结核分枝杆菌ATP依赖的丝氨酸蛋白酶蛋白水解亚基1基因的原核表达

Prokaryotic expression of ATP-dependent caseinolytic protease proteolytic subunit 1 gene of Mycobacterium tuberculosis

目的构建结核分枝杆菌(Mycobacterium tuberculosis,MTB)ATP依赖的丝氨酸蛋白酶蛋白水解亚基1(ATP-dependent Clp proteolytic subunit 1,ClpP1)基因重组原核表达质粒,并在大肠埃希菌中表达重组蛋白。方法从结核分枝杆菌H37Rv株基因组DNA中PCR扩增ClpP1基因,插入原核表达载体pET-32a(+)中,构建重组表达质粒pET-32a(+)-ClpP1,转化大肠埃希菌B21(DE3),IPTG诱导表达。表达产物经SDS-PAGE和Western blot进行鉴定。结果重组表达质粒pET-32a(+)-ClpP1经双酶切和测序鉴定,证明构建正确;表达的重组蛋白相对分子质量约35 000,可与鼠抗His单克隆抗体特异性结合。结论成功构建了重组表达质粒pET-32a(+)-ClpP1,并在大肠埃希菌中表达了重组蛋白,为进一步研究ClpP1蛋白在MTB中的生物学特性奠定了基础。

Objective To construct a prokaryotic expression vector for ATP-dependent caseinolytic protease proteolytic subunit 1（ClpP1） gene of Mycobacterium tuberculosis（MTB） and express recombinant protein in E.coli.Methods ClpP1gene was amplified by PCR from the genome of MTB H37Rv strain and inserted into prokaryotic expression vector pET-32a（＋）.The constructed recombinant plasmid pET-32 a（＋）-ClpP1 was transformed to E.coli B21（DE3）and induced with IPTG.The expressed product was identified by SDS-PAGE and Western blot.Results Restriction analysis and sequencing proved that recombinant plasmid pET-32a（＋）-ClpP1 was constructed correctly.The expressed recombinant protein,with a relative molecular mass of about 35 000,showed specific binding to mouse monoclonal antibody against His tag.Conclusion Recombinant plasmid pET-32a（＋）-ClpP1 was constructed correctly,and recombinant protein was expressed in E.coli,which laid a foundation of biological characters of ClpP1 protein in MTB.