旋毛虫保护性抗体靶向抗原ATPA在幼虫侵入肠上皮细胞中的作用及机制

The key steps for pathogensis of Trichinella spiralis infective larvae are that they recognize and invade the host’s intestinal epithelia cells (IECs). However, the mechanism by which larvae recognize and invade the host’s IECs are unknown. In our previous study, a serine protease (ATPA) from the excretory-secretory (ES) proteins of T. spiralis muscle larvae was screened by immunoproteomics, and it is speculated that the ATPA might be associated with invasion of IECs by T. spiralis larvae, which was probably mediated by hydrolyzing cytoskeletal proteins. In this project, we will detect the catalytic and hydrolytic activity of rATPA using substrate, collagen and fibronectin by zymographic analysis. The transcription, expression and location of the ATPA gene in different development stages of T. spiralis will be investigated by Real-time PCR, Western blot and IFT, respectively. Serum anti-rATPA antibodies (total IgG and the subtypes) in immunized mice will be determined by ELISA. Protective immunity against T. spiralis infection induced by rATPA will be evaluated by animal experiments. The binding activity of rATPA with IECs will be observed by IFT, confocal laser scanning microscope and Far-Western. An in vitro invasion assay will be performed to observe the inhibition of anti-ATPA serum on the larval invasion of IECs and their development. The function of ATPA gene was analyzed by RNAi. This study will be very helpful to better understand the molecular mechanism of invasion by T. spiralis infective larvae and search potential high protective vaccines for trichinellosis.

旋毛虫感染性幼虫对宿主小肠上皮细胞（IEC）的侵入是其致病的关键，但侵入机制尚不清楚，可能是由幼虫表面或分泌性蛋白作用于IEC所介导。我们前期应用免疫蛋白组学从旋毛虫幼虫排泄分泌蛋白中筛选出丝氨酸蛋白酶（ATPA），推测其可能通过水解细胞骨架蛋白而介导了幼虫侵入IEC。该项目将通过明胶酶谱分析，应用底物、胶原蛋白及粘连蛋白等检测rATPA的催化和水解活性，应用Real-time PCR、Western blot及IFT分析其在不同期虫体的转录、表达及定位，ELISA检测免疫血清总IgG抗体及亚型，通过动物实验观察其对小鼠的免疫保护作用，应用IFT、共聚焦显微镜及Far-Western观察其与IEC的结合，体外观察免疫血清对幼虫侵入IEC及发育的抑制作用，并应用RNAi研究其功能。该项目对进一步阐明旋毛虫侵入宿主肠上皮细胞的分子机制及寻找高保护性旋毛虫病疫苗具有重要的科学意义。

旋毛虫感染性幼虫对宿主小肠上皮细胞（IECs）的侵入是其致病的关键，但侵入机制尚不清楚，可能是由幼虫表面或分泌性蛋白作用于IEC所介导。该项目的主要研究内容是表达旋毛虫保护性抗体靶向抗原（ATPA；即31kDa旋毛虫丝氨酸蛋白酶，Ts31），分析其在旋毛虫不同发育期的表达与虫体定位，观察rATPA与小鼠IECs的结合、rATPA免疫血清对幼虫侵入IECs及发育的抑制作用；明确rATPA对小鼠的免疫保护作用及抗rATPA抗体通过ADCC对新生幼虫的杀伤作用。该项目应用qPCR分析显示，ATPA在旋毛虫不同发育期均有转录，但在ML期的转录水平明显高于肠道感染性幼虫（IIL）、成虫及新生幼虫期。免疫荧光试验（IFT）结果显示，ATPA在4个发育期均有表达，主要定位于虫体表皮与杆状体部位。Far-Western、ELISA及IFT结果显示，rATPA能够与IECs蛋白特异性结合并进入胞浆内。抗rATPA抗体可显著抑制幼虫对IEC单层的侵入，且这种抑制作用具有抗rATPA抗体剂量依赖性(P<0.001)；ADCC结果显示，抗ATPA抗体可显著促进小鼠巨噬细胞对旋毛虫新生幼虫的杀伤，ADCC对新生幼虫的这种细胞毒性具有抗ATPA抗体剂量依赖性（P<0.001）且与作用时间有关（r=0.978）。将rATPA皮下接种免疫小鼠，诱导产生了高水平的特异性抗rATPA抗体IgG，IgG1水平显著高于IgG2a（P<0.001），表明rATPA免疫接种小鼠激发了Th2型为主的体液免疫应答。rATPA免疫小鼠在幼虫攻击感染后6d和42d，成虫和肌幼虫的减虫率分别为56.93%和53.5%（P<0.001）。结果表明，ATPA在旋毛虫侵入IECs中发挥了重要作用。该项目为进一步阐明旋毛虫侵入宿主IECs的机制及寻找高保护性抗旋毛虫疫苗等奠定了基础。