【作者】 李敏；

【导师】 朱冠；

【作者基本信息】 吉林大学 ， 兽医公共卫生， 2024， 硕士

【摘要】 微小隐孢子虫为顶复门肠道寄生原虫,具有复杂的生活史,通常寄生在宿主小肠上皮细胞中,通过粪口途径传播,感染后引起宿主严重腹泻甚至死亡,是引起人兽共患隐孢子虫病的重要病原体之一。与其他顶复门寄生虫不同的是,隐孢子虫属于细胞膜内但细胞质外寄生。目前研究已经确定了微小隐孢子虫极其精简的代谢途径和对宿主的依赖,因其无法从头合成任何营养物质,必须从宿主细胞中摄取所需营养,所以转运蛋白在此过程中发挥极其重要的作用。作为原虫类寄生虫必需的营养物质之一,氨基酸同样也需要寄生虫从摄取。对于任何给定的膜转运蛋白,要充分了解其功能,就需要了解其亚细胞定位和底物特异性。在微小隐孢子虫上,尽管有一些相应的氨基酸转运蛋白已经被预测出来,但领域内针对这些蛋白的定位和相应的功能研究仍旧欠缺。本研究以微小隐孢子虫氨基酸转运蛋白cgd3＿4350作为研究对象,并将其命名为微小隐孢子虫氨基酸转运蛋白1(Cryptosporidium parvum Amino acid transporter 1,CpAAT1)。首先对其进行了生物信息学的分析,预测了该蛋白的理化性质、跨膜区、信号肽、磷酸化位点和蛋白结构等,并对其系统发育进行了分析。其次,针对该蛋白,我们设计了一段免疫原性良好的特异性多肽序列,偶联KLH蛋白后免疫家兔获得了多克隆抗体,利用间接ELISA的方法对免疫后血清的抗体效价进行了检测,同时利用Western blot的方法对该抗体的特异性进行了验证。最后,通过间接免疫荧光实验对CpAAT1蛋白在虫体不同发育时期的定位情况进行了分析。生物信息学分析显示,CpAAT1蛋白大小为60 kDa,由539个氨基酸组成,是一个不含信号肽,拥有11个跨膜区的疏水蛋白;预测蛋白的二级结构包含220个α螺旋、107个β折叠、22个β转角和190个无规则卷曲;含有50个磷酸化位点和13类蛋白激酶结合位点;系统进化分析提示与该蛋白亲缘关系最近的是泰泽隐孢子虫(Cryptosporidium tyzzeri)氨基酸转运蛋白(Gen Bank基因号为TRY52725.1)。间接ELISA检测第四次免疫后的血清抗体效价为1:16,000,可用于后续其他实验。同时,Western blot结果表明抗CpAAT1多克隆抗体能特异性识别虫体天然蛋白。IFA结果显示CpAAT1在微小隐孢子虫生活史的各个时期均有表达,但分布位置不同:子孢子和卵囊时期,CpAAT1定位于子孢子膜;入侵过程中,该蛋白逐渐向子孢子尾端聚集,在尾端形成荧光光斑亮点,并且在完全入侵后仍然存在;无性生殖时期该蛋白定位在成熟的裂殖体膜上;在有性生殖阶段,雌雄配子体均可被该抗体标记。综上所述,本研究确定了微小隐孢子虫氨基酸转运蛋白CpAAT1的亚细胞定位,为进一步探索微小隐孢子虫氨基酸转运蛋白的转运机制奠定了基础。更多还原

【Abstract】 Cryptosporidium parvum,an Apicomplexan parasite transmitted by fecal-oral route,leading to severe diarrhea or even death in immunocompromised hosts.Distinct from other apicomplexan parasites,Cryptosporidium occupies an intracellular but extracytoplasmic niche within the enterocytes of the gastrointestinal tract.The parasite has highly streamlined metabolic pathway,which relies heavily on the host for nutrition..Due to it cannot synthesize any nutrients novo,it must obtain the required nutrients from the host.Transporters play important roles in this process.As one of the essential nutrients for protozoa,amino acids are similarly required for uptake from the host.For any given membrane transport protein,a full understanding of its function requires knowledge of its subcellular localization and substrate specificity.In Cryptosporidium parvum,although a number of corresponding amino acid transport proteins have been predicted,the localization and corresponding functions of these proteins remain poorly investigated in the field.This study focus on the Cryptosporidium parvum amino acid transporter 1(Gene ID: cgd3＿4350,named CpAAT1).First,a comprehensive bioinformatics analysis was conducted to predict various aspects of the protein,including its physical and chemical properties,transmembrane regions,signal peptides,phosphorylation sites,and structural features.Subsequently,a specific polypeptide with good immunogenicity was designed and synthesized.After coupled with the KLH,peptides were immunized rabbits to obtain polyclonal antibodies.Then indirect ELISA was used to evaluate the titer of the antibody after immunization,and the specificity of the antibody was verified by Western blot.Finally,CpAAT1 at at different developmental stages were localized by IFA.Bioinformatics analysis shows that CpAAT1 is 60 kDa protein with 539 amino acids.It is a hydrophobic protein with no signal peptide but has 11 transmembrane domains.The predicted secondary structure of the protein contains 220 α-helices and107 β-sheet,22 β-turns and 190 random coils.In addition,the protein contains 50 phosphorylation sites and 13 protein kinase binding sites.Phylogenetic analysis indicates that the protein exhibits the closest genetic relationship to an amino acid transporter found in C.tyzzeri(Gen Bank ID: TRY52725.1).The indirect ELISA results showed the titer of immune sera reach to 1:16,000 after the fourth immunization.Then it was used for subsequent IFA.At the same time,Western blot results showed that the anti-CpAAT1 polyclonal antibody could specifically recognize the native protein of the parasite.IFA results showed that CpAAT1 was expressed at all stages of the life history of Cryptosporidium parvum,but at different locations: At the sporozoite stage and oocyst stage,CpAAT1 was localized in the sporozoite membrane.During the invasion process,the protein gradually accumulated to the tail of the sporozoite,forming a bright spot in the tail,and it still existed after the complete invasion.During asexual reproduction,the protein was located on the mature merozoite membrane.During sexual reproduction,both male and female gametophytes could be labeled with this antibody.In conclusion,the subcellular localization of CpAAT1 was determined,which lays the foundation for further exploration of the transport mechanism of the amino acid transporter protein in Cryptosporidium parvum.更多还原