旋毛虫肠道感染性幼虫表面蛋白与宿主肠上皮细胞相互作用及机制研究

The critical step for establishing Trichinella infection in host is that muscle larvae in intestine are activated to the intestinal infective larvae (IIL) and penetrate intestinal mucosa to develop further. The surface proteins (SP) of IIL were exposed to the host's immune system, directly contacted with intestinal epithelial cells (IEC), and interact at the interface between the parasite and the IEC. Our previous studies indicated that some new proteins were produced by the IIL co-cultured with IEC and entered into the IEC. These new proteins were mainly located on the larval surface. However, until now the mechanisms by which T. spiralis IIL invade the intestinal mucosa have not ever been elucidated. The larval invasion of intestinal mucosa is possibly mediated by the larval surface proteins. The genes of T. spiralis IIL surface protein will be cloned, expressed and purified. The antigenicity and enzymatic activity of recombinant surface proteins (rSP) will be identified by Western blot, ELISA and biochemical methods. The specific binding of the rSP with IEC will be confirmed by immunofluorescence test (IFT),ELISA and Far-Western blotting. The promotion or suppression and blocking effects of rSP and anti-rSP antibodies on the larval invasion of IEC and intestinal mucosa and development are observed by using the in vitro larval invasion model and in animal experiment. The functions of the SP genes will be studied by using RNA interference to silence the expression of the SP mRNA and protein in the parasite.Finally, the regulation role of the SP on the intestinal worm expulsion and immune reaction (serum IgG and intestinal sIgA antibodies, the percentage of Th1/Th2 and Th17/Treg cells, and the transcription as well as expression levels of their cytokines) will be investigated in the mice infected with T. spiralis. This project will elucidate the interaction between T. spiralis intestinal infective larval surface protein and the intestinal epithelial cells and its mechanism, lay the foundation for searching the high protective target antigens for a vaccine against trichinellosis.

旋毛虫肌幼虫在小肠内被激活为肠道感染性幼虫（IIL）并侵入肠粘膜是感染宿主的关键。IIL表面蛋白（SP）暴露于宿主免疫系统、直接与肠上皮细胞（IEC）接触并相互作用。我们的研究发现，与IEC共培养后IIL产生了一些新蛋白，主要定位于幼虫表面并可进入IEC内。IIL侵入肠粘膜的机制尚不清楚，很可能是SP介导的。本项目将克隆表达IIL的SP，鉴定SP的抗原性与酶活性，免疫荧光与Far-Western blotting等鉴定SP 与IEC结合的特异性；体外与动物实验观察SP 及抗SP 抗体对幼虫侵入IEC与肠粘膜的影响；应用RNAi明确SP基因功能；研究SP对旋毛虫感染小鼠肠道排虫与免疫反应（IgG与sIgA，Th1/Th2和Th17/Treg细胞百分比及其细胞因子水平）的调节作用。该项目将阐明旋毛虫肠道感染性幼虫表面蛋白与肠上皮细胞相互作用及机制，为寻找旋毛虫病疫苗的强保护性抗原等奠定基础。

旋毛虫肌幼虫在小肠内被激活为肠道感染性幼虫（IIL）并侵入肠粘膜是感染宿主的关键。IIL表面蛋白暴露于宿主免疫系统、直接与肠上皮细胞（IEC）接触并相互作用。IIL侵入肠粘膜的机制尚不清楚，很可能是其表面蛋白介导的。该项目的主要研究内容是明确IIL表面蛋白的特性、功能及其与IEC的相互作用等。该项目克隆与表达了IIL表面蛋白丝氨酸蛋白酶（TsSP）。应用qPCR、Western blot与IFT发现，TsSP在旋毛虫肌幼虫、IIL、3d和6d成虫及新生幼虫均有表达，主要定位在虫体皮层、杆状体及胚胎等。RNAi抑制了旋毛虫TsSP的表达。Far-Western、ELISA、IFT及共聚焦显微镜检查发现，rTsSP能与IEC特异性结合, 结合部位在IEC膜与胞质。体外侵入实验表明抗rTsSP抗体可明显抑制幼虫侵入IEC，且这种抑制作用具有抗rTsSP抗体剂量依赖性。ADCC对肌幼虫和新生幼虫具有杀伤作用，降低了幼虫的感染性及其在宿主体内的发育。rTsSP 皮下免疫小鼠诱导了Th2型为主的免疫应答，攻击感染后5d与42d成虫与肌幼虫减虫率分别为52.70%和52.1%；rTsSP经鼻免疫小鼠后诱导了肠道特异性sIgA应答及全身混合型Th1/Th2应答，攻击感染后9d与42d成虫与幼虫减虫率分别为71.10%和62.1%。该项目还克隆表达了旋毛虫谷胱甘肽S-转移酶（TspGST），TspGST在旋毛虫各期均有表达，主要定位在虫体皮层及杆状体等；rTspGST具有天然GST的酶活性。抗rTspGSP抗体可抑制幼虫侵入IEC；抗rTspGST抗体依赖的ADCC对新生幼虫具有明显的杀伤作用。rTspGST皮下接种免疫小鼠诱导了明显的抗体应答及免疫保护。结果表明，旋毛虫IIL表面蛋白TsSP与TspGST是旋毛虫侵入与发育过程中的必需蛋白，可作为抗旋毛虫疫苗潜在的候选靶分子。