伪旋毛虫丝氨酸蛋白酶抑制剂在寄生虫免疫逃逸中的功能分析

发布时间：2018-03-06 22:08

本文选题：伪旋毛虫　切入点：丝氨酸蛋白酶抑制剂　出处：《吉林大学》2015年硕士论文　论文类型：学位论文

【摘要】：旋毛虫病是由旋毛虫属（Trichinella spp.）线虫引起对人类健康构成巨大威胁的食源性人兽共患寄生虫病。据目前统计，在世界范围内约有1000多万人被感染，在我国该病仍有发生。在旋毛虫中已报道十二个种中，伪旋毛虫是唯一一种既可以感染哺乳动物也可以感染鸟类的虫种，感染宿主范围广，具有异于其它种的特殊感染特点。丝氨酸蛋白酶抑制剂是能够抑制丝氨酸蛋白酶及半胱氨酸蛋白酶的蛋白酶抑制剂超家族，参与调控凝集、炎症和凋亡等生理过程，广泛存在于哺乳动物、植物、蠕虫、昆虫、微生物乃至某些病毒之中。伪旋毛虫的肌幼虫由于无包囊保护，更容易受到宿主免疫攻击，但是伪旋毛虫却能成功寄生于宿主肌细胞内，造成整个肌细胞感染引发强烈炎症。因此，伪旋毛虫必然产生更强的免疫抑制来逃避宿主的攻击。迄今为止伪旋毛虫免疫逃逸相关分子的调控机制仍然未知，研究伪旋毛虫丝氨酸蛋白酶抑制剂在免疫调节过程中的作用具有极其重要的意义。首先，本研究根据GenBank中伪旋毛虫丝氨酸蛋白酶抑制剂（Trichinellapseadospiralis serine proteinase inhibitor, Tp-Serpin）基因序列设计特异性引物，RT-PCR克隆得到Tp-Serpin基因，构建重组表达质粒pET-28a-Tp-Serpin。转化大肠埃希菌中，通过优化表达纯化条件获得高纯度的可溶性重组蛋白，利用生物化学方法即底物特异性反应对其抑制宿主的丝氨酸蛋白酶活性进行分析。研究发现rTp-Serpin对小鼠肥大细胞糜蛋白酶，人中性粒细胞弹性蛋白酶以及猪胰脏弹性蛋白酶均有明显的抑制作用。我们推测伪旋毛虫通过分泌Serpin来抵抗源自宿主肠道消化酶对其损伤，发挥抗炎作用，，逃避宿主免疫攻击而达到长期寄生。其次，通过rTp-Serpin重组蛋白与J774A.1小鼠巨噬细胞的共培养并经脂多糖（LPS）刺激，通过采用CCK-8、SYBR Green I实时荧光定量PCR、ELISA分析rTp-Serpin对Toll样受体介导的对巨噬细胞活性的影响。结果表明，rTp-Serpin降低了LPS刺激的巨噬细胞分泌促炎性细胞因子及效应分子iNOS的表达，促进抗炎性细胞因子及替代性活化的巨噬细胞（AAM）标记物Arg1分泌表达。另外我们通过Western blot技术检测rTp-Serpin对JAK2/STAT3信号通路的磷酸化情况，结果表明，JAK2/STAT3信号通路可能介导rTp-Serpin诱导巨噬细胞向AAM型转化，发挥抗炎功效。我们推测伪旋毛虫可以通过丝氨酸蛋白酶抑制剂抑制炎症反应，诱导巨噬细胞向可替代激活表型转换。最后，利用半定量RT-PCR技术对Tp-Serpin抗原基因在伪旋毛虫不同发育时期中的转录水平进行鉴定。制备不同感染时期的裸虫切片，利用特异性多克隆抗体，采用Western blot、免疫组织化学技术对Tp-Serpin抗原基因在伪旋毛虫生活史中的表达特性进行鉴定，探讨此抗原基因作为伪旋毛虫诊断抗原的可能性，为深入研究伪旋毛虫宿主入侵、免疫逃避及发育机制奠定了基础。半定量RT-PCR结果显示，Tp-Serpin基因在伪旋毛虫各个时期均有表达。Western blot分析显示抗rTp-Serpin血清能够识别伪旋毛虫虫体粗抗原而与ES产物反应较弱，此外免疫组化结果表明，Tp-Serpin蛋白主要分布于虫体表皮，由此推测Tp-Serpin蛋白很可能是一种由虫体表皮细胞合成分泌的功能蛋白，发挥细胞外作用，从而能够抵抗宿主肠道环境中消化酶对虫体的损伤，参与抗炎及逃避宿主对虫体的免疫效应，达到在宿主体内的长期寄生。
[Abstract]:Trichinosis is by Trichinella (Trichinella spp.) caused by nematodes pose a huge threat to human health food borne parasitic disease. According to the statistics, in the world there are about about 10000000 people infected, the disease in China still occur. In Trichinella has been reported in the twelve species, t. pseudospiralis is the only one both species infect mammals can also infect birds, with a wide range of hosts, are different from other kinds of special infection characteristics.
Serine protease inhibitor is a serine protease and protease inhibitors can inhibit the cysteine protease superfamily, involved in the regulation of coagulation, inflammation and apoptosis and other physiological processes, widely exist in mammals, plants, worms, insects, microorganisms and some viruses. The muscle larvae of Trichinella pseudospiralis without cyst protection, more susceptible to immune attack, but false it can successfully parasitize host of Trichinella spiralis muscle cells, cause the strong inflammation whole muscle cells. Therefore, immune t.pseudospiralis must have stronger inhibition to evade the host attack. So far the regulation mechanism of Trichinella pseudospiralis immune escape related molecules remains unknown, has very important significance to study the effect of Trichinella pseudospiralis serine protease inhibitor in immune in the regulation process.
