恙虫病东方体Karp株56kDa与47kDa外膜蛋白基因的嵌合和嵌合基因在大肠杆菌的表达

Fusion of gene encoding 56-kDa protein with 47-kDa protein gene of Orientia tsutsugamushi Karp strain and expression of the fusion gene in E.coli

目的 将恙虫病东方体 (Orientiatsutsugamushi,Ot)Karp株的 5 6kDa和 4 7kDa外膜蛋白基因嵌合 ,并使嵌合基因在大肠杆菌细胞内表达 ,产生双抗原融合蛋白。方法 采用PCR方法 ,从OtKarp株基因组DNA中扩增 5 6kDa蛋白基因片段 ,将该片段分别与原核表达载体pQE30及 4 7kD蛋白基因重组质粒 pQE30 / 4 7连接 ,构建 pQE30 / 5 6及pQE30 / 5 6 - 4 7重组质粒 ;用IPTG诱导转入大肠杆菌内的重组质粒的目的基因表达。结果 SDS -PAGE显示 ,pQE30 / 5 6转化的大肠杆菌产生一约 4 5kDa的融合蛋白和 pQE30 / 5 6 - 4 7转化大肠杆菌产生一约 90kDa融合蛋白 (5 6 - 4 7融合蛋白 ) ,免疫印迹分析显示两融合蛋白均与OtKarp株感染鼠血清产生特异性反应 ,5 6 - 4 7融合蛋白分别与 4 7kDa、5 6kDa重组蛋白免疫的小鼠血清产生特异性反应 ,以及 5 6 - 4 7融合蛋白免疫血清与 5 6kDa和 4 7kDa重组蛋白反应。结论 OtKarp株的 5 6kDa与 4 7kDa外膜蛋白基因嵌合后在大肠杆菌细胞内实现了表达 ,表达的融合蛋白具有 5 6kDa和 4 7kDa外膜蛋白的抗原特性。

To fuse the gene encoding 56-kDa outer membrane protein (OMP) with 47kDa OMP gene of Orientia.tsutsugamushi (Ot) Karp,and to express the fusion gene in E.coli cells to produce a fusion protein of the two antigens.The 56-kDa OMP gene fragment was amplified from Ot Karp strain genomic DNA by PCR.And the fragment was ligated into prokaryotic expression vector pQE30 and pQE30/47 recombined with 47-kDa OMP gene,respectively.The recombinant plasmids,pQE30/56 and pQE30/56-47,were used to transform E.coli cells.And the E.coli cells transformed with pQE30/56 or pQE30/56-47 were induced to produce the target proteins with IPTG.In this way,a50-kDa recombinant protein was found in cells transformed with pQE30/56 and a 90-kDa (56-47) fusion protein was found in cells transformed with pQE30/56-47 in SDS-PAGE assay.Both 50-kDa and 90-kDa protein reacted to sera from mice infected with Ot Karp and the 90-kDa fusion protein reacted to sera from the mice immunized with 47-kDa protein or 56-kDa protein as well as 47-kDa and 56-kDa proteins reacted to the 90-kDa fusion protein-immunized serum in immunoblot assay.It is concluded the fusion gene encoding 56-and 47-kDa OMPs recombined with pQE30 may efficiently express in E.coli cells and the expressed fusion protein has the antigenic properties of 56-kDa and 47-kDa OMP of Ot Karp.