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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 select and construct pET32a-ypo1996 based on the comparison of proteome results and to express Yersinia pestis specific hypothetical protein YPO1996 recombinant protein (rYPO1996) for the research of Yersinia pestis diagnostic reagent Antigen selection. Methods According to the whole genome sequence of Yersinia pestis CO92 strain, the target gene YPO1996 was amplified by PCR and inserted into the expression vector pET32a (+). The recombinant plasmid was transformed into E. coli BL21 (DE3) and induced by IPTG. Plasmids stably and efficiently express the histidine-tagged fusion protein in the engineered bacteria and affinity-purified the protein of interest. The expressed protein product was analyzed by SDS-PAGE and its antigenicity was analyzed by Western blot. The results of single restriction enzyme digestion and DNA sequencing showed that the target gene YPO1996 was successfully ligated into the expression vector pET32a (+), and the molecular mass unit of the expressed product was about 53.11 ku by SDS-PAGE, which was consistent with the expected value. The recombinant protein was identified by Western blot and could be recognized by rabbit anti-Yersinia pestis EV strain sera. Conclusion The prokaryotic expression system of pET32a-ypo1996 recombinant was constructed successfully. The expressed recombinant protein rYPO1996 has good solubility and antigenicity and can be used as a candidate antigen for the development of a new Yersinia pestis diagnostic reagent.