抗微小隐孢子虫TSP3蛋白质单克隆抗体的制备及鉴定

发布时间：2018-08-06 16:44

【摘要】：顶器门原虫可引起一些在医学和兽医学方面危害非常严重的疾病。这些寄生虫利用一些相似的亚细胞结构、细胞器和相关分子侵入宿主细胞。含有1个或多个重复的TSP1结构域的TRAP家族蛋白在虫体的侵入和运动过程中发挥重要作用。目前已在各种疟原虫、柔嫩艾美尔球虫、巨型艾美尔球虫、弓形虫、隐孢子虫中发现了该蛋白家族的成员。这些蛋白质大都定位于虫体头部的微线体上。目前已发现恶性疟原虫的TRAP蛋白和弓形虫的MIC2蛋白是虫体侵入过程中的重要分子。通过对微小隐孢子虫的全基因组比对分析，发现有12个含有TSP结构域的蛋白质编码基因，但目前仅发现其中2个（TRAP-C1和TSP8）定位在虫体的顶端微线体，其它的蛋白质的定位及功能尚不清楚。本研究通过制备针对TSP3的单克隆抗体来定位该蛋白质，为深入研究该蛋白质的功能奠定基础。本研究首先对隐孢子虫TSP家族蛋白TSP3蛋白质的编码基因的结构进行分析，，发现其含有一个信号肽和跨膜区，其胞外区含有4个TSP1结构域和2个apple结构域，发现其与已确定的隐孢子虫微线体蛋白TRAP-C1在结构上相似。分别设计2对PCR引物将其胞外区编码基因分为两段，其中第1段含有2个apple结构域和1个TSP1结构域，第2段含有3个串联重复的TSP1结构域。将扩增产物克隆到pMD-18T载体，测序正确后克隆到pGEX-4T-1表达载体，在BL21中诱导表达，然后用谷胱甘肽磁珠纯化。获得了分段表达的TSP3重组蛋白TSP3-1和TSP3-2。将纯化后的重组蛋白质腹腔免疫BALB/C小鼠获得脾细胞，与Sp2/0细胞融合，成功获得了杂交瘤细胞，经过筛选、克隆后成功获得了2株强阳性的杂交瘤细胞株，进一步成功获得了抗TSP3的单克隆抗体，亚类鉴定结果表明所获得的单克隆抗体均为IgG1亚型，通过间接免疫荧光法对该TSP3蛋白质进行子孢子定位，发现该蛋白质位于子孢子的头部，由于与侵入相关的蛋白质均存在位于虫体的头部顶器部位，因此推测TSP3可能与虫体的侵入功能相关，很可能是隐孢子虫的另一个微线体蛋白。
[Abstract]:Protozoa can cause some very serious diseases in medicine and veterinary medicine. These parasites use similar subcellular structures, organelles and related molecules to invade host cells. TRAP family proteins containing one or more repeated TSP1 domains play an important role in the invasion and movement of the insect. At present, members of the protein family have been found in various Plasmodium, Eimeria tenella, giant Eimeria, Toxoplasma gondii and Cryptosporidium. Most of these proteins are located in the microsomes of the head of the worm. The TRAP protein of Plasmodium falciparum and the MIC2 protein of Toxoplasma gondii have been found to be important molecules in the process of body invasion. By analyzing the whole genome of Cryptosporidium minimus, we found that there are 12 protein coding genes containing TSP domain, but only 2 (TRAP-C1 and TSP8) are located at the top of the microline of Cryptosporidium microphylla, but only two of them (TRAP-C1 and TSP8) are located at the top of the parasite. The location and function of other proteins are unclear. In this study, monoclonal antibodies against TSP3 were prepared to locate the protein, which laid a foundation for further study of the function of the protein. In this study, the structure of the coding gene of TSP3 protein of Cryptosporidium TSP family was analyzed. It was found that it contained a signal peptide and a transmembrane region, and its extracellular domain contained four TSP1 domains and two apple domains. It was found that its structure was similar to that of the identified Cryptosporidium microline protein TRAP-C1. Two pairs of PCR primers were designed to divide the gene encoding extracellular domain into two segments, the first one containing two apple domains and one TSP1 domain, and the second one containing three tandem repeat TSP1 domains. The amplified product was cloned into pMD-18T vector, sequenced correctly, cloned into pGEX-4T-1 expression vector, induced in BL21, then purified by glutathione magnetic beads. TSP3 recombinant proteins TSP3-1 and TSP3-2 were obtained. After the purified recombinant protein was immunized intraperitoneally with BALB/C mice to obtain spleen cells and fused with Sp2/0 cells, hybridoma cells were successfully obtained. After screening, two strongly positive hybridoma cell lines were obtained. Furthermore, monoclonal antibodies against TSP3 were successfully obtained. The results of subclass identification showed that the obtained monoclonal antibodies were all IgG1 subtypes. The TSP3 protein was located by indirect immunofluorescence assay. It was found that the protein was located at the head of the sporozoite, and that the invasion-related proteins were located in the acrosome of the head of the parasite. Therefore, it was speculated that TSP3 might be related to the invading function of the parasite and might be another microline protein of Cryptosporidium.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392

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