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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 recombinant prokaryotic expression plasmid of ATP-dependent Clp proteolytic subunit 1 (ClpP1) of Mycobacterium tuberculosis (MTB) and express the recombinant protein in Escherichia coli. ClpP1 gene was amplified by PCR from genomic DNA of Mycobacterium tuberculosis strain H37Rv and inserted into prokaryotic expression vector pET-32a (+) to construct recombinant plasmid pET-32a (+) - ClpP1. The recombinant plasmid was transformed into E. coli B21 ), IPTG induced expression. The expressed product was identified by SDS-PAGE and Western blot. Results The recombinant plasmid pET-32a (+) - ClpP1 was verified by double enzyme digestion and sequencing. The recombinant plasmid pET-32a (+) - ClpP1 was constructed correctly and its relative molecular mass was about 35 000. It could specifically bind to mouse anti-His monoclonal antibody. Conclusion The recombinant plasmid pET-32a (+) - ClpP1 was successfully constructed and the recombinant protein was expressed in Escherichia coli, which laid the foundation for further study on the biological characteristics of ClpP1 protein in MTB.