%0 Journal Article
%T 微小隐孢子虫黏蛋白CGD5_2060 原核表达及其黏附功能
%A 阳毅敏
%A 潘灵韬
%A 庄浩瀚
%A 孙洪超
%A 杨怡
%A 陈学秋
%A 杜爱芳
%J 浙江大学学报(农业与生命科学版)
%D 2018
%R 10.3785/j.issn.1008-9209.2018.03.123
%X 从微小隐孢子虫cDNA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pET32a-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。 通讯作者: 杜爱芳（https://orcid.org/0000-0002-3796-6621） E-mail: afdu@zju.edu.cn</br>Abstract: The gene cgd5_2060 was cloned from Cryptosporidium parvum cDNA, and its function-related motifs were predicted. The prokaryotic expression vector pET32a-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 byWestern 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 pET32a-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 ELISArevealed 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. Key words: Cryptosporidium parvum mucin gene cgd5_2060 prokaryotic expression cell adhesion
%U http://www.zjujournals.com/agr/CN/10.3785/j.issn.1008-9209.2018.03.123