First of all, according to the GenBank of Trichinella pseudospiralis serine protease inhibitor (Trichinellapseadospiralis serine proteinase inhibitor, Tp-Serpin) gene sequence specific primers, cloned RT-PCR Tp-Serpin gene, the recombinant plasmid pET-28a-Tp-Serpin. was transformed into E. coli and purified to obtain high purity of soluble recombinant protein by optimizing the expression, by using biochemical method special substrate the specific reaction to analyze the inhibition of host serine protease activity. The study found that rTp-Serpin protease on mouse mast cell chymase, inhibited human neutrophil elastase and porcine pancreatic elastase were. We speculate that Serpin secreted by Trichinella pseudospiralis to resistance from the host digestive enzyme of injury, exerts anti-inflammatory effects, to evade the host immune attack Click to send long Students.
Secondly, through the co culture of recombinant rTp-Serpin protein and J774A.1 in murine macrophages by lipopolysaccharide (LPS) stimulation by CCK-8, SYBR Green I real-time fluorescence quantitative PCR ELISA analysis of rTp-Serpin mediated by Toll like receptors on macrophage activity effect. The results showed that rTp-Serpin reduced LPS stimulated macrophages secrete inflammation promoting expression of iNOS cytokines and effector molecules that promote anti-inflammatory cytokines and alternatively activated macrophages (AAM) marker Arg1 expression. We also detected by rTp-Serpin Western blot on the phosphorylation of JAK2/STAT3 signaling pathway, the results show that the JAK2/STAT3 signaling pathway may mediate the transformation of rTp-Serpin to AAM induced macrophage, anti-inflammatory effect. We speculate that Trichinella pseudospiralis can inhibit inflammatory reaction by serine protease inhibitor, induced macrophage cells to be replaced It activates phenotypic conversion.
Finally, to identify the transcription level of Tp-Serpin gene in Trichinella pseudospiralis in different developmental stages by semi quantitative RT-PCR method. The preparation of different infection period naked insect slices, using specific polyclonal antibody by Western blot immunohistochemical technique was used to identify the expression characteristics of Tp-Serpin gene in Trichinella pseudospiralis in life history to investigate this possibility, as the antigen gene of Trichinella pseudospiralis antigens for diagnosis, for the further study of t.pseudospiralis host intrusion, immune evasion and developmental mechanism of the foundation. The semi quantitative RT-PCR results showed that Tp-Serpin gene in.Western blot analysis showed that anti rTp-Serpin serum can identify pseudo Trichinella spiralis crude antigen and reacted with ES expressed in each product is weak during the period of t.pseudospiralis in addition, immunohistochemistry results showed that Tp-Serpin protein mainly distributed in the body surface, by hypothesizing Tp-Serpin Protein is likely to be a parasite by epidermal cells synthesis and secretion of extracellular protein, which can play a role on the worm damage the digestive enzyme resistance of intestinal environment, participate in anti inflammation and evade the host immune effect on the parasite in the host, to send the long term.

【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.7

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