沙眼衣原体新的免疫优势抗原CT143生物学特性研究

Chlamydia trachomatis is an obligate intracellular prokaryotic parasite which can cause various diseases in humans. It is considered a major contributor to chlamydial intracellular replication and pathogenesis to synthesize and secret chlamydial proteins, interact with host cells and then interfere with their biological behavior. We previously established the humoral immune response profile to Chlamydia trachomatis infection in humans and identified 15 novel chlamydial immunodominant antigens which were recognized by ≥50% antisera from a whole genome scale by using a proteome array of Chlamydia trachomatis which was set up by us. CT143 is a novel chlamydial specific protein with the highest immunogenic which were strongly recognized by 84% antisera. The observation that CT143 is immunodominantly recognized by human antibodies suggests that this protein can be dominantly presented to the host immune system and induce immune response. This research will further study the biological characteristics of CT143 by detecting the expression characteristics of CT143, clarifiing the mechanisms of CT143 interaction with host cells and its biological functions, and evaluating the characteristics of the immune response induced by CT143 and its protection efficacy. This study will provide new experimental evidence for clarifying the mechanisms of Chlamydia trachomatis interaction with host cells and new research strategies for preventing and controlling Chlamydia infectious disease.

沙眼衣原体是一类专性细胞内寄生原核微生物，具有广泛的致病谱。合成分泌自身蛋白与宿主细胞相互作用，继而干扰宿主细胞的生物学行为，是衣原体在宿主细胞中生长和致病的关键。申请者前期建立了沙眼衣原体蛋白质组数据库及衣原体感染人体后的体液免疫应答谱，从全基因组范围内发现了15个新的衣原体免疫优势抗原（能被50％以上患者的免疫血清识别）。其中CT143是一个新的免疫原性最强的衣原体特异蛋白，84％的患者血清中均检测到高浓度抗CT143抗体，提示CT143蛋白能被有效提呈给宿主细胞识别并诱导产生免疫应答。本课题拟进一步探讨CT143的生物学特性，深入研究CT143在衣原体感染细胞内的表达特征，与宿主细胞的相互作用机制及其生物学功能，并进一步评估CT143 所诱导的免疫应答的特点及其保护效果。本课题将为沙眼衣原体与宿主细胞相互作用机制研究提供新的实验依据，从而为衣原体感染性疾病的防治提供新的研究策略。

沙眼衣原体(Chlamydia trachomatis,简称Ct)是一类专性细胞内寄生原核微生物，具有广泛的致病谱。目前有关衣原体致病因子与保护性因子知识的缺乏已经成为了当前衣原体致病机制研究及疫苗研究的瓶颈。我们前期建立了沙眼衣原体蛋白质组数据库及衣原体感染人体后的体液免疫应答谱，从全基因组范围内发现了15个新的衣原体免疫优势抗原（能被50％以上患者的免疫血清识别）。其中CT143是一个新的免疫原性最强的衣原体特异蛋白，本课题应用杂交瘤技术成功获得了稳定分泌抗CT143蛋白单克隆抗体的杂交瘤细胞株并制备了相应单克隆抗体。间接免疫荧光法及Western Blot法检测了内源性CT143蛋白表达的时空特征，发现衣原体CT143蛋白定位于包涵体内，属于衣原体中晚期表达基因产物，是原体（Elementary body，EB）相关蛋白。构建CT143过表达慢病毒表达载体，通过包装获得过表达CT143基因的慢病毒颗粒，感染Hela229细胞，用嘌呤霉素进行筛选，获得稳定过表达CT143的Hela229细胞株，间接免疫荧光法检测发现CT143稳定过表达组细胞中衣原体包涵体数目显著少于Hela229细胞组和空载体转染阴性对照细胞组，提示CT143蛋白可能对Ct感染和生长有一定的抑制作用。ELISA检测发现CT143蛋白能够刺激THP-1分化的巨噬细胞分泌促炎症细胞因子IL-1β、IL-8和TNF-α。给小鼠接种CT143蛋白＋佐剂以评估外源性CT143蛋白诱导的免疫应答。ELISA法检测小鼠脾细胞培养上清中IFN-γ和IL-4的水平、小鼠血清中抗体的滴度和亚类、以及小鼠阴道冲洗液中sIgA的水平，发现CT143重组蛋白在小鼠体内诱导产生了以Th1应答为主的细胞免疫、体液免疫及黏膜免疫，TC0420（鼠肺炎衣原体MoPn的CT143同源体）重组疫苗具有部分保护作用，其诱导产生的免疫有利于小鼠生殖道内衣原体感染的清除，但不能减轻衣原体感染后小鼠上生殖道组织的病理损伤。这些结果为进一步明确免疫优势抗原CT143蛋白的生物学特性及探讨衣原体的致病机制和疫苗的研制提供了实验依据。