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

作　　者：李紫玥 苏晓悦 谢晓婷 胡欣 陈佳琪 董凤 黄钰 张莉 张雷 LI Ziyue;SU Xiaoyue;XIE Xiaoting;HU Xin;CHEN Jiaqi;DONG Feng;HUANG Yu;ZHANG Li;ZHANG Lei(Institute of Pathogens and Vectors,Yunnan Provincial Key Laboratory for Zoonosis Control and Prevention,Dali University,Dali,Yunnan,671000,China;Laboratory of Pathogenic Biology,Dali University School of Basic Medicine)

机构地区：[1]大理大学病原与媒介生物研究所,云南省自然疫源性疾病防控技术重点实验室,云南大理671000 [2]大理大学基础医学院病原生物学综合实验室

出　　处：《中国病原生物学杂志》2023年第5期508-512,共5页Journal of Pathogen Biology

基　　金：国家自然科学基金项目(No.81760303)。

摘　　要：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.

关 键 词：淋病奈瑟菌 外膜蛋白H.8 原核表达及纯化 表位分析 

分 类 号：R378.16[医药卫生—病原生物学]