鼠疫耶尔森杆菌YPO0388蛋白的原核表达与鉴定

Prokaryotic expression and identification of fusion protein YPO388 of Yersinia pestis

目的构建表达鼠疫耶尔森杆菌(Yersinia Pestis)的假想蛋白YPO0388的重组质粒,为鼠疫耶尔森杆菌的诊断方法的发展提供支持。方法用PCR方法扩增出ypo0388基因,将其克隆到表达载体pET32a(+)中转化到大肠杆菌(E.co-liBL21(DE3)),用IPTG诱导目的基因表达,用SDS-PAGE方法对表达产物进行分析,用WesternBlot鉴定其免疫原性,并纯化出目的蛋白。结果根据单、双酶切鉴定和DNA测序结果显示,目的基因ypo0388已成功连接到表达载体pET32a(+)上,SDS-PAGE结果显示表达产物rYPO0388相对分子质量约为69.71kDa。重组蛋白经Western blot鉴定,能被兔抗鼠疫菌EV株血清识别。结论成功克隆并构建了pET32a-ypo0388重组基因原核表达系统,所表达的重组蛋白rYPO0388具有较好的可溶性与抗原性,有可能做为研制新型鼠疫耶尔森杆菌诊断试剂的备选抗原。

To construct the recombinant plasmid expressing fusion protein YPO388 of Yersinia pestis in order to establish a diagnostic method to detect this organism, the ypo388 gene was amplified by PCR, cloned into prokaryotic expression plasrnid pET43a（＋） and then transformed to E. coli BL21（DE3） through IPTG induction. The fusion protein was purified and analyzed by SDS-PAGE and Western blotting, It was demonstrated that the target gene ypo0388 cotild be successively inserted to plasmid pET32a（＋）. As demonstrated by SDS-PAGE, the size of the expressed product was about 69.7 kDa as predicted. The fusion protein rYPO0388 performed well in solubility and could be recognized by antibodies against Y. pestis EV strain. It is apparent that this recombinant protein may be used the preferable candidate antigen for the diagnosis of Y. pestis infection.