论文部分内容阅读
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caf1及p H6抗原基因。方法利用分子克隆技术克隆后,在原核系统中表达鼠疫菌重要功能蛋白caf1及p H6蛋白,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p ET-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。结果根据酶切鉴定和PCR产物测序结果显示,目的基因caf1及p H6已成功连接到表达载体p ET-32a(+)上,SDS-PAGE结果显示表达产物的相对分子质量约为34.2 ku和35.5 ku,与理论分子量一致。结论成功克隆并构建了p ET32a-caf1及p ET32a-p H6重组基因原核表达系统,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 the fusion gene of Yersinia pestis caf1 and p H6 antigen in Escherichia coli using DNA recombination technology. Methods After cloning by molecular cloning technique, caf1 and p H6 proteins were expressed in prokaryotic system. The primers were designed according to the whole genome sequence of Yunnan Yulong strain (D106004), and the target gene fragment was amplified by PCR. +) As the expression vector, the target gene fragment was inserted into the vector by double enzyme digestion and ligation to construct a recombinant expression plasmid; IPTG was induced to express the recombinant plasmid in its host strain E. coli BL21 (DE3). Results According to the restriction enzyme digestion and PCR product sequencing, the target genes caf1 and p H6 were successfully ligated into the expression vector p ET-32a (+). SDS-PAGE showed that the relative molecular mass of the expressed product was about 34.2 ku and 35.5 ku, consistent with the theoretical molecular weight. Conclusion The prokaryotic expression system of p ET32a-caf1 and p ET32a-p H6 recombinant genes was successfully cloned and constructed, which lays the foundation for the potential diagnosis of plague and the possibility of new vaccine selection.