摘要
目的构建表达肠血性大肠杆菌(EHEC)O157∶H7Ⅲ型分泌蛋白EspA与紧密粘附素C-端免疫保护性片断(IntiminC300)的融合蛋白(EspA-IntiminC300)。方法采用PCR技术从EHEC O157∶H7基因组中扩增EspA的编码基因espA及IntiminC300的编码基因eaeC300,T-A克隆后依次构建至表达载体pET-28a(+),转化宿主细胞E.coliBL21(DE3),测序鉴定,IPTG诱导表达,SDS-PAGE检测其表达量及表达形式,亲和层析法纯化目的蛋白,免疫印迹分析免疫反应性。结果PCR法自EHEC O157∶H7基因组中分别扩增出了约580bp(espA)和900bp(eaeC300)的目的片段,将二者融合构建了重组质粒pET-28a(+)-espA-eaeC300,测序结果与理论预测值一致性为99.9%(1501/1502)。融合蛋白在工程菌中表达量约40%,PAGE初步测定目的蛋白的相对分子量(Mr)约54×103Da,破菌后电泳证实目的蛋白主要以包涵体形式表达,包涵体洗涤后纯化,获得目的蛋白纯度约90%。免疫印迹显示融合蛋白能分别与兔抗EspA和IntiminC300血清发生免疫反应。结论高效表达了融合蛋白(EspA-IntiminC300),此融合蛋白具有良好的免疫反应性和免疫原性,为研制EHECO157∶H7多亚单疫苗奠定了基础。
To construct and prokaryotically express the fusion protein of EspA and IntinminC300 from EHEC O157 , H7,the coding genes of EspA （espA） and IntinminC300 （eaeC300） was amplified by PCR from EHEC O157 , H7 chromosome and then cloned into pMD-18T vector. Thereafter target genes cloned into pET-28a（＋） vector in turn by recombinant technology, and transferred to host cells E. coll BL21 strain （DE3）. Then, the protein was induced and expressed with induction of IPTG. The expression quantities and style of fusion protein were determined by SDS-PAGE, and purified by affinity chromatography. Finally, its immunoreactivity was analyzed by Western blotting. The determination of the sequence encording of the espA-eaeC,300 fusion gene has 99. 90% （1501/1502） of consistency with the sequence from GenBank Sakai strain and contrivable linker. The fusion protein EspA-IntinminC300 was expressed as inclusion body formation and the percentage was approximately 40 %. The molecular weight of the expressed product was 54kD. Western blotting demontrated the was excellent immunoreactivity of the fusion protein. It is evident that the fusion protein EspA-IntinminC300 of EHEC O157 t H7 was successfully constructed and well expressed in prokaryotic expression system, and the fusion protein has excellent immunoreactivity. These results may provide the foundation for the further development on EHEC O157 , H7 multivalent subunit vaccine.
出处
《中国人兽共患病学报》
CAS
CSCD
北大核心
2007年第2期106-109,共4页
Chinese Journal of Zoonoses
基金
军队杰出人才基金资助项目(No.04J009)
