微小隐孢子虫黏蛋白CGD5_2060原核表达及其黏附功能  

Prokaryotic expression of Cryptosporidium parvum mucin CGD5_2060 and its role in adhesion

作　　者：阳毅敏 潘灵韬 庄浩瀚 孙洪超 杨怡 陈学秋[1] 杜爱芳[1] YANG Yimin;PAN Lingtao;ZHUANG Haohan;SUN Hongchao;YANG Yi;CHEN Xueqiu;DU Aifang(Institute of Preventive Veterinary Medicine, College of Animal Sciences, Zhejiang University, Hangzhou 310058, China)

机构地区：[1]浙江大学动物科学学院动物预防医学研究所,杭州310058

出　　处：《浙江大学学报（农业与生命科学版）》2018年第2期230-236,共7页Journal of Zhejiang University:Agriculture and Life Sciences

基　　金：浙江省重大科技专项重点农业项目(2012C12009-2)

摘　　要：从微小隐孢子虫c DNA中克隆黏蛋白cgd5_2060基因,并对其蛋白功能相关基序进行预测;构建原核表达载体p ET32a-cgd5_2060,转化至大肠埃希菌Rosetta中,诱导表达并纯化重组蛋白CGD5_2060,制备鼠源多克隆抗体,并通过间接酶联免疫吸附测定(enzyme-linked immunosorbent assay,ELISA)方法测定其抗体效价;将纯化的重组蛋白CGD5_2060及载体对照蛋白与Caco2细胞共孵育,通过蛋白质印迹法(Western blotting)与间接ELISA方法检测其细胞黏附特性。结果表明,CGD5_2060蛋白的1~19个氨基酸为信号肽,说明该蛋白是分泌型蛋白。使用PROSITE数据库分析发现:该蛋白含有1个富含苏氨酸的区域TTTTTTKSTTTTTTAVTT,位于510~527个氨基酸处;此外,还有1个与细胞黏附有关的序列RGD(精氨酸-甘氨酸-天冬氨酸),位于69~71个氨基酸处。上述结果表明,cgd5_2060基因可能与微小隐孢子虫黏附宿主细胞相关。原核表达的重组蛋白CGD5_2060具有良好的免疫原性,获得了较高效价的多克隆抗体。重组蛋白与Caco2细胞共孵育,通过蛋白质印迹法与间接ELISA方法检测发现,CGD5_2060可以与Caco2细胞黏附。本试验为揭示微小隐孢子虫黏附细胞的机制提供了一定的理论基础。The gene cgd5_2060 was cloned from Cryptosporidium parvum c DNA, and its function-related motifs were predicted. The prokaryotic expression vector p ET32 a-cgd5_2060 was constructed and transformed into Escherichia coli Rosetta. The recombinant protein CGD5_2060 was expressed and purified, with which the murine polyclonal antibody was prepared, and its antibody titers were determined by indirect enzyme-linked immunosorbent assay（ELISA）. The purified recombinant protein CGD5_2060 and control protein were co-cultured with Caco2 cells, and their cell adhesion properties were detected by Western blotting and indirect ELISA. The prediction of CGD5_2060 protein function-related motifs revealed that the 1-19 amino acids of the protein are signal peptides, indicating that the protein is a secreted protein. Using PROSITE database for analysis, the CGD5_2060 protein contains a threonine-rich region, TTTTTTKSTTTTTTAVTT, at 510-527 amino acids; in addition, CGD5_2060 has a cell-attachment related RGD（Arg-Gly-Asp） at 69-71 amino acids. The above results indicated that the cgd5_2060 gene might be associated with C. parvum adhesion to host cells. The recombinant plasmid p ET32 a-cgd5_2060 was successfully constructed, and a high-quality recombinant protein was successfully purified by nickel column affinity chromatography. The recombinant protein CGD5_2060 had good immunogenicity and polyclonal antibodies with higher titer were successfully prepared. After coincubation of recombinant proteins and Caco2 cells, Western blotting and indirect ELISA revealed that CGD5_2060 could adhere to Caco2 cells. This study provides a theoretical basis for revealing the mechanism of C. parvum adhesion to host cells through preliminary study of cgd5_2060 gene adhesion function.

关 键 词：微小隐孢子虫 黏蛋白基因cgd5_2060 原核表达 细胞黏附 

分 类 号：S855.9[农业科学—临床兽医学]