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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。
Objective To clone the gene encoding the MIC3 mature peptide of Toxoplasma gondii RH strain microtubule protein MIC3 and construct prokaryotic expression vector pET MMIC3 and express it in E. coli BL21 strain. Methods The gene encoding MIC3 mature peptide was amplified from GenBank DNA of Toxoplasma gondii RH strain by PCR and cloned into T vector. After sequencing, the target gene was subcloned into the expression vector pET30a (+ ) To construct plasmid pET MMIC3. The expression product was further confirmed by Western blot. Results The recombinant plasmid was identified by PCR and identified by restriction enzyme digestion. The sequence of inserted T vector was confirmed to be the desired sequence. The recombinant plasmid pET-MMIC3 was subcloned into E. coli BL21, and the molecular weight was 37.2 kDa recombinant protein, Westernblot results consistent with the prediction. Conclusion The cloning and fusion expression of the MIC3 mature peptide of T. gondii RH strain micronaire protein was successfully cloned and laid the foundation for further study on its role in the adhesion and invasion of Toxoplasma gondii.