刚地弓形虫RH株微线体蛋白MIC3成熟肽的克隆与表达

Cloning and expression of gene encoding the mature peptide of microneme protein MIC3 from Toxoplasma gondii RH straimn

目的 克隆编码刚地弓形虫(Toxoplasmagondii)RH株微线体蛋白MIC3成熟肽的基因,构建原核表达载体pET MMIC3,并在大肠杆菌BL2 1株中表达。方法 采用PCR技术从刚地弓形虫RH株基因组DNA中扩增编码微线体蛋白MIC3成熟肽的基因,并克隆到T载体上,经测序鉴定后,将目的基因亚克隆到表达载体pET30a(+)上,构建质粒pET MMIC3。表达产物用Westernblot进行进一步确认。结果 经初步菌液PCR鉴定,提取质粒双酶切鉴定并测序,确认插入T载体的序列为所需的目的序列;亚克隆构建pET -MMIC3,转化到宿主菌大肠杆菌BL2 1,得到分子量为37.2kDa的重组表达蛋白,Westernblot的结果与预测相符。结论 成功的克隆并融合表达了刚地弓形虫RH株微线体蛋白MIC3成熟肽,为进一步研究其在弓形虫粘附和入侵中的作用奠定了基础。

To clone the gene encoding the mature peptide of microneme preotein MIC3 from Toxoplasma gondii RH strain, constract its prokaryotic expression vector pET-MMIC3 to express on E.coli BL21 strain in order to investigate the mechanism of adhesion of T.gondii and its invasion to the host cells as well as the role of the MIC3 protein in these processes, the sequence encoding the mature microneme protein of MIC3 was amplified by PCR by using the genomic DNA from the tachyzoites of T.gondii RH strain as template.The amplified product was cloned into T-vector, identified and then subcloned into expression vector pET-30a(+), thus obtaining a recombinant plasmid pET-MMIC3. This recombinant vector was then transferred to E.coli BL21 strain (DE3). The induction was optimized, and a recombinant protein of 37.2 kDa was obtained, and the result of Western blotting was consistent with the calculated molecular weight. A prokaryotic recombinant plasmid to express the fusion protein of mature MIC3 was successfully constructed in the present study, and these results may provide the foundation for the further studies on the mechanism of T.gondii adhesion and invasion to host cells as well as the role of MIC3 in these processes.