微小隐孢子虫Ⅰ型跨膜蛋白Cgd7＿4560的亚细胞定位及其对HCT-8细胞的黏附特性  

Subcellular Localization and HCT-8 Cell Adhesion of Type Ⅰ Transmembrane Protein Cgd7＿4560 of Cryptosporidium parvum

作　　者：邵清延 王科杰 王东强 张天宇 焦新 尹继刚 SHAO Qingyan;WANG Kejie;WANG Dongqiang;ZHANG Tianyu;JIAO Xin;YIN Jigang(Key laboratory of Zoonosis Research,Ministry of Education,Institute of Zoonosis,Jilin University,Changchun 130062,China)

机构地区：[1]吉林大学人兽共患病研究所人兽共患病研究教育部重点实验室

出　　处：《吉林农业大学学报》2019年第3期357-362,共6页Journal of Jilin Agricultural University

基　　金：国家自然科学基金项目(31772731,31272549)

摘　　要：Ⅰ型跨膜蛋白在顶复门寄生虫入侵宿主细胞过程中发挥重要作用.试验对微小隐孢子虫Ⅰ型跨膜蛋白Cgd7_4560进行生物信息学分析,选取抗原性强的序列,采用PCR方法扩增出微小隐孢子虫DNA Cgd7_4560部分基因.将该片段分别连接到pET-28a和pGEX-4T-1载体,构建重组表达质粒pET-28a-Cgd7_4560和pGEX-Cgd7_4560,并将其转化到大肠埃希氏杆菌BL21(Condon plus),IPTG诱导表达获得重组蛋白.SDS-PAGE和Western blot分析,His标签重组蛋白为31 ku,GST标签重组蛋白为53 ku.用His标签重组蛋白免疫家兔,间接ELISA方法检测血清抗体效价达到1:16000,成功制备多克隆抗体.采用Western blot方法验证该抗体能够特异性的识别虫体天然蛋白,间接免疫荧光分析该蛋白在子孢子头端,间接ELISA结果表明GST-Cgd7_4560能与HCT-8细胞黏附,为其功能研究奠定了基础.TypeⅠtransmembrane proteins play an important role during the invasion of phylum Apicomplexa parasites. Bioinformatics analysis of typeⅠtransmembrane proteins Cgd74560 of Cryptosporidium parvum was carried out to select the highly antigenic part of Cgd74560. The PCR fragments were inserted to the vectors of pET-28 a and pGEX-4 T-1,respectively,to construct the recombinant expression plasmids pET-Cgd74560 and pGEX-Cgd74560,and they were transformed into Escherichia coli BL21( Condon plus). IPTG was used to induce the expression of recombinant protein. The recombinant proteins were analyzed by SDS-PAGE and Western blot,and a His-tagged31 ku recombinant protein and a 53 ku GST-tagged recombinant protein were identified. Rabbit was immunized with His-tagged recombinant protein,and the titer of antibody in serum was 1 ∶ 16 000 by indirect ELISA. Native Cgd74560 protein could be recognized at 340 ku by Western blot. Indirect immunofluorescence analysis of the protein suggested that it is localized in the apical end of sporozoites. Cell binding ELISA results showed that recombinant protein can attach to HCT-8 cells in dose-dependent manner. These findings lay foundation for future functional research of the Cgd7 4560 in Cryptosporidium parvum.

关 键 词：微小隐孢子虫 Ⅰ型跨膜蛋白 原核表达 亚细胞定位 

分 类 号：R38[医药卫生—医学寄生虫学] S852.7[医药卫生—基础医学]