贝氏柯克斯体表面抗原基因p1与hspB的融合基因在大肠杆菌中的表达

目的　将贝氏柯克斯体 (Coxiellaburnetii)表面抗原P1蛋白基因与热休克蛋白B(HspB)基因片段融合 ,并使该融合基因在大肠杆菌细胞内表达 ,产生P1 HspB融合蛋白。 方法　采用PCR方法 ,从贝氏柯克斯体新桥株基因组DNA中扩增P1蛋白基因 (p1)和HspB蛋白基因 (hspB)片段 ,将两基因片段分别与原核表达载体 pQE30连接 ,分别构建重组表达质粒pQE30 / p1和pQE30 /hspB。用BamHI和SacIDNA内切酶将 p1基因片段从 pQE30 /p1切下并与 pQE30 /hspB的hspB连接 ,构建重组质粒 pQE30 / p1 hspB。用IPTG诱导pQE30 / p1 hspB转化的大肠杆菌表达融合基因 p1 hspB。 结果　SDS PAGE分析发现 pQE30 /p1 hspB转化的大肠杆菌表达一 83kDa融合蛋白 ;经薄层扫描分析 ,该融合蛋白约占到全菌体蛋白的 12 .1% ;免疫印迹分析发现该融合蛋白能被贝氏柯克斯体感染小鼠血清所识别。结论　构建的 p1 hspB融合基因在大肠杆菌中得到了有效表达 ,表达的P1 HspB融合蛋白能与抗贝氏柯克斯体抗体特异反应 ,P1 HspB融合蛋白为Q热亚单位疫苗的研制提供了一种新型融合蛋白

Expression of surface antigen gene p 1 fused with hspB of Coxiella burnetii in E.coli

To fuse the gene encoding the surface antigen P1 with hspB of Coxiella burnetii and to express the p1 hspB fusion gene in E. coli cells. The p1 and hspB fragment was amplified from C. burnetii genomic DNA by PCR and the gene fragments were ligated into the prokaryotic expression vector pQE30 to construct pQE30/p1 and pQE30/hspB, respectively. The p1 from pQE30/p1 was fused with hspB of pQE30/hspB to construct pQE30/p1 hspB. The expression of p1 hspB fusion gene of pQE30/p1 hspB was induced with IPTG in E. coli cells.The P1 HspB fusion protein was observed to be about 83 kDa in SDS PAGE and 12.1% of total cell protein in scanning analysis; the fusion protein was recognized by the serum from mice infected with C. burnetii in immunoblot assay.The p1 hspB fusion gene of pQE30/p1 hspB is expressed in E. coli cells by inducing of IPTG and the resulting P1 HspB fusion protein has ability to react to the antibodies to C. burnetii; the P1 HspB may be a new candidate of subunit vaccines against Q fever.