日本血吸虫Cystatin的分子特征及对小鼠的免疫调节机制研究
发布时间：2018-10-11 15:53
【摘要】：目前,对日本血吸虫的免疫逃避机制还不十分了解。现有研究表明,寄生虫的半胱氨酸蛋白酶抑制剂(Cystatin)具有抑制宿主免疫应答的作用,可能是参与免疫逃避的重要分子。本研究应用生物信息学与分子生物学方法获得了日本血吸虫一个新的半胱氨酸蛋白酶抑制剂基因SjCystatin,通过研究该基因及其重组蛋白的结构与功能,特别是对宿主的免疫调节功能,旨在初步阐明日本血吸虫Cystatin分子在免疫逃避中的作用及其机制。 首先,在NCBI中搜索到日本血吸虫半胱氨酸蛋白酶抑制剂基因部分序列,再结合日本血吸虫基因组数据库,利用PCR与3'RACE技术,得到了SjCystatin的全长cDNA序列和genomic DNA序列。利用生物信息学方法分析发现,该基因cDNA开放阅读框全长306个碱基,编码101个氨基酸,预测蛋白质理论分子量为11.3 kD,等电点为6.95。其基因组DNA全长376个碱基,由两个内含子和三个外显子组成。通过比较基因组学分析发现,SjCystatin与脊椎动物的stefins家族和cystatins家族的基因组结构类似,推测它们可能是从共同的祖先基因进化而来。系统发育分析显示该预测蛋白与Genebank序列号为XP 002572115的曼氏血吸虫半胱氨酸蛋白酶抑制剂的进化距离最近,相似性为77%。生物信息学分析显示预测蛋白质没有信号肽和跨膜区,因此推测该蛋白应可能定位在细胞内。通过GeneDoc软件分析发现该预测蛋白存在三个保守区,分别是N末端的Gly6,Q49VVAG53序列和C末端的L76P77,与脊椎动物stefins家族和cystatins家族的成员具有相同的保守区,并且无二硫键。通过对以上特征的分析,认为SjCystatin应属于stefins家族的新成员。 本研究成功构建和高效原核表达了SjCystatin基因,并经镍离子鳌合亲和层析柱法被纯化,SDS-PAGE证实其分子量为12.1 kD；其免疫血清能特异性识别日本血吸虫成虫及虫卵可溶性蛋白中11.3 kD分子；经酶活性测定证明其具有抑制蛋白水解酶的生物学活性,在37℃、pH值为7.4的反应条件下,10μg重组SjCystatin每分钟可抑制0.0517木瓜蛋白酶活力单位。半定量RT-PCR的结果表明,日本血吸虫虫卵、童虫和成虫阶段均有该基因的表达。免疫组化和免疫荧光共聚焦实验显示SjCystatin基因编码的蛋白质主要定位在日本血吸虫虫卵的毛蚴中和成虫的肠道表皮和体表。免疫保护性实验结果显示,重组SjCystatin免疫小鼠的减虫率和减卵率与PBS对照组相比,无显著性差异,说明重组SjCystatin对小鼠无明显的保护性效果。免疫荧光共聚焦检测证实,重组SjCystatin可以进入到小鼠DC细胞中,并且只进入到DC细胞的胞浆中。MTS法证明重组SjCystatin具有抑制DC细胞活性和代谢水平的作用。通过流式细胞术检测小鼠DC细胞表面MHC-Ⅱ类分子的平均荧光强度,间接证实了重组SjCystatin的确可以降低DC细胞对抗原的提呈作用。重组SjCystatin体内诱导小鼠CD4+ CD25+ Foxp3+ T细胞增殖的实验表明,重组SjCystatin可显著提高感染小鼠体内CD4+ CD25+ Foxp3+免疫调节性T细胞的比例。以上结果表明SjCystatin基因编码的蛋白分子具有抑制抗原提呈及诱导抑制性免疫反应的作用,提示其可能是一个血吸虫虫源性的免疫抑制因子,在血吸虫免疫逃避中发挥作用。
[Abstract]:At present, the immune evasion mechanism of Schistosoma japonicum is not very well understood. It has been shown that cystatin has an important role in inhibiting the host immune response and may be an important molecule involved in immune evasion. In this study, a new cysteine protease inhibitor gene named Cystatin was obtained by bioinformatics and molecular biology method, and the structure and function of the gene and its recombinant protein, especially the immune regulation function of the host, were studied. The purpose of this study is to clarify the role and mechanism of cystatin in immune evasion of Schistosoma japonicum. Firstly, the gene partial sequence of cysteine protease inhibitor of Schistosoma japonicum was searched in NCBI and then combined with the genome database of Schistosoma japonicum. The full-length cDNA sequence and genomic DNA of Cystatin were obtained by PCR and 3 'RACE technique. Sequence analysis showed that the cDNA open reading frame had 306 bases full length, 101 amino acids were coded, the molecular weight of predicted protein was 11. 3 kD, and the isoelectric point was 6. 95. Its genomic DNA has a total length of 376 bases consisting of two introns and three exons. Composition. By comparative genomics analysis, Cystatin is found to be similar to the genome structure of the stews family and the cysteins family of vertebrates, suggesting that they may be evolved from common ancestor genes. The phylogenetic analysis showed that the predicted protein was closest to Genebank serial number XP 0025721115, and the similarity was 7. 