人空肠弯曲菌FlaA蛋白重组表达与免疫血清制备以及间接ELISA检测血清抗体的方法建立及初步应用OA

Objective To express and purify the Campylobacter jejuni(C.jejuni)FlaA protein and to develop an ELISA diagnos-tic kit for detecting C.jejuni antibodies in humans.Methods Bioinformatic analysis of the C.jejuni FlaA protein was performed,and the recombinant plasmid pET-28a-flaA was constructed.The recombinant protein was expressed by transforming BL21(DE3)competent cells.Female New Zealand white rabbits were immunized to prepare the corresponding polyclonal antiserum.The op-timal coating concentration was screened,experimental conditions were optimized,and an indirect ELISA assay was established.The assay was then applied to serum samples from 58 patients with diarrhea and 697 healthy blood donors to evaluate the sensi-tivity and specificity of the method.Results The pET-28a-flaA recombinant plasmid was successfully constructed.The recom-binant protein obtained via the prokaryotic expression system exhibited high expression levels.Western blot analysis confirmed that the recombinant protein reacted strongly with positive sera and could serve as a target antigen.An optimized indirect ELISA protocol was established,and the test results were generally consistent with clinical findings,with specificity and sensitivity of 75%and 99.86%,respectively.Conclusions The C.jejuni FlaA recombinant protein is successfully expressed and purified,and an indirect ELISA method using FlaA as the coating antigen is established.