肠出血性大肠杆菌毒力因子Z1444的原核表达及其丝/苏氨酸激酶活性验证

目的:在大肠杆菌中表达肠出血性大肠杆菌(EHEC)毒力岛上的毒力因子Z1444并纯化,对其丝/苏氨酸激酶活性进行初步检测。方法:根据GenBank中Z1444基因序列及pET-28a(+)载体的多克隆位点设计引物,以EHECO157∶H7全菌裂解液为模板,经PCR钓取1047 bp的目的片段,与表达载体pET-28a(+)连接,构建重组表达质粒pET-28a(+)-Z1444,将其转化至大肠杆菌BL21(DE3)中,IPTG诱导蛋白表达并经SDS-PAGE鉴定,利用体外反应体系鉴定重组蛋白的丝/苏氨酸激酶活性。结果:双酶切和测序鉴定表明,pET-28a(+)-Z1444原核表达质粒构建正确;诱导表达后经纯化,获得纯度在90%以上的可溶性重组Z1444,相对分子量约为38×103;体外酶活实验验证了Z1444的丝/苏氨酸激酶活性。结论:Z1444在大肠杆菌中获得高效可溶性表达,为后续功能验证奠定了基础。

Prokaryotic Expression of EHEC Virulence Factor Z1444 and its Serine/Tllreonine Kinase Activity Detection

Objective： To prokaryotic express the virulence factor Z1444 in enterohemorrhagic E.coli （EHEC） pathogenicity island, and detect its activity. Methods： Based on the Z1444 gene sequence in GenBank and multiple clone sites in pET-28a（＋） vector, a pair of primers was designed. Z1444 coding region was amplified by PCR from the bacterial lysate of EHEC O157：H7 and cloned into pET-28a（＋） vector. The plasmid pET-28a（＋）- Z1444 was transformed into E.coli BL21（DE3） for expression. Induced by IPTG, the expression protein was detected by SDS-PAGE, and the conformation was validated by Western blotting. In vitro kinase assay was used to determine the activity of Z1444 serine/threonine kinase. Results： The expression plasmid pET-28a（＋）-Z1444 was constructed correctly identified by digestion and sequencing. The recombinant Z1444 was expressed in E.coli as shown by SDS-PAGE. The purity of the target protein with relative molecular weight about 38×10^3 was higher than 90%. The activity of Z1444 serine/threonine kinase was determined by in vitro kinase assay. Conclusion： The recombinant soluble Z1444 has been expressed, which had laid foundation for further study on its function.