猪弓形虫体内诱导抗原的鉴定及功能分析

项目名称： 猪弓形虫体内诱导抗原的鉴定及功能分析

项目编号： No.30800818

项目类型： 青年科学基金项目

立项/批准年度： 2009

项目学科： 医药、卫生

项目作者： 周艳琴

作者单位： 华中农业大学

项目金额： 19万元

中文摘要： 弓形虫（Toxoplasma gondii）是一种寄生于所有有核细胞的人兽共患原虫。由于其复杂的生活史和多样的形态结构，弓形虫成为学者研究的焦点。本研究对弓形虫RH 株进行体外培养，收集虫体，提取总RNA，反转录，构建弓形虫cDNA 文库，用体内诱导抗原技术（in vivo induced antigen technology, IVIAT），利用吸附的弓形虫病康复猪血清,对已经建立的弓形虫cDNA文库进行筛选，得到体内诱导抗原基因31个，并对TgCaM, TgPI, TgGRA5, TgMIC11和TgCyp五个基因进行体内外表达差异的验证和体外表达。对TgCaM基因进行了转录水平的RNAi, mRNA转录水平抑制效率可达到88.04%。同时，克隆TgMIC3和TgSAG1基因，分别构建了重组自杀性DNA质粒和重组杆状病毒，免疫活性和保护力实验证实重组弓形虫自杀性DNA质粒和杆状病毒能刺激巴贝西小鼠产生体液免疫和细胞免疫，对致死性弓形虫攻击有一定的免疫力，可作为疫苗候选，预防弓形虫病。

中文关键词： 弓形虫; 体内诱导抗原; 基因功能;弓形虫钙调蛋白

英文摘要： Toxoplasma gondii is a zoonotic protozoan which can infects all the eukaryocytes. Because of the complex life cycle and various structures, T.gondii becames the focus of exploration. In order to provide molecule foundation of pathogenesis, the present study aims to screening and identify some in vivo induced antigens of T.gondii for pig, which related with the pathogenesis directly, using in vivo induced antigen technology(IVIAT). Thirty one in vivo induced antigens were screened. The verification of differential expression between in vivo and in vitro and prokaryotic expression of TgCaM, TgPI, TgGRA5, TgMIC11 and TgCyp were performed. RNAi were carried out in TgCaM, the suppression of mRNA transcription on this gene was 88.04%. Based on TgMIC3 and TgSAG1, two recombinant suicidal DNA plasmid and pseudorabies viruses were constructed. The results of immune and protection assays showed that both two recombinant plasmid and viruses were competent to become the vaccine candidates against toxoplasmosis.

英文关键词： Toxoplasma gondii; in vivo induced antigen; gene fuction;TgCaM