肠出血性大肠埃希菌O157∶H7外膜蛋白A的表达、鉴定和纯化

Identification and purification of prokaryotic expression of outer membrane protein A (OmpA) from enterohaemorrhagic Escherichia coli(EHEC) O157∶H7

目的构建肠出血性大肠埃希菌O157∶H7外膜蛋白A基因重组质粒,研究其在大肠埃希菌中的表达情况。方法用RT-PCR法从肠出血性大肠埃希菌O157∶H7菌株克隆OmpA基因,构建重组质粒pET-30a(+)-OmpA,经双酶切及基因测序鉴定后在E.coli BL21(DE3)原核表达系统中诱导OmpA的表达,通过蛋白质电泳技术检测蛋白的表达,用Western blot法测定和分析免疫反应性。结果获得的目的基因为1041bp,与预期值相符,测序结果与GenBank公布序列的同源性达100%,重组质粒pET-30a(+)-OmpA在原核系统下可表达OmpA,Western blot分析His-Tag单抗能与OmpA(His-Tag)发生免疫反应。结论OmpA重组质粒构建成功,表达产物具有抗原性。

Objective To construct recombinant plasmid pET-30a（＋ ）-OmpA and investigate transientexpression of vectors in E. coli BL21（DE3）. Methods The OmpA gene was amplified from EHEC O157 ： H7 strain by RT-PCR to construct recombinant plasmid pET-30a （＋）-OmpA, after identified by double restriction endonucleases digestion and DNA sequencing analysis, the recombinants were transformed into E. coli BL21 （DE3）, the OmpA was induced and expressed in a protokaryotic expression system. With SDS-PAGE the expressed protein was detected. The immunological reaction of the OmpA was determined by Western blot assay. Results The acquired gene is 1 041 bp, in accordance with the expected result. DNA sequence analysis showed that the OmpA gene homology of EHEC O157 ： H7 and reported in GenBank was 100%. Recombinant plasmid pET-30a（＋ ）-OmpA was able to express OmpA within protokaryotic expression system. Western blot test demonstrated that the OmpA （His-Tag） showed immunological reaction with the His-Tag monoclonal antibody. Conclusion Recombinant plasmid pET-30a（ ＋）-OmpA has been constructed successfully and the expression product showes immunological reaction with according antibody.