旋毛虫抗原基因Ts88原核表达及重组蛋白鉴定  

Prokaryotic Expression of an Antigen Gene of Trichinella spiralis and Identification of the Recombinant Protein

作　　者：雷丽萍[1] 诸欣平[1] 杨静[1] 杨雅平[1] 丁利[1] 

机构地区：[1]首都医科大学基础医学院人体寄生虫学教研室,北京100054

出　　处：《中国寄生虫学与寄生虫病杂志》2004年第5期257-261,共5页Chinese Journal of Parasitology and Parasitic Diseases

基　　金：美国中华医学会基金(CMB) (Parasitology 98 674);国家教委留学回国人员科研基金(1 998~ 2 0 0 1 );北京市教委科技发展基金(0 1KJ 0 91 );北京市跨世纪优秀人才工程基金(2 0 0 1~ 2 0 0 4 )~~

摘　　要：目的　原核表达旋毛虫抗原基因Ts88,并对重组蛋白的抗原性进行鉴定。　方法　免疫筛选cDNA文库得到旋毛虫抗原新基因Ts88,克隆入原核表达载体pET 2 8c(+ ) ,异丙基 β D 硫代半乳糖苷诱导表达。重组蛋白免疫小鼠制备免疫血清 ,ELISA检测抗体滴度。蛋白质印迹法检测重组蛋白的抗原性。免疫荧光试验确定Ts88蛋白在虫体的分布。　结果　经原核表达的Ts88重组蛋白 ,用其免疫小鼠可得到高滴度的抗体血清。蛋白质印迹法结果表明该重组蛋白能与旋毛虫病患者血清、旋毛虫病猪血清、人工感染及人工免疫兔血清反应 ,不与其他蠕虫病患者血清发生交叉反应 ,并能识别旋毛虫虫体天然抗原成分。免疫荧光试验显示Ts88蛋白主要分布于虫体体壁。　结论　成功制备Ts88基因的重组蛋白 。Objective To obtain the recombinant protein of an antigen gene Ts88 of Trichinella spiralis and identify the characteristics of the recombinant protein. Methods Ts88 cDNA obtained by immunoscreening the cDNA library of adult T. spiralis was subcloned into the pET 28c(+) expression vector and expressed in E.coli . Mice were immunized with the fusion protein incorporated into Freund’s adjuvant and the immune sera were collected. The titers of the Ts88 immune sera and the antigenicity of the recombinant protein were detected by ELISA and Western blotting. Immuno fluorescence test was performed in order to confirm the distribution of Ts88 protein in the worm. Results The fragment of Ts88 gene was expressed successfully in E.coli and a highly purified fusion protein was obtained. Immunization with the recombinant protein in mice produced high titers of antibodies, which recognized some components of native antigens of soluble proteins from adult worm of T. spiralis. Western blotting analysis showed that Ts88 recombinant antigen was recognized by all the positive sera, such as the sera from infected or immunized rabbits, from infected swine and from patients of trichinosis. Immuno fluorescence test confirmed that Ts88 protein mainly distributed in the cuticle surface of the worm. Conclusion The Ts88 antigen gene from T. spiralis was successfully expressed. The recombinant protein presented antigenicity.

关 键 词：重组蛋白 旋毛虫 血清 抗原性 抗原基因 蛋白质印迹法 原核表达 虫体 免疫荧光试验 病猪 

分 类 号：R383.15[医药卫生—医学寄生虫学] S858[医药卫生—基础医学]