弓形虫致密颗粒蛋白4的原核细胞表达与纯化  

Expression and Purification of Toxoplasma gondii GRA4 Gene in Prokaryotic System

作　　者：林绮萍[1] 吴少庭[1] 翁亚彪[2] 雷明军[3] 潘晖榕[3] 袁仕善[1] 温见翔[4] 秦莉[3] 黄达娜[1] 张仁利[1] 高世同[1] 

机构地区：[1]深圳市疾病预防控制中心,深圳518020 [2]华南农业大学兽医学院,广州510642 [3]华中科技大学同济医学院,武汉430030 [4]广东医学院微生物学教研室,湛江524023

出　　处：《中国寄生虫学与寄生虫病杂志》2005年第6期419-423,共5页Chinese Journal of Parasitology and Parasitic Diseases

基　　金：深圳市科技项目基金(编号:1997-15)~~

摘　　要：目的构建弓形虫致密颗粒蛋白4(GRA4)的pET原核细胞表达系统,并对其表达产物进行纯化及其免疫学活性测定。方法利用PCR扩增弓形虫GRA4基因片段,定向亚克隆入原核细胞表达载体pET-23a(+),转化大肠埃希菌(E.coliBL21 DE3),以组氨酸结合柱亲和层析法纯化表达产物,并对其进行抗原活性分析。将纯化的重组蛋白免疫小鼠,用ELISA检测其特异性抗体滴度。结果成功构建了弓形虫pET-GRA4原核细胞表达系统,其表达产物相对分子质量(Mr)约为40 000,主要以包涵体形式存在,纯化后的重组蛋白能被人工感染弓形虫RH株速殖子的兔血清识别。免疫小鼠后可诱导产生高滴度的特异性IgG抗体。结论利用pET原核细胞表达系统成功表达和纯化了弓形虫GRA4重组蛋白,该蛋白具有一定的免疫学活性。Objective To construct a prokaryotic expression system containing the dense granule protein 4 （GRA4） of Toxoplasrna gondii, purify the expressed protein and detect its immunogenicity. Methods The specific fragment of GRA4 gene was amplified by PCR. After subcloning the prokaryotic expression recombinant pET-GRA4, the expressed product was purified with His.Bind^TM affinity chromatography and analyzed by Western blot. BALB/c mice were immunized with the GRA4 recombinant protein, and the antibody IgG titer was detected by ELISA. Results The pET-GRA4 prokaryotic expression system was obtained. The MW of the expressed protein was Mr 40 000 and formed in inclusion body. After purification, the recombinant protein could be specifically recognized by the T.gortclii infected rabbit serum. Mice immunized with the purified recombinant protein elicited high titer of IgG antibody. Conclusion The pETGRA4 recombinant protein was successfully expressed and purified, which shows the immunogenicity.

关 键 词：弓形虫 致密颗粒蛋白4 原核细胞 基因表达 蛋白纯化 

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]