牙龈卟啉单胞菌菌毛蛋白的重组及表达

Recombination and expression of fimbriae protein for Porphyromonas gingivalis

目的克隆Ⅱfi m A型牙龈卟啉单胞菌菌毛fi m A基因并使其在结肠埃希菌中正确表达。方法利用聚合酶链反应技术克隆Ⅱ型fi m A基因,经酶切和测序验证后构建原核表达质粒p ET-Fim A,使其在结肠埃希菌中表达,十二烷基硫酸钠聚丙烯酰胺凝胶电泳和Western blot鉴定表达产物。结果克隆基因测序结果与Gene Bank数据库中的序列呈现95%同源性,成功构建fi m A基因原核表达载体p ET-Fim A。结论高效表达出Ⅱfi m A型重组菌毛蛋白,并获得较高纯度,且具有免疫原性和免疫反应性。

Objective This study aimed to clone the type U fimA gene ofPorphyromonas gingivalis（P, gingivalis） and detect its expression in Escherichia coli（E, co10. Methods The type Ⅱ fimA gene was obtained by polymerase chain reaction（PCR） from the genome ofP. gingivalis to construct a prokaryotic expression plasmid pET-FimA, pET-FimA was transformed into E. coli BL21（DE3） competent cells, and the recombination protein was characterized via sodium dodecyl sulfate polyacrylamide gel electrophoresis（SDS-PAGE） and Western blot analysis. Results DNA sequencing showed that the fragment was 95% consistent with that of published literature. We successfully constructed prokaryotic expression plasmid pET-FimA. Conclusion The plasmid efficiently expressed the recombinant fimbriae protein of the type Ⅱ fimA gene ofP. gingivalis. A high purity of protein FimA was obtained. FimA has immunogenicity and immune reactivity.