本文選題：偽旋毛蟲　切入點：絲氨酸蛋白酶抑制劑　出處：《吉林大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

【摘要】：旋毛蟲病是由旋毛蟲屬（Trichinella spp.）線蟲引起對人類健康構(gòu)成巨大威脅的食源性人獸共患寄生蟲病。據(jù)目前統(tǒng)計，在世界范圍內(nèi)約有1000多萬人被感染，在我國該病仍有發(fā)生。在旋毛蟲中已報道十二個種中，偽旋毛蟲是唯一一種既可以感染哺乳動物也可以感染鳥類的蟲種，感染宿主范圍廣，具有異于其它種的特殊感染特點。 絲氨酸蛋白酶抑制劑是能夠抑制絲氨酸蛋白酶及半胱氨酸蛋白酶的蛋白酶抑制劑超家族，參與調(diào)控凝集、炎癥和凋亡等生理過程，廣泛存在于哺乳動物、植物、蠕蟲、昆蟲、微生物乃至某些病毒之中。偽旋毛蟲的肌幼蟲由于無包囊保護(hù)，更容易受到宿主免疫攻擊，但是偽旋毛蟲卻能成功寄生于宿主肌細(xì)胞內(nèi)，造成整個肌細(xì)胞感染引發(fā)強(qiáng)烈炎癥。因此，偽旋毛蟲必然產(chǎn)生更強(qiáng)的免疫抑制來逃避宿主的攻擊。迄今為止偽旋毛蟲免疫逃逸相關(guān)分子的調(diào)控機(jī)制仍然未知，研究偽旋毛蟲絲氨酸蛋白酶抑制劑在免疫調(diào)節(jié)過程中的作用具有極其重要的意義。 首先，本研究根據(jù)GenBank中偽旋毛蟲絲氨酸蛋白酶抑制劑（Trichinellapseadospiralis serine proteinase inhibitor, Tp-Serpin）基因序列設(shè)計特異性引物，RT-PCR克隆得到Tp-Serpin基因，構(gòu)建重組表達(dá)質(zhì)粒pET-28a-Tp-Serpin。轉(zhuǎn)化大腸埃希菌中，通過優(yōu)化表達(dá)純化條件獲得高純度的可溶性重組蛋白，利用生物化學(xué)方法即底物特異性反應(yīng)對其抑制宿主的絲氨酸蛋白酶活性進(jìn)行分析。研究發(fā)現(xiàn)rTp-Serpin對小鼠肥大細(xì)胞糜蛋白酶，人中性粒細(xì)胞彈性蛋白酶以及豬胰臟彈性蛋白酶均有明顯的抑制作用。我們推測偽旋毛蟲通過分泌Serpin來抵抗源自宿主腸道消化酶對其損傷，發(fā)揮抗炎作用，，逃避宿主免疫攻擊而達(dá)到長期寄生。 其次，通過rTp-Serpin重組蛋白與J774A.1小鼠巨噬細(xì)胞的共培養(yǎng)并經(jīng)脂多糖（LPS）刺激，通過采用CCK-8、SYBR Green I實時熒光定量PCR、ELISA分析rTp-Serpin對Toll樣受體介導(dǎo)的對巨噬細(xì)胞活性的影響。結(jié)果表明，rTp-Serpin降低了LPS刺激的巨噬細(xì)胞分泌促炎性細(xì)胞因子及效應(yīng)分子iNOS的表達(dá)，促進(jìn)抗炎性細(xì)胞因子及替代性活化的巨噬細(xì)胞（AAM）標(biāo)記物Arg1分泌表達(dá)。另外我們通過Western blot技術(shù)檢測rTp-Serpin對JAK2/STAT3信號通路的磷酸化情況，結(jié)果表明，JAK2/STAT3信號通路可能介導(dǎo)rTp-Serpin誘導(dǎo)巨噬細(xì)胞向AAM型轉(zhuǎn)化，發(fā)揮抗炎功效。我們推測偽旋毛蟲可以通過絲氨酸蛋白酶抑制劑抑制炎癥反應(yīng)，誘導(dǎo)巨噬細(xì)胞向可替代激活表型轉(zhuǎn)換。 最后，利用半定量RT-PCR技術(shù)對Tp-Serpin抗原基因在偽旋毛蟲不同發(fā)育時期中的轉(zhuǎn)錄水平進(jìn)行鑒定。制備不同感染時期的裸蟲切片，利用特異性多克隆抗體，采用Western blot、免疫組織化學(xué)技術(shù)對Tp-Serpin抗原基因在偽旋毛蟲生活史中的表達(dá)特性進(jìn)行鑒定，探討此抗原基因作為偽旋毛蟲診斷抗原的可能性，為深入研究偽旋毛蟲宿主入侵、免疫逃避及發(fā)育機(jī)制奠定了基礎(chǔ)。半定量RT-PCR結(jié)果顯示，Tp-Serpin基因在偽旋毛蟲各個時期均有表達(dá)。Western blot分析顯示抗rTp-Serpin血清能夠識別偽旋毛蟲蟲體粗抗原而與ES產(chǎn)物反應(yīng)較弱，此外免疫組化結(jié)果表明，Tp-Serpin蛋白主要分布于蟲體表皮，由此推測Tp-Serpin蛋白很可能是一種由蟲體表皮細(xì)胞合成分泌的功能蛋白，發(fā)揮細(xì)胞外作用，從而能夠抵抗宿主腸道環(huán)境中消化酶對蟲體的損傷，參與抗炎及逃避宿主對蟲體的免疫效應(yīng)，達(dá)到在宿主體內(nèi)的長期寄生。
[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.

【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S852.7

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