目的 本研究以微小隐孢子虫含有纤维连接蛋白Ⅲ型结构域蛋白(Cryptosporidium parvum fibronectin type Ⅲ domain-containing protein,CpFN3)为研究对象,研究其亚细胞定位和黏附特性.方法 该蛋白由cgd4_640 基因编码、全长为含有2 430 个氨基酸的I型跨膜蛋白.设计特异性抗原多肽并免疫家兔,获得多克隆抗体,利用亲和纯化法获特异性抗体;经 Western blot 方法验证该抗体特异性,再通过间接免疫荧光法(IFA)进行蛋白定位.通过原核表达获得重组蛋白(His-CpFN3),通过ELISA确定重组蛋白与HCT-8细胞和肝素的结合情况.结果 成功获得纯化的抗CpFN3 抗体,Western blot 分析显示该抗体可识别条带大小约为 280 kDa;IFA结果表明该蛋白定位于子孢子的表膜,呈点状分布.该蛋白在子孢子入侵阶段及胞内无性繁殖和有性繁殖阶段均有表达.利用重组蛋白,确定了CpFN3 能够与HCT-8细胞及肝素结合(结合常数Kd 分别为 0.23 和 1.21 μmol/L),并呈剂量依赖性和可饱和性.结论 本研究确定CpFN3 参与了虫体与宿主细胞的黏附过程.
SUBCELLULAR LOCALIZATION AND ADHESION CHARACTERISTICS OF FIBRONECTIN TYPE Ⅲ DOMAIN-CONTAINING PROTEIN IN CRYPTOSPORIDIUM PARVUM
Objective This study was performed to investigate the subcellular localization and adhesion properties of the Cryptosporidium parvum fibronectin type Ⅲ domain-containing protein(CpFN3).CpFN3 is a 2 430-aa single-pass type Ⅰ membrane protein encoded by the cgd4_640 gene.Methods A CpFN3-epitope short peptide was designed to immunize two specific pathogen-free rabbits,from which polyclonal antibodies were affinity-purified.As expected,this antibody detected a 280 kDa band on Western blot analysis of a crude sporozoite extract.In an immunofluorescence assay,the antibody labeled the surface of C.parvum sporozoites and trophozoites mainly in granular form.The antibody labeled all lifecycle stages,from sporozoites to intracellular asexual and sexual stages.A protein fragment spanning the FN3 domain was expressed as a His-tagged recombinant protein(His-CpFN3)in bacteria and purified to determine binding kinetics by ELISA.Results In congruence with the presence of the FN3 domain,His-CpFN3 displayed high binding affinity to fixed HCT-8 with an apparent Kd of 0.23 μmol/L and to heparin with a Kd of 1.21 μmol/L.Conclusions Binding kinetics for both targets showed dose-dependence and saturability,indicating that binding is specific in both cases.The data suggest that CpFN3 may play a role in parasite-host adhesion.
Cryptosporidium parvumFibronectin type Ⅲ domainsHeparinAdhesion
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