日本血吸虫胰岛素样多肽基因的真核表达及生物学功能研究

发布时间：2018-01-26 21:55

本文关键词： 日本血吸虫胰岛素样多肽真核表达多克隆抗体免疫共沉淀　出处：《甘肃农业大学》2017年硕士论文　论文类型：学位论文

【摘要】：日本血吸虫是一种寄生于恒温脊椎动物体内的雌雄异体寄生性蠕虫,由于其生活史的复杂性以及终末宿主的多样性,使其导致的血吸虫病防治困难,严重危害公众的健康。胰岛素样多肽(Insulin-like peptide,ILP)在无脊椎动物中具有基础且重要的功能。早期的研究中未鉴定到日本血吸虫内源性的ILP编码基因,人们认为血吸虫通过利用宿主的胰岛素进行生长代谢的调节。但最近的研究表明,日本血吸虫基因组中存在唯一的ILP编码基因,同时也鉴定到了唯一的胰岛素生长因子结合蛋白(IGFBP)。血吸虫可以利用宿主胰岛素,而自身又存在ILP,那么这一ILP具有什么功能?日本血吸虫胰岛素样生长因子结合蛋白(Sj-IGFBP)与自身ILP和宿主胰岛素的对应结合关系又如何?这些问题的研究对我们理解血吸虫的胰岛素信号利用机制及其与宿主互作机制具有重要意义,同时也为血吸虫病的治疗和预防提供新的思路。本研究对日本血吸虫胰岛素样多肽(Sj-ILP)进行了克隆表达,并对其与Sj-IGFBP的相互作用关系进行了研究。主要取得以下结果:1、日本血吸虫胰岛素样多肽基因的克隆与序列分析。根据日本血吸虫基因组注释信息,设计特异性引物,利用RT-PCR从日本血吸虫成虫mRNA中成功扩增出长度为588 bp的Sj-ILP基因,其CDS序列长度为393 bp,由131个氨基酸残基构成。理论分子质量为15.53 ku,等电点为7.49。三级结构预测显示,Sj-ILP基因与人胰岛素基因的空间结构非常相似。2、Sj-ILP的酵母表达及组织定位。基于毕赤酵母密码子的偏好性,对Sj-ILP编码基因进行密码子优化,构建重组表达质粒pPIC9K-Sj-ILP,并在毕赤酵母GS115中进行诱导表达。SDS-PAGE与Western-blot检测结果显示,重组蛋白Sj-ILP获得高效表达,其大小约为15 ku,与预期结果一致。利用合成肽技术,设计并合成得到两条特异性的肽段,偶联KLH,免疫新西兰大白兔,制备多克隆抗体,间接ELISA检测其抗体效价达到1:51200,Western-blot鉴定特异性多肽具有良好的免疫原性。免疫组化实验结果表明,Sj-ILP蛋白在日本血吸虫成虫的基底膜和卵巢部位表达量相对其他部位较高,预测Sj-ILP可能与营养摄取及生殖发育有关。3、Sj-IGFBP与Sj-ILP、人胰岛素(Human insulin,HI)的相互作用研究。分别构建pCMV-HA-Sj-IGFBP与pCMV-myc-Sj-ILP、pCMV-myc-HI重组质粒,共转染CHO细胞。间接免疫荧光分析,三者均在CHO细胞中成功表达;免疫荧光共聚焦试验表明,发绿色荧光的pCMV-HA-Sj-IGFBP可与发红色荧光的pCMV-myc-Sj-ILP、pCMV-myc-HI共表达呈黄色荧光,可以共定位于细胞的同一层面;免疫共沉淀证实Sj-IGFBP可以沉淀Sj-ILP、HI,反之Sj-ILP、HI也可以沉淀Sj-IGFBP,表明它们之间存在潜在的相互作用。
[Abstract]:Schistosoma japonicum is a parasite on the temperature of vertebrates dioecious parasitic worms, because of the complexity of its life history and end host diversity, which leads to the difficulty of schistosomiasis control, serious harm to public health. Insulin like peptide (Insulin-like peptide ILP) is a fundamental and important function in marine invertebrates. Early studies have not identified the ILP gene encoding Schistosoma japonicum endogenous, people think that regulate the growth and metabolism of the host through the use of insulin. But recent studies show that ILP gene encoding only the genome of Schistosoma japonicum, also identified that the only insulin-like growth factor binding protein (IGFBP) of Schistosoma japonicum can. The use of insulin and its host, and ILP, then this ILP has what function? Japanese blood sucking insect insulin-like growth factor Sub binding protein (Sj-IGFBP) combined with ILP and its relationship with the corresponding host insulin how? The study of these problems in our understanding of the mechanism of insulin signal by schistosome and has important significance and host interaction mechanism, but also provide new ideas for the prevention and treatment of schistosomiasis. The pre study on insulin like peptide of Schistosoma japonicum (Sj-ILP) were cloned, and its interaction with Sj-IGFBP were studied. The main results are as follows: 1. Cloning and sequence analysis of Schistosoma japonicum insulin-like polypeptide gene. According to Schistosoma genome annotation, specific primers were designed by RT-PCR mRNA from Schistosoma japonicum was successfully amplified by Sj-ILP the gene is 588 BP in length, the length of the CDS sequence was 393 BP, consists of 131 amino acids. The theoretical molecular weight of 15.53 Ku and an isoelectric point of 7.4 9. the three stage structure prediction showed that the spatial structure of Sj-ILP gene and human insulin gene is very similar to.2, yeast expression and tissue localization of Sj-ILP. Pichia pastoris codon bias based on codon optimization of Sj-ILP encoding gene, recombinant expression plasmid pPIC9K-Sj-ILP, and induced the expression of.SDS-PAGE and Western-blot results showed that Pichia pastoris GS115, recombinant protein Sj-ILP was expressed, its size is about 15 Ku, consistent with the expected results. By using the synthetic peptide technique, designed and synthesized two peptides, a specific coupling of KLH, New Zealand white rabbits were immunized to prepare polyclonal antibody, indirect ELISA to detect the antibody titer reached 1:51200 Western-blot, identification of specific peptide has good immunogenicity. The immunohistochemical results showed that the expression of Sj-ILP protein in the basement membrane of adult Schistosoma japonicum eggs and nest site The relative amount of other parts is high, may predict Sj-ILP and nutritional intake and reproductive development of.3, Sj-IGFBP and Sj-ILP, human insulin (Human insulin, HI). The interaction of pCMV-HA-Sj-IGFBP and pCMV-myc-Sj-ILP were constructed, pCMV-myc-HI recombinant plasmids were transfected into CHO cells. Indirect immunofluorescence analysis, three were successfully expressed in CHO cells show; confocal immunofluorescence test, pCMV-myc-Sj-ILP green fluorescence pCMV-HA-Sj-IGFBP with red fluorescence, yellow fluorescence co expression of pCMV-myc-HI, can set the same level in the cell; confirmed that Sj-IGFBP can precipitate Sj-ILP, CO immunoprecipitation and HI, Sj-ILP, HI can also precipitate Sj-IGFBP, indicate the existence of potential interactions between them.

【学位授予单位】：甘肃农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.7

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