7%. Bioinformatics analysis showed that the predicted protein had no signal peptide and transmembrane region, so it was presumed that the protein should be located in fine It is found that there are three conserved regions in the predicted protein, which are the N-terminal Gly6, Q49VVVF AG53 sequence and the C-terminal L76P77, respectively, and the members of the vertebrate stefins family and the cysteins family have the same conserved region, and no two. According to the analysis of the above features, it is considered that Cystatin should belong to the Stefins family. Its molecular weight was 12. 1kD, and the immune serum was able to specifically identify the soluble protein of Schistosoma japonicum and egg soluble protein. The enzyme activity assay showed that it had the biological activity of inhibiting proteolytic enzyme. Under the reaction conditions of 37 鈩，
本文编号：2264584
[Abstract]:At present, the immune evasion mechanism of Schistosoma japonicum is not very well understood. It has been shown that cystatin has an important role in inhibiting the host immune response and may be an important molecule involved in immune evasion. In this study, a new cysteine protease inhibitor gene named Cystatin was obtained by bioinformatics and molecular biology method, and the structure and function of the gene and its recombinant protein, especially the immune regulation function of the host, were studied. The purpose of this study is to clarify the role and mechanism of cystatin in immune evasion of Schistosoma japonicum. Firstly, the gene partial sequence of cysteine protease inhibitor of Schistosoma japonicum was searched in NCBI and then combined with the genome database of Schistosoma japonicum. The full-length cDNA sequence and genomic DNA of Cystatin were obtained by PCR and 3 'RACE technique. Sequence analysis showed that the cDNA open reading frame had 306 bases full length, 101 amino acids were coded, the molecular weight of predicted protein was 11. 3 kD, and the isoelectric point was 6. 95. Its genomic DNA has a total length of 376 bases consisting of two introns and three exons. Composition. By comparative genomics analysis, Cystatin is found to be similar to the genome structure of the stews family and the cysteins family of vertebrates, suggesting that they may be evolved from common ancestor genes. The phylogenetic analysis showed that the predicted protein was closest to Genebank serial number XP 0025721115, and the similarity was 7. 7%. Bioinformatics analysis showed that the predicted protein had no signal peptide and transmembrane region, so it was presumed that the protein should be located in fine It is found that there are three conserved regions in the predicted protein, which are the N-terminal Gly6, Q49VVVF AG53 sequence and the C-terminal L76P77, respectively, and the members of the vertebrate stefins family and the cysteins family have the same conserved region, and no two. According to the analysis of the above features, it is considered that Cystatin should belong to the Stefins family. Its molecular weight was 12. 1kD, and the immune serum was able to specifically identify the soluble protein of Schistosoma japonicum and egg soluble protein. The enzyme activity assay showed that it had the biological activity of inhibiting proteolytic enzyme. Under the reaction conditions of 37 鈩，
本文编号：2264584
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