恙虫病东方体47kDa蛋白基因部分片段的克隆与表达

Clone and expression of partial 47kDa gene of Orientia tsutsugamushi

目的 表达恙虫病东方体 (Orientiatsutsugamushi,Ot) 4 7kDa保护性抗原蛋白。 方法 采用PCR方法 ,从OtKarp株基因组DNA中扩增出 4 7kDa蛋白基因片段 ,鉴定后将该片段克隆于原核表达载体 pBV2 2 0 ,构建成重组质粒pBV -4 7。用该重组质粒转化E coli,转化子在 4 2℃诱导表达并对其鉴定。结果 (1)获得长约 14 30bp的PCR片段 ,序列分析结果与已知 4 7kDa基因序列相同 ;(2 )SDS -PAGE检测表达产物 ,在相对分子量 4 0× 10 3 处有表达带 ;(3)经薄层扫描分析 ,目的蛋白占全菌蛋白的 19% ;(4)免疫印迹实验证明其具有免疫反应性。结论 获得了 4 7kDa基因片段 ,并在大肠杆菌中实现了表达 ,表达产物具有免疫反应性。

Aim To express 47kDa protective antigen protein of O tsutsugamushi Methods 47kDa gene fragment was amplified from the genomic DNA of O tsutsugamushi Karp strain by PCR The fragement was cloned into a prokaryotic expression vector pBV220 to construct a recombinant plasmid pBV 47 The Escherichia coli cells were transformed with the recombinant plasmid and the transformants were induced to expressed the recombinant protein at 42℃and the target protein was identified Results (1)A 1430bp length PCR product was obtained and the sequence of the fragment was the same as known 47kDa gene sequence (2)The expression product was detected by SDS PAGE and an expression band about 40×10 3MW was found (3)The target protein represented 19% of total cell protein by scanning map analysis (4)Immunoblotting analysis testified its immunogenicity Conclusions We have obtained the 47kDa gene of O tsutsugamushi which can be efficiently expressed in the E coli cells and the expressed protein has immunoreactivity