幽门螺杆菌flaA基因在大肠杆菌中的表达与鉴定

目的：观察幽门螺杆菌(H．pylori)flaA基因在大肠杆菌中的表达。方法：用PCR方法扩增flaA基因,然后插入原核表达载体pMAL-c2x进行融合表达。采用蛋白印迹法对表达产物进行鉴定。结果：特异PCR和酶切鉴定证实flaA基因克隆入表达载体,并以融合蛋门形式MBP-FLA高效表达,约占细胞总蛋白的28％。该融合蛋白可与H.pylori阳性病人血清和小鼠H.pylori全菌抗体发生特异反应。结论：成功构建了能高效表达H．pylori FlaA蛋白的重组质粒,为H．pylori多价基因工程疫苗的研制奠定了基础。

Expression and identification of flaA gene from Helicobacter pylori in Escherichia coli

Aim: To express flagellar protein A(FlaA) from H.pylori in E.coli and study its immunoreactivity.Methods: The flaA gene were amplified from H.pylori by PCR and cloned directly into expression vector pMAL-C2x, and the recombinant plasmid was then transformed into E.coli TB1. The immunoreactivity of expressing product was identified by the method of Western blot. Results: It was confirmed that the flaA gene had been cloned into pMAL-C2x by specified PCR and restriction enzyme digestion. SDS-PAGE and optical density scanning indicated that the fusion protein BMP-FLA was expressed in TB1(PMFA) as a protein with 95 000 dolton of molecular weight and was 28 % of the total bacterial protein. The fusion protein could be recognized by patient serum and mouse serum infected with H.pylori. Conclusions: A recombinant plasmid expressing FlaA of H.pylori was constructed and identified successfully, and this study may help develop gene recombinant vaccine against H.pylori infection.