Eg Innexin-unc9重组蛋白的表达纯化及诊断价值  

Study on expression,purification and diagnostic value of Eg Innexin-unc9(Eg Inx)recombinant protein

作　　者：吴军[1] 范宇 黎飞海 李军 张文宝 郭宝平 杨梅 WU Jun;FAN Yu;LI Feihai;LI Jun;ZHANG Wenbao;GUO Baoping;YANG Mei(Department of Labor Hygiene and Environmenta Health,School of Public Health,School of Basic Medical Sciences,Xinjiang Medical University,Urumqi 830017,China;Department of Biochemistry and Molecular Biology,School of Basic Medical Sciences,Xinjiang Medical University,Urumqi 830017,China;State Key Laboratory of Causes and Prevention of Highly Disease-Prone Diseases in Central Asia,Provincial-Ministerial Jointly Constructed by the Institute of Clinical Medical Research,the First Affiliated Hospital,Xinjiang Medical University;Xinjiang Key Laboratory of Endemic Diseases and Key Laboratory of Molecular Biology,Urumqi 830054,China)

机构地区：[1]新疆医科大学公共卫生学院劳动卫生与环境卫生教研室,乌鲁木齐830017 [2]新疆医科大学基础医学院生物化学与分子生物学教研室,乌鲁木齐830017 [3]新疆医科大学第一附属医院临床医学研究院省部共建中亚高发病成因与防治国家重点实验室 [4]新疆地方病及分子生物学重点实验室,乌鲁木齐830054

出　　处：《新疆医科大学学报》2025年第4期421-425,431,共6页Journal of Xinjiang Medical University

基　　金：新疆维吾尔自治区自然科学基金项目(2023D01C46)。

摘　　要：目的构建并表达纯化细粒棘球绦虫缝隙连接通道蛋白(Eg Innexin unc9,Eg Inx)重组蛋白,制备多抗评估其诊断潜力及组织定位,为功能研究和犬用疫苗开发奠定基础。方法通过NdeI/HindⅢ双酶切构建pET-30a-Eg Inx原核表达质粒;将重组质粒转化至大肠杆菌BL21(DE3),通过异丙基-β-D-硫代半乳糖苷(Isopropylβ-D-1-thiogalactopyranoside,IPTG)在不同温度下诱导表达,利用镍亲和层析柱纯化重组Eg Inx蛋白(r Eg Inx),并通过蛋白质免疫印迹(Western blotting,WB)验证其特异性;制备多克隆抗体进行免疫组化;采用生物素-亲和素放大系统酶联免疫吸附实验(Biotin-avidin system-enzyme linked immunosorbent assay,BAS-ELISA)检测22份健康人血清及7份囊型棘球蚴病患者血清,初步评估其诊断价值。结果成功表达并纯化r Eg Inx,WB检测显示其可被组氨酸标签抗体特异性识别。免疫组化表明,在胆盐诱导后Eg Inx在原头蚴头节及体表皮层高表达,成虫则表达在生殖系统,主要位于孕节。BAS-ELISA检测敏感度为85.71%(6/7),特异度95.45%(21/22)。结论r Eg Inx具有高诊断特异性,其发育阶段差异性定位提示其参与虫体发育及繁殖调控,可作为囊型棘球蚴病(Cystic echinococcosis,CE)新型诊断标志物及犬用疫苗潜在靶点。Objective This study constructed and expressed the purified recombinant protein of Eg Innexin unc9(Eg Inx)from Echinococcus granulosus(Eg),and prepared polyantibodies to evaluate its diagnostic potential and tissue localization,so as to lay the foundation for the functional study and the development of canine vaccine.Methods The pET-30a-Eg Inx prokaryotic expression plasmid was constructed by NdeI/HindIII double digestion;the recombinant plasmid was transformed into Escherichia coli BL21(DE3),and the expression of the recombinant plasmid was induced by Isopropylβ-D-1-thiogalactopyranoside(IPTG)at different temperatures.The recombinant Eg Inx protein(r Eg Inx)was purified using a nickel affinity chromatography column and its specificity was verified by western blotting(WB);polyclonal antibodies were prepared for immunohistochemistry;and a Biotin-Avidin System-Enzyme Linked Immunosorbent Assay was used.Linked Immunosorbent Assay(BAS-ELISA)was used to detect 22 healthy human sera and 7 sera from patients with cystic echinococcosis,and its diagnostic value was preliminarily evaluated.Results r Eg Inx was successfully expressed and purified,and WB showed that it was specifically recognized by His-tagged antibodies.Immunohistochemistry showed that Eg Inx was highly expressed in the cephalic nodes and body surface cortex of protozoa after bile salt induction,while adults were expressed in the reproductive system,mainly located in the gestational nodes.r Eg Inx was detected with a sensitivity of 85.71%(6/7)and a specificity of 95.45%(21/22)by BAS-ELISA.Conclusion r Eg Inx has high diagnostic specificity,and its differential localization in developmental stages suggests that it is involved in the regulation of worm development and reproduction,and itcan be used as a novel diagnostic marker for CE and a potential target for canine vaccines.

关 键 词：细粒棘球绦虫 重组蛋白 免疫诊断 

分 类 号：R383.33[医药卫生—医学寄生虫学]