利用枯草芽孢杆菌芽孢展示人APPsw蛋白  

Surface display of human APPsw on Bacillus subtilis spore

作　　者：李志辉[1] 张莹莹[1] 杜萍萍[1] 申培立 于妍[1] 徐大庆[2] 马雯[1] 李英军[1] 檀建新[1] 

机构地区：[1]河北农业大学食品科技学院/河北省农产品加工工程技术中心,河北保定071001 [2]河北农业大学生命科学学院,河北保定0710001

出　　处：《河北农业大学学报》2014年第5期95-99,共5页Journal of Hebei Agricultural University

基　　金：国家自然科学基金资助项目(31071494);河北省留学回国人员科技活动项目(冀人社字[2010]195号)

摘　　要：为了获得经济高效的人淀粉样前体样蛋白(APPsw)的抗原,本试验将枯草芽孢杆菌的芽胞衣壳蛋白CotG的启动子及其编码序列与穿梭载体pDG150进行重组,构建了芽孢表面展示载体pDG150-cotg。克隆了绿色荧光蛋白(GFP)基因gfp和人淀粉样前体蛋白基因APPsw,并插入pDG150-cotg的多克隆位点构建重组质粒,分别转化枯草芽孢杆菌168,获得工程菌G168和A168。G168芽孢在荧光显微镜下可观察到绿色荧光,表明GFP得到展示;A168的芽孢通过western bolt检测,检测到102kDa大小条带,表明本研究构建的表面展示载体pDG150-cotg能将APPsw展示在芽孢表面,为后续的免疫试验提供了疫苗材料。To efficiently prepare human amyloid protein（APPsw）as antigen,the promoter and coding sequence of spore coat protein CotG fromBacillus subtilis were recombined with a shuttle vector pDG150,leading to the construction of a vector pDG150-cotg for spore surface display.The green fluorescent protein（gfp）gene and APPsw was amplified by PCR and inserted into pDG150-cotg,respectively.Each of the resultant recombinant plasmids was then transformed into Bacillus subtilis 168 to construct the engineered B.subtilis strains G168 and A168.The green fluorescence was observed on the spore of B.subtilis strain G168 under a fluorescence microscope and a protein band with molecular weight about 102 kDa of B.subtillis strain A168 spore was detected by western bolt analysis,which indicated that both GFPand APPsw were displayed on the spore surface through the vector pDG150-cotg.The expression and display of APPsw on the spore surface of B.subtillis will provide sufficient antigen or vaccine for the subsequent immunology studies.

关 键 词：枯草芽胞杆菌 芽胞表面展示 淀粉样前体蛋白 CotG GFP 

分 类 号：Q78[生物学—分子生物学]