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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调节亚基C2(ATP-dependent Clp regulatory subunit C2,ClpC2)基因的原核表达质粒,并在大肠埃希菌中表达重组蛋白。方法以MTB H37Rv基因组DNA为模板,PCR扩增ClpC2基因,插入表达载体pET32a(+)中,构建重组原核表达质粒pET32a(+)-ClpC2,经双酶切及测序鉴定正确后,转化大肠埃希菌BL21(DE3),IPTG诱导表达。表达产物经SDS-PAGE和Western blot进行鉴定。结果重组表达质粒经双酶切和测序鉴定,证明构建正确;表达的重组融合蛋白相对分子质量约46 000,可与鼠抗组氨酸单克隆抗体特异性结合。结论已成功构建了重组表达质粒pET32a(+)-ClpC2,并在大肠埃希菌中表达了重组蛋白,为进一步研究ClpC2蛋白的生物学功能奠定了基础。
Objective To construct prokaryotic expression plasmid of ATP-dependent Clp regulatory subunit C2 (ClpC2) gene of Mycobacterium tuberculosis (MTB) and express the recombinant protein in Escherichia coli. Methods ClpC2 gene was amplified by PCR using genomic DNA of MTB H37Rv as a template and inserted into expression vector pET32a (+) to construct recombinant prokaryotic expression plasmid pET32a (+) - ClpC2. After double-digestion and sequencing, ClpC2 gene was transformed into E. coli Strain BL21 (DE3), induced by IPTG. The expressed product was identified by SDS-PAGE and Western blot. Results The recombinant plasmid was identified by double enzyme digestion and sequencing. The recombinant plasmid was constructed correctly. The expressed recombinant fusion protein has a relative molecular mass of about 46 000 and can specifically bind to mouse anti-histidine monoclonal antibody. Conclusion The recombinant plasmid pET32a (+) - ClpC2 was constructed successfully and the recombinant protein was expressed in Escherichia coli, which laid a foundation of further study on the biological function of ClpC2 protein.