鼠疫耶尔森杆菌蛋白YPO1996的融合表达与鉴定

Expression and identification of Yersinia pestis fusion protein YPO1996

目的在比较蛋白质组结果的基础上选择并构建pET32a-ypo1996,表达鼠疫耶尔森杆菌(Yersinia pestis)特异的假想蛋白YPO1996重组蛋白(rYPO1996),为新鼠疫耶尔森杆菌诊断试剂的研究提供备选抗原。方法根据鼠疫菌CO92株全基因组序列设计引物,用PCR方法扩增目的基因YPO1996,将其定向插入表达载体pET32a(+)上,转化至大肠埃希菌BL21(DE3),用IPTG诱导,使重组质粒在工程菌中稳定高效地表达带组氨酸标签的融合蛋白,并亲和纯化该目的蛋白。通过SDS-PAGE方法对表达的蛋白产物进行初步分析,并用Western blot分析其抗原性。结果单、双酶切鉴定及DNA测序显示,目的基因YPO1996成功连接到表达载体pET32a(+)上,SDS-PAGE显示表达产物分子质量单位约为53.11 ku,与预期值相符。重组蛋白经Western blot鉴定,能被兔抗免疫鼠疫菌EV株血清识别。结论成功构建了pET32a-ypo1996重组基因原核表达系统,表达的重组蛋白rYPO1996具有较好的可溶性及抗原性,可作为研发新型鼠疫耶尔森杆菌诊断试剂的备选抗原。

Objective To construct the recombinant plasmid pET32a-ypo1996 based on Yersinia pestis comparative proteome and express the protein rYPO1996 in order to develop a new method of diagnosing Y.pestis.Methods Primers were designed according to the YPO1996 gene sequence of Y.pestis strain CO92.The target DNA fragments of YPO1996 were amplified by polymerase chain reaction（PCR）,and the products were inserted directionally into a pET32a（＋） vector.The recombinant plasmid was transformed into E.coli BL21（DE3） and inducted by IPTG.The fusion protein was effectively expressed and purified.The expressed protein was analyzed by SDS-PAGE and Western blot. Results The target gene YPO1996 had been inserted successfully into pET32a（＋）,and the results of SDS-PAGE showed that the expression product was about 53.11 ku as predicted.The fusion protein rYPO1996 performed well in terms of solubility and can be recognized by antibodies against the Y.pestis strain（EV strain） in Western blot.Conclusion The new pET32a-ypo1996 plasmid is successfully constructed in a prokaryotic expression system,and the protein rYPO1996 showed satisfactory solubility and antigenicity.It may be an acceptable candidate antigen for the diagnosis of Y.pestis infection.