【摘要】 目的 :表达恙虫病东方体 (Orientiatsutsugamushi)Karp株 5 6 0 0 0表面蛋白 ,探索其作为诊断抗原的可能性。方法 :采用PCR方法 ,从Ot.Karp株菌种基因组DNA中扩增出 5 6 0 0 0成熟蛋白基因片段 ,将该片段克隆于原核表达载体pQE30 ,构建重组质粒pQE30 / 5 6 ,转化大肠杆菌 ,经IPTG诱导表达、SDS_PAGE电泳和免疫印迹检测目的蛋白的表达。结果 :(1)获得长约 15 0 0bp的Ot.Karp株 5 6 0 0 0外膜蛋白基因 ;(2 )SDS_PAGE电泳和免疫印迹显示该重组质粒转化的大肠杆菌有一相对分子质量约为 5 6 0 0 0的独特蛋白带 ;(3)表达后菌体超声破碎后 ,目的蛋白主要以包涵体形式存在 ;(4)重组表达质粒序列分析结果显示 ,pQE30 / 5 6中插入的基因片段的序列与已报道的Ot.Karp株5 6 0 0 0外膜蛋白基因序列基本一致。结论 :获得了Ot.Karp 5 6 0 0 0蛋白基因 ,并在大肠杆菌中实现了表达 ,表达的重组蛋白具有免疫反应性。更多还原

【Abstract】 Objective:To express 56?000 protective antigen protein of Orientia tsutsugamushi and explore its possibility for preparation of diagnosis antigen.Methods: 56?000 gene fragment was amplified from O.tsutsugamushi Karp strain genomic DNA through nest-PCR. The fragment was identified and cloned into prokaryotic expression vector pQE30 and the recombinant plasmid pQE30/56 was constructed. Then IPTG inducement expression was used to express 56?000 protein and the expression product was examined by immunoblot assay with polyclonal antiserum to Ot.Karp.Results:(1) PCR product of about 1?550?bp was obtained and the sequence was same as known Ot.Karp 56?000 gene sequence. (2) An expression band about 56?000 was found and was verified to react to polyclonal antiserum to Ot.Karp. (3)Most of the target protein existed in the form of inclusion.Conclusions:56?000 gene was obtained and expressed successfully in Escherichia coli.更多还原