旋毛虫侵入宿主肠上皮细胞关键蛋白分子的进一步筛选、鉴定及功能研究

Trichinellosis is a serious food-borne parasitic zoonosis. Following ingestion, Trichinella muscle larvae in intestine are activated to the intestinal infective larvae by bile. The invasion of intestinal mucosa by the infective larvae is the key steps for establishing Trichinella infection. Because the larvae do not possess oral appendices or a spike, the invasion of intestinal epithelial cells by larvae can't be a result of simple mechanical penetration, and possibly is mediated by some of the larval secretory proteins. The intestinal epithelial cells as a model for the in vitro invasion of Trichinella spiralis infective larvae was established and more than 200 kinds of the invasion-related proteins were screened by using this model during our previous studies. But, which proteins are the crucial invasion proteins of Trichinella has not been clarified yet. Based on the previous studies, the interacting protein genes of T. spiralis with enterocytes will be screened from the T7 phage display library and subtractive cDNA library of the intestinal infective larvae. Then, these genes will be cloned, expressed and purified. The recombinant proteins and antibodies to recombinant proteins are prepared. The proteins that can obviously promote larvae to invade into epithelial cells are screened by using intestinal epithelial cells in vitro and animal experiments, these proteins are the crucial Trichinella invasion proteins. The expression, location and function of the crucial invasion proteins are identified by the immunofluorescence and RNA interference. This project will lay the foundation for illustrating the molecular mechanism of invasion of intestinal mucosa by Trichinella larvae, searching the high protective antigens for trichinellosis vaccine, and screening the new drugs against Trichinella.

旋毛虫病是一种严重的食源性人兽共患寄生虫病，肌幼虫经口感染宿主后，在小肠内被胆汁激活为肠道感染性幼虫并侵入肠黏膜是建立感染的关键。由于幼虫口孔无齿状和矛状结构，幼虫侵入肠黏膜不是简单的机械性损伤，很可能是幼虫分泌的某些蛋白介导的。我们的前期研究建立了旋毛虫-肠上皮细胞体外侵袭模型并筛选出了200多种侵入相关蛋白，但目前不清楚哪些蛋白是旋毛虫侵入的关键蛋白。该项目将在前期研究的基础上，进一步应用旋毛虫肠道感染性幼虫T7噬菌体展示文库及消减cDNA文库，筛选出旋毛虫与肠上皮细胞互作蛋白基因进行克隆、表达及纯化，制备重组蛋白与抗重组蛋白抗体，通过体外与动物实验鉴定出对幼虫侵入肠上皮细胞有明显促进作用的蛋白，即为旋毛虫侵入关键蛋白；应用免疫荧光及RNA干扰等技术研究侵入关键蛋白的表达、定位及功能。该项目为阐明旋毛虫侵入肠黏膜的分子机制、寻找旋毛虫病疫苗的强保护性抗原及筛选抗旋毛虫新药等奠定基础。

旋毛虫病是一种危害严重的食源性寄生虫病，但旋毛虫对宿主肠黏膜的侵入机制尚不清楚。该项目建立了旋毛虫肠道感染性幼虫（IIL）T7噬菌体展示文库及消减cDNA文库（SSH），从T7噬菌体展示文库中筛选到10个能与肠上皮细胞（IEC）特异性结合的IIL基因；从SSH-1文库中选出IIL特异性表达或表达明显上调的33个基因，从中选取10个基因应用real time-PCR分析，发现这10个基因在IIL的转录水平比肌幼虫分别上调了1.11~2.32倍。在2个文库中均存在的4个基因（Tsp3、Tsp4、Tsp5及Tsp10）是在IIL表达明显上调且与IEC特异性结合的基因, 为旋毛虫的关键侵入蛋白基因。将具有nudix水解酶活性的Tsp10免疫小鼠后产生了Th2型为主的免疫应答，Western blot分析表明Tsp10可被旋毛虫感染小鼠及ES免疫小鼠血清识别，抗Tsp10抗体可特异性识别旋毛虫排泄分泌（ES）抗原与可溶性抗原中天然的Tsp10。IFT显示Tsp10基因在旋毛虫IIL与成虫期高表达，主要定位在虫体表皮及杆状体。Tsp10免疫小鼠用旋毛虫攻击感染后3d与42d成虫和肌幼虫的减虫率分别为62.8%与78.7%，表明Tsp10免疫可诱导小鼠产生明显的抗旋毛虫感染的保护性免疫。体外侵入试验结果表明，抗Tsp10抗体可阻止部分幼虫对IEC的侵入与发育，证明Tsp10是旋毛虫的关键侵入蛋白。该项目还进行了旋毛虫氨基肽酶（TsAP）、谷胱甘肽转移酶（TsGST）及蛋白酶体β7亚基（Tspst）的克隆表达、特性与功能及其免疫保护作用的研究，结果表明，TsAP、TsGST及Tspst在旋毛虫的不同发育期均有转录与表达，将TsAP、TsGST及Tspst分别免疫小鼠后均产生了部分免疫保护作用；应用抗重组蛋白抗体或RNA干扰可部分阻断旋毛虫幼虫对IEC的侵入及幼虫在小鼠体内的发育与存活，表明这3种旋毛虫蛋白亦参与了旋毛虫对宿主IEC的侵入。该项目为进一步阐明旋毛虫侵入宿主肠黏膜的分子机制、研制抗肠道期旋毛虫病疫苗等奠定了基础。