弓形虫主要表面抗原1单链抗体与绿色荧光蛋白的融合表达及其生物活性初步观察  

Expression and Bioactivity of S1 scFv Antibody against SAG1 of Toxoplasma gondii Fused with Green Fluorescent Protein

作　　者：司进[1] 朱荫昌[1] 曹利民[2] 王晓婷[1] 梁幼生[1] 管晓虹[3] 

机构地区：[1]江苏省寄生虫病防治研究所,无锡214064 [2]华中科技大学同济医学院免疫学系,武汉430030 [3]南京医科大学分子生物学研究所,南京210019

出　　处：《中国寄生虫学与寄生虫病杂志》2006年第3期161-165,共5页Chinese Journal of Parasitology and Parasitic Diseases

基　　金：江苏省寄生虫病重点学科;江苏省寄生虫分子生物学实验室开放课题资助项目(No.WK005-008)~~

摘　　要：目的构建、表达和纯化刚地弓形虫(Toxoplasmagondii)主要表面抗原1(SAG1)单链抗体S1与绿色荧光蛋白(GFP)的融合蛋白,体外观察其与弓形虫速殖子的特异性结合能力。方法分别设计引物,构建原核表达质粒pET-26b-GFP和pET-26b-GFPS1,测序验证后转化大肠埃希菌BL21(DE3),以异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达,荧光显微镜观察融合基因中GFP的表达情况。用Ni2+螯合柱亲和纯化融合蛋白GFPS1,十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)检测融合蛋白GFPS1。荧光显微镜观察融合蛋白GFPS1与弓形虫速殖子体外孵育后单链抗体S1对弓形虫速殖子的靶向作用。结果测序结果显示,弓形虫SAG1单链抗体S1与GFP的融合基因构建到原核表达载体pET-26b(+)中;IPTG诱导表达后,在荧光显微镜下可观察到E.coliBL21发出的特异性绿色荧光。SDS-PAGE检测到表达的融合蛋白GFPS1相对分子质量(Mr)约为53000,多以包涵体形式存在。免疫荧光检测可见弓形虫速殖子表膜周围发出特异性的绿色荧光,表明纯化的GFPS1可特异性地结合到弓形虫速殖子表膜。结论弓形虫主要表面抗原1(SAG1)单链抗体S1与弓形虫速殖子表膜有较强的结合能力。Objective To construct, express and purify human scFv antibody （S1） against the recombinant SAG1 of Toxoplasma gondii and fused to green fluorescent protein＂ （GFP）, and observe its binding capacity to tachyzoite of Toxoplasma gondii. Methods The GFP gene amplified from vector pEGFP-N1 was subcloned into procaryotic expression vector pET-26b （＋）, then the S1 scFv antibody gene amplified from phagmid pIT-2-S1 was cloned into downstream of GFP gene. The recombinant plasmid pET-26b-GFPS1 proved by DNA sequencing was transformed into E.coli BI21, and induced for fusion expression of GFPS1 with IPTG, the green fluorescence of E.coli BL21 harboring plasmid pET-26b- GFPS1 was observed under the fluorescence microscope. The expressed GFPS1 was purified with Ni^2＋ chelating HiTrap HP column, and detected with SDS-PAGE. Toxoplasma tachyzoites were incubated with the recombinant GFPS1, and the binding bioactivity was observed under the fluorescence microscope. Results The fused gene of S1 and GFP was successfully cloned into procaryotic expression vector pET-26b proved by DNA sequencing. The green fluorescence of E.coli BL21 harboring plasmid pET-26b-GFPS1 was catched under the fluorescence microscope. The recombinant GFPS1 protein about Mr 53 000 was expressed in E.coli as inclusion body. The immunofluorescence detection verified that anti-rSAG1 scFv antibody S1 could specifically bind outer membrane of Toxoplasma tachyzoite. Conclusions The purified rGFPS1 shows a strong binding capacity to outer membrane of Toxoplasma tachyzoite using GFP as a labeling protein, and the constructed pET-26b-GFP can also be used for research on other targeting molecules.

关 键 词：刚地弓形虫 单链抗体 绿色荧光蛋白 融合表达 

分 类 号：R382.5[医药卫生—医学寄生虫学] R392.11[医药卫生—基础医学]