【作者】 孙婷；

【导师】 尹继刚；

【作者基本信息】 吉林大学 ， 预防兽医学， 2015， 硕士

【摘要】 微小隐孢子虫（Cryptosporidium parvum）是一种经水传播的顶复门原虫,无论在医学还是兽医学都具有重要的意义。在免疫功能正常的人群表现为自限性感染,然而对于艾滋病患者和免疫缺陷患者,表现为慢性感染甚至威胁生命,目前尚无有效治疗方案。虽然微小隐孢子虫侵入过程的分子机制尚不完全清楚,但已有证据表明其入侵同样依赖顶端细胞器如微线体和棒状体的分泌调节,且超微结构观察已证实棒状体参与侵入过程。目前报道的隐孢子虫分泌蛋白主要是微线体蛋白,还没有棒状体蛋白功能的研究报道。通过生物信息学发现微小隐孢子虫基因组含有两段序列cgd4₂420和cgd8₂530与编码弓形虫棒状体蛋白RON9和RON10的基因存在较高的同源性,且均含有N-末端信号肽、C-末端跨膜区、sushi结构域及重复的ankyrin结构域,因此推测其可能为微小隐孢子虫棒状体蛋白,并可能参与虫体的侵入过程。本研究首先通过荧光定量PCR检测微小隐孢子虫感染HCT-8细胞后不同阶段,棒状体蛋白候选基因Cp cgd4₂420与Cp cgd8₂530的转录规律。结果显示Cp cgd4₂420与Cp cgd8₂530在入侵细胞后转录水平逐渐增高,在24 h达到峰值,随后降低。然后,对Cp cgd4₂420与Cp cgd8₂530含有保守结构域的片段进行克隆,构建原核表达载体,亲和层析纯化重组蛋白质,并免疫家兔制备多克隆抗体。然后通过Western blot与间接免疫荧光对虫体蛋白质cgd4₂420与cgd8₂530表达情况进行鉴定,结果显示二者在微小隐孢子虫的卵囊裂解产物中均有表达且均定位于子孢子上。最后,通过Western blot与间接免疫荧光检测GST标签重组蛋白质与HCT-8细胞结合特性实验,结果显示重组蛋白质Cp cgd4₂420能结合HCT-8细胞,而重组蛋白质Cp cgd8₂530不能与HCT-8细胞结合,说明Cp cgd4₂420可能参与微小隐孢子虫入侵宿主细胞的过程。基于棒状体蛋白质在其它顶复门寄生虫的入侵、复制、毒力相关过程起重要作用,因此了解微小隐孢子虫疑似棒状体蛋白质的生物学功能是非常重要的。本实验为深入研究cgd4₂420与cgd8₂530的特性功能和揭示微小隐孢子虫的棒状体蛋白质成员奠定基础。更多还原

【Abstract】 The apicomplexan parasite Cryptosporidium parvum is a waterborne enteric protozoan of significant medical and veterinary importance. In immunocompetent individuals C.parvum determines a self-limiting watery diarrhea, whereas in AIDS patients and other immunocompromised subjects cryptosporidiosis is a chronic and potentially life-threatening disease still lacking effective therapies. Although the molecular mechanisms underlying this peculiar invasion process are poorly understood, there is evidence that C.parvum also relies for invasion on the regulated secretion of apical organelles, i.e., micronemes and rhoptries.The implication of this organelle in C.parvum invasion is supported by ultrastructural observations While a few microneme proteins were described in C.parvum, the repertoire of molecules contained in the single parasite rhoptry is largely unknown. Through bioinformatic analysis, which rhoptry protein candidates cgd4₂420 and cgd8₂530 are found to have conserved domain with T.gondii RON9 and RON10, including N-terminal signal peptide, C-terminus transmembrane region, ankyrin repeat domains and a sushi domain. They are supposed to be rhoptry proteins and may be involved in the invasion process.Firstly, expression of cgd4₂420 and cgd8₂530 were evaluated by realtime PCR in infected HCT-8 cell monolayers following infection by Cryptosporidium parvum.The results showed that both of them have a upward trend of transcription gradually post-infection. It peaked at 24 h and then decreased. Seconly, fragments of cgd4₂420 and cgd8₂530 were amplified by PCR, then cloned into p ET-28 a and p GEX-4T-1 plasmid for expression. The recombinant proteins were purified through affinity chromatography. The polyclonal antibody against these recombinant proteins were prepared in rabbits. Thirdly, the expression of cgd4₂420 and cgd8₂530 in Cryptosporidium parvum were verified by Western blot and IFA. The results showed that both on them were expreesed in the lysate of the occysts and located on the sporozoites. Finally, the binding of GST-tag recombinant proteins to HCT-8 cells was assessed by Western blot and IFA. The results showed that Cp cgd4₂420 could bind to HCT-8 cells while cgd8₂530 could not. It indicated that Cp cgd4₂420 was associated with the invasion of host cells in C.parvum.As the importance of rhoptries in other Apicomplexan invasion, reproduction and virulence-related processes, the study on the biological function of Cryptosporidium parvum putative rhoptry protein is very essential. This study was the foundation for studying on the feature and function of cgd4₂420 and cgd8₂530, revealling the rhoptry proteins family of Cryptosporidium parvum.更多还原