肠炎沙门菌菌毛重组蛋白对其细胞黏附的竞争性阻断效应

Competitive Blocking Effect of Salmonella Enteritis Fimbriae Recombinant Protein on Its Cell Adhesion

鸡沙门菌通过其菌毛、鞭毛黏附宿主小肠上皮细胞是其致病过程不可或缺的重要环节。本研究旨在分析原核表达的菌毛重组蛋白FimA-sefA对肠炎沙门菌黏附小肠上皮细胞(IPEC-J2)竞争性阻断效应。将FimA、sefA基因片段克隆至pET28a(+),构建重组质粒pET28a-FimA-sefA,转化至菌株BL21(DE3)经镍柱亲和层析法(Ni-NTA)纯化获得重组蛋白FimA-sefA;利用间接免疫荧光法(IFA)、平板计数法分析肠炎沙门菌对IPEC-J2细胞的黏附。结果显示:表达的重组蛋白FimA-sefA分子量为36 ku;重组蛋白FimA-sefA、重组菌及肠炎沙门菌均能黏附IPEC-J2细胞;重组蛋白FimA-sefA及重组菌均能竞争性阻断肠炎沙门菌对IPEC-J2细胞的黏附,重组菌的阻断效应强于重组蛋白。因此,我们认为重组蛋白FimA-sefA能够在一定程度上竞争性阻断肠炎沙门菌对IPEC-J2细胞的黏附,为预防及减轻肠炎沙门菌感染策略提供了新的思路。

Adhesion of host small intestinal epithelial cells through fungal hairs and flagella is an indispensable and important link in pathogenic processes of Salmonella Enterison.This study aims to analyze the competitive blocking effect of the hair recombinant protein FimA-sefA expressed by prokaryotes on the adhesion of Salmonella Enteritis to intestinal epithelial cells(IPEC-J2).FimA and sefA gene fragments were cloned into pET28a(+),resulting in the construction of a recombinant plasmid pET28a-FimA-sefA,which was then transformed into the strain BL21(DE3).The recombinant protein FimA-sefA was purified using nickel-column affinity chromatography(Ni-NTA).The adhesion of enteritis to IPEC-J2 cells was analyzed using indirect immunofluorescence(IFA)and plate counting.The molecular weight of the expressed recombinant protein FimA-sefA was 36 ku;the recombinant protein FimA-sefA,recombinant bacteria,and Salmonella Enteritis can adhere to IPEC-J2 cells.Recombinant protein FimA-sefA and recombinant bacteria can competitively block the adhesion of Salmonella Enteritis to IPEC-J2 cells,and the blocking effect of recombinant bacteria was stronger than that of the recombinant protein.We believe that the recombinant protein FimA-sefA can competitively block the adhesion of Salmonella Enteritis to IPEC-J2 cells to a certain extent,providing a new strategy for preventing and reducing enteritis infection.