微小隐孢子虫含有纤维连接蛋白Ⅲ型结构域蛋白的亚细胞定位及其细胞黏附特性研究  

SUBCELLULAR LOCALIZATION AND ADHESION CHARACTERISTICS OF FIBRONECTIN TYPEⅢDOMAIN-CONTAINING PROTEIN IN CRYPTOSPORIDIUM PARVUM

作　　者：王自强 王东强[1] 吴婷婷 朱冠 尹继刚[1] WANG Zi-qiang;WANG Dong-qiang;WU Ting-ting;ZHU Guan;YIN Ji-gang(State Key Laboratory for Diagnosis and Treatment of Severe Zoontic Infectious Diseases,Institute of Zoonioses/College of Veterinary Medicine,Jilin University,Changchun 130062,Jilin,China)

机构地区：[1]人畜共患传染病重症诊治全国重点实验室,吉林大学人兽共患病研究所/动物医学学院,长春130062

出　　处：《寄生虫与医学昆虫学报》2024年第3期129-136,共8页Acta Parasitologica et Medica Entomologica Sinica

基　　金：“十四五”国家重点研发计划资助(2022YFD1800200)。

摘　　要：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 typeⅢdomain-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2430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1.21μmol/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。Objective This study was performed to investigate the subcellular localization and adhesion properties of the Cryptosporidium parvum fibronectin typeⅢdomain-containing protein(CpFN3).CpFN3 is a 2430-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 K d of 0.23μmol/L and to heparin with a K d of 1.21μmol/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.

关 键 词：微小隐孢子虫 纤维连接蛋白Ⅲ型结构域 肝素 黏附 

分 类 号：R382.3[医药卫生—医学寄生虫学]