体中。重组质粒pET32a(+)IP10经酶切、PCR及测序鉴定后转化大肠杆菌BL21(DE3)中进行表达。结果所获得的大鼠IP10克隆,经测序与GenBank报道的完全一致。所表达的产物经SDSPAGE和Westernblot分析获得证实。结论已成功构建原核表达载体pET32a(+)IP10,并使其在大肠杆菌中获得了融合表达。

Objective To express and identify rat inducible protein-10 （IP-10） in E. coli. Methods The rat splenecytes were used for extraction of RNA. The cDNA of rat IP-10 was cloned from RNA by RT-PCR, and then the PCR products were cloned into prokaryote expression vector pET-32a（ ＋ ） after being digested with two restrictive enzymes of Kpn Ⅰ and EcoR Ⅰ Recombinant plasmid pET-32a（ ＋ ）- IP10 was confirmed by restrictive enzymes analysis, PCR, and sequencing, and then expressed in E. coli BL21（DE3） induced by IPTG. Resalts The sequence of obtained rat IP-10 clones was completely identical to that of IP-10 in GenBank. SDS-PAGE and Western blotting proved that the expressed protein was IP-10. Conclusion A prokaryotic expression vector pET-32a（ ＋ ）-IP-10 is constructed successfully, and IP-10 fusion protein is expressed in E. coli.