微小隐孢子虫Ⅰ型跨膜蛋白Cgd7_1430的亚细胞定位及其HCT-8细胞黏附特性研究

Subcellular localization and adhesion characteristics of Cryptosporidium parvum type I transmembrane protein Cgd7_1430 in HCT-8 cells

目的对微小隐孢子虫Ⅰ型跨膜蛋白Cgd7_1430进行亚细胞定位,研究其对HCT-8细胞的黏附特性及肝素结合特性,为深入研究微小隐孢子虫的侵入机制提供依据。方法对Cgd7_1430序列进行生物信息学分析,合成特异性多肽并制备抗多肽抗体,通过间接免疫荧光对该蛋白质在子孢子进行亚细胞定位;通过PCR扩增Cgd7_1430全长基因并克隆至PGEX-4T-1载体,用大肠埃希菌BL21(DE3)菌株表达重组蛋白;通过细胞亲和ELISA和流式细胞术确定重组蛋白与HCT-8细胞的黏附特性;通过Pull-down确定重组蛋白的肝素结合特性。结果成功获得抗Cgd7_1430多肽抗体及其重组蛋白质,间接免疫荧光表明该蛋白定位于子孢子表膜,ELISA、流式细胞术表明该重组蛋白能够与HCT-8细胞结合,并呈现剂量依赖性和可饱和性,Pull-down实验表明重组蛋白能够与肝素结合。结论 Cgd7_1430蛋白质是一种子孢子表膜蛋白,能够与HCT-8细胞特异性结合,该蛋白为肝素结合蛋白,可能与宿主细胞表面的硫化肝素受体结合介导虫体的侵入过程。本研究为深入研究隐孢子虫的侵入机制提供依据。

Objective Type I transmembrane proteins play an important role during invasion by parasitic alveolates.This study examined the subcellular localization and preliminary adhesion characteristics of Cryptosporidium parvum type I transmembrane protein Cgd71430 in order to provide evidence for the study of the parasite’s mechanism of invasion.Methods Cgd71430 was bioinformatically analyzed,and a specific peptide was designed and synthesized to prepare an anti-peptide antibody.Subcellular localization of the protein in sporozoites was identified using indirect immunofluorescence.The full-length Cgd71430 gene was cloned into a PGEX-4 T-1 vector and expressed in E.coli BL21(DE3).The binding of the recombinant protein to HCT-8 cells was identified using cell-binding ELISA and flow cytometry.The binding of the recombinant protein to heparin was investigated using a pull-down assay.Results The anti-peptide antibody and recombinant protein were successfully prepared.An indirect immunofluorescence assay indicated that the protein was localized to the membrane of sporozoites.ELISA and flow cytometry indicated that the recombinant protein bound to HCT-8 cells in a dose-dependent and saturable manner.A pull-down assay indicated that the recombinant protein bound to heparin.Conclusion Cgd71430 protein is located on the membrane of sporozoites and can specifically bind to HCT8 cells and heparin,which may mediate the invasion process by adhesion to heparin sulfate on host cells.This study may provide some evidence for further study of the parasite’s mechanism of invasion.