【摘要】 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。更多还原

【Abstract】 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 multi-ple clone sites in pET-28a(+) vector, a pair of primers was designed. Z1444 coding region was amplified byPCR 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 detect-ed by SDS-PAGE, and the conformation was validated by Western blotting. In vitro kinase assay was used to de-termine the activity of Z1444 serine/threonine kinase. Results: The expression plasmid pET-28a(+)-Z1444 wasconstructed correctly identified by digestion and sequencing. The recombinant Z1444 was expressed in E.coli asshown by SDS-PAGE. The purity of the target protein with relative molecular weight about 38×103was higherthan 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.更多还原