恙虫病东方体Karp株47kDa蛋白成熟肽的克隆与表达

Cloning and Expression of 47kDa Protein Gene of Orientia tsutsugamushi Karp Strain

目的 :克隆表达恙虫病东方体 (Orientiatsutsugamushi,Ot)Karp株 4 7kDa表面蛋白的成熟肽 ,探索其作为疫苗及诊断抗原的可能性。方法 :采用PCR方法 ,从 OtKarp株菌种基因组DNA中扩增出 4 7kDa成熟蛋白基因片段 ,将该片段克隆于原核表达载体pQE30 ,构建成重组质粒pQE30 4 7,转化大肠杆菌 (E coli) ,经IPTG诱导表达 ,SDS_PAGE和Western_blotting检测目的蛋白的表达。结果 :①获得长约 12 72bp的OtKarp株 4 7kDa外膜蛋白基因 ;②SDS_PAGE和免疫印迹显示该重组质粒转化的E coli有一相对分子量约为 4 3× 10 4 的独特蛋白带 ;③重组表达质粒序列分析结果显示pQE30 4 7中插入的基因片段的序列与已报告的OtKarp株 4 7kDa外膜蛋白基因序列基本一致 ,并与已知序列相同。结论 :获得了OtKarp 4 7kDa蛋白基因 ,并在E coli中实现了表达 。

Objective:To express 47kDa outer membrane protein of Orientia tsutsugamushi(Ot),and explore the possibility of using it as vaccine and diagnosis antigen.Methods:47kDa gene fragment was amplified from Ot Karp strain genomic DNA by PCR.The fragement was identified and cloned into prokaryotic expression vector pQE30 and constructed recombinant plasmid pQE30/47.The E.coli cells transformed with pQE30/47 were induced to express the target protein.Results:A 1272bp length PCR product was obtained and the sequence was the same as known Ot Karp 47kDa gene sequence.An expression band about 43×10 3MW was found and was reacting to antiserum to Ot Karp by SDS-PAGE and Western-blotting assay.Conclusion:47kDa gene is obtained and successfully expressed in E.coli.The expressed protein can specifically react to antiserum to Ot Karp.