淋病奈瑟菌外膜蛋白H.8抗原表位分析及其原核表达与纯化

Antigen epitopes analysis,prokaryotic expression and purification of out membrane protein H.8 from Neisseria gonorrhoeae

目的分析淋病奈瑟菌外膜蛋白H.8的抗原表位,利用原核表达系统表达并纯化该蛋白。方法运用生物信息学相关软件对目的蛋白进行理化性质分析及B细胞表位、T细胞表位的预测。将外膜蛋白H.8基因克隆至pET32a(+)质粒,并在Rosetta(DE3)宿主菌中构建原核表达系统,利用镍柱亲和层析纯化重组蛋白,采用SDS-PAGE和Western blot鉴定重组蛋白的表达及其免疫反应性。结果生物信息学分析显示外膜蛋白H.8的优势B细胞表位位于20-57、66-73、76-81、93-99、111-117、120-132、141-147、157-160肽段,优势T细胞表位位于125-139、132-146肽段。构建的含有外膜蛋白H.8基因的pET32a(+)重组质粒转化DE3后表达重组蛋白,以IPTG浓度为0.5mmol/L时重组蛋白在上清中的表达效果最佳;Western blot检测该重组蛋白能分别被His标签抗体和淋病奈瑟菌多克隆抗体识别。结论生物信息学方法预测淋病奈瑟菌外膜蛋白H.8含有优势B细胞表位和优势T细胞表位,原核表达的外膜蛋白H.8具有免疫反应性,为淋病奈瑟菌疫苗及其致病机制的研究提供了实验基础。

Objective Analysis of the antigenic epitope of the outer membrane protein H.8,expression and purification of the protein using a prokaryotic expression system.Methods The target protein was analyzed by bioinformatics technology to obtain its corresponding physical,chemical properties and prediction of B cell and T cell epitopes.The out membrane protein H.8 gene was cloned into pET32a(+),and its prokaryotic expression system was constructed using the E.coli expression hosts,Rossetta(DE3).The fusion protein was detected by SDS-PAGE,and isolated and purified by nickel column affinity chromatography to verify its immunoreactivity by Western blot assay.Results Peptides19-57,63-70,73-83,90-100,108-135,140-150 and 153-163 of the out membrane protein H.8 were the dominantB cell epitopes,while peptides 125-139 and 132-146 were the dominantT cell epitopes.The recombinant protein was significantly expressed in the supernatant at IPTG concentration of 0.5 mmol/L after transformation of DE3 with the constructed pET32a(+)recombinant plasmid containing the outer membrane protein H.8 gene.The results of Western blot showed that the recombinant protein can recognize by His-tag antibody andpolyclonal antibody of N.gonorrhoeae respectively.Conclusion Bioinformatics methods predicted that N.gonorrhoeae outer membrane protein H.8 contains dominant B-cell epitopes and dominant T-cell epitopes,and that the outer membrane protein H.8 expressed in prokaryotic cellshas immunoreactive,which providing an experimental basis for the study of N.gonorrhoeae vaccines and its pathogenic mechanisms.