鼠疫耶尔森氏菌 YopD 抗原基因在大肠杆菌中的克隆表达

摘要

目的：利用 DNA 重组技术，在大肠杆菌中获得融合表达的鼠疫耶尔森氏菌 YopD 抗原基因。方法根据查找文献及基因比对选取了鼠疫菌重要功能蛋白-YopD 蛋白。利用分子克隆技术克隆后，在原核系统中进行表达。根据云南玉龙菌株（D106004）全基因组序列设计引物，PCR 扩增目的基因片段。采用 pET-32a（＋）作为表达载体，通过双酶切和连接反应，将目的基因片段定向插入载体中，构建重组表达质粒。IPTG 诱导，使重组质粒在其宿主菌 E ．coli BL21（DE3）中表达。结果在大肠杆菌中成功获得了融合表达蛋白，即重组 YopD 蛋白。结论以质粒 pET-32a（＋）作为表达载体，鼠疫菌重要功能蛋白 YopD 能够在大肠杆菌 E ．coli BL21（DE3）中稳定高效地表达，为鼠疫潜在诊断靶点及新型疫苗选择的可能性奠定了基础。%Objective To obtain YopD protein of Yersinia pestis in vitro by DNA recombination techniques. Methods According to the literature review and genes comparison,the important functional protein of Yersinia pestis,YopD was selected,and cloned by using molecular cloning techniques,in prokaryotic sys-tems for expression.According to the complete gcnome sequence of Yersinia pestis strain D106004,detec-tion primers were designed.The target DNA fragments were amplificated by polymerase chain reaction. Plasmid DNA pET-32a(+)acted as expression vector.By two different restriction enzymes and T4 DNA Ligase,the PCR products were cloned into pET-32a(+)in correct direction.The reconstructed plasmid was then transformed into E.coli BL 21.The fusion proteins were induced by IPTG to be expressed in E.coli BL21.Result In E.coli BL21 one fusion protein,namely restructured YopD was successfully obtained. Conclusion Y.pestis important functional protein YopD was stably and effectively expressed in E.coli BL21 (DE3)by means of the expression vector of Plasmid pET-32a (+),and it was of good point for the plague potential diagnostic targets and new vaccines choice.