恙虫病东方体58kDa热休克蛋白与47kDa外膜蛋白的基因嵌合及58-47嵌合基因在大肠杆菌中的表达

采用PCR方法 ,从恙虫病东方体Karp株基因组DNA中扩增 5 8kDa热休克蛋白的基因 ,将该基因分别与原核表达载体pQE30及 4 7kDa外膜蛋白的基因重组质粒 (pQE30 4 7)连接 ,构建pQE30 5 8及pQE30 5 8 4 7重组质粒 ,用重组质粒转化大肠杆菌。SDS PAGE显示 ,pQE30 5 8和pQE30 5 8 4 7转化的大肠杆菌分别产生一 5 8kDa重组蛋白和一 90kDa的双抗原 (5 8- 4 7)融合蛋白。免疫印迹分析显示Karp株免疫血清能特异识别 5 8kDa重组蛋白和 90kDa融合蛋白 ,90kDa融合蛋白既与 4 7kDa重组蛋白也与 5 8kDa重组蛋白的免疫血清特异反应 ,5 8kDa与 4 7kDa重组蛋白也被 90kDa融合蛋白免疫血清所识别。研究结果证明 5 8 4 7融合蛋白具有恙虫病东方体Karp株 4 7kDa外膜蛋白和 5 8kDa热休克蛋白的抗原特性

FUSION OF GENE ENCODING 58-kDa HSP WITH GENE ENCODING 47-kDa OMP OF ORIENTIA TSUTSUGAMUSHI AND EXPRESSION OF THE FUSION GENE IN E.COLI

The gene fragment encoding 58-kDa heat shock protein(Hsp)was amplified from genomic DNA of Orientia tsutsugamushi(Ot) Karp strain by PCR.The gene fragment was ligated into prokaryotic expression vectors pQE30 and pQE30/47 recombined with 47-kDa outer membrane protein(Omp)of Ot,respectively.The E.coli cells were transformed with pQE30/58 or pQE30/58-47.A 58-kDa recombinant protein and a 90-kDa(58-47)fusion protein were found in cells transformed with pQE30/58 and in cells transformed with pQE30/58-47,respectively,in SDS-PAGE assay. Both 58-kDa recombinant protein and 58-47kDa fusion protein were recognized with sera from the mice immunized with Ot Karp and the 58-47 fusion protein were recognized with sera from the mice immunized with 47-kDa recombinant protein or 58-kDa recombinant protein as well as 47-kDa and 58-kDa proteins reacted to the 58-47kDa fusion protein-immunized serum in immunoblot assay.These results demonstrated that the 58-47kDa fusion protein has the antigenic characteristics of 58-kDa Hsp and 47-kDa Omp of Ot Karp.