摘要
目的构建以尿路致病性大肠杆菌(UPEC)Ⅰ型菌毛编码基因fimH和fimC为目的基因的原核重组表达质粒,诱导其在E.coliBL-21中表达,并对其免疫原性进行分析。方法PCR法自UPEC标准菌株J96获取fimH和fimC基因,分别插入原核表达载体pGEX-4T-2;将重组表达质粒转染感受态E.coliBL-21,经IPTG诱导表达、SDS-PAGE和Westernblot鉴定并纯化目的蛋白fimH和fimC蛋白,定量后免疫BALB/c小鼠,动态检测抗体产生水平。结果PCR法克隆出全长为903bp的fimH和720bp的fimC基因,构建的原核表达质粒pGEX-4T-2-fimH及pGEX-4T-2-fimC经诱导可分别表达出60KD和48KD左右的GST融合蛋白;蛋白经纯化后免疫动物能诱导产生高效价的IgG抗体。结论成功获取了UP-ECⅠ型菌毛基因fimH和fimC,所构建的原核表达质粒在BL-21中成功表达;fimH有免疫原性。
To construct the prokaryotic expression plasmid encoding fimH and fimC genes from type I pilus of uropathogenic Escherchia coli and to induce their expression in E.coli BL21 in order to analyze their immunogenicity,the fimH and fimC genes were amplified by PCR from a standard strain UPECJ96 and then inserted to a prokaryotic expression vector pGEX-4T-2.The fusion proteins GST-fimH and GST-fimC were expressed in E.coli BL21 with induction by IPTG and identified by Western blotting.Afterwards,BALB/c mice were immunized with fusion proteins to confirm if the antibodies would be produced as demonstrated by ELISA.In these ways,the full-length of 903 bp fimH gene and 720 bp fimC gene were cloned,and the GST-fusion proteins with molecular weight of 60 kDa and 48 kDa were expressed after induction from the constructed prokaryotic expression plasmids pGEX-4T-2-fimH and pGEX-4T-2-fimC respectively.Mice immunized with these fusion proteins could elicit high tittered IgG antibodies against FimH protein.These results indicate the successful construction of the prokaryotic expression plasmids for fimH and fimC genes and the expression of the constructed plasmds in E.coli BL21,and the expressed fusion proteins shows immunogenicity to FimH.
出处
《中国人兽共患病学报》
CAS
CSCD
北大核心
2007年第11期1131-1134,共4页
Chinese Journal of Zoonoses
基金
河北省卫生厅资助项目(02076)
