弓形虫LAMP检测方法的建立与ROP18的免疫保护作用-预防兽医学专业论文.docxVIP

I II 小鼠存活时间，从而研究 ROP18 对小白鼠的抗弓形虫免疫保护力。结果：构建了原 核表达质粒 pET-32a-ROP18 和真核表达质粒 pcDNA3.1-ROP18-Flag；原核表达质粒 在 E.coil Rosseta 中以包涵体形式表达，经纯化后得到浓度为 2.3mg/mL 重组蛋白，符 合免疫要求；真核表达质粒 pcDNA3.1-ROP18-Flag 在 BHK-21 细胞中成功表达，表 达产物经间接免疫荧光试验显示出特异性的绿色荧光；Western-blot 结果表明两种表 达方式的表达产物均具有良好的反应原性；间接 ELISA 法测得重组质粒和重组蛋白 均可诱导小鼠产生抗弓形虫抗体，其中重组质粒组平均效价为 1:15360，重组蛋白组 平均效价为 1:12800；重组蛋白组小鼠平均存活 9.5 天、重组质粒组小鼠平均存活 9.7 天，均与对照组差异显著，但两组之间以及其他各对照组之间无显著差异。 本课题成功建立了快速、特异、敏感的弓形虫 LAMP 检测方法，这对于人畜共 患弓形虫病的快速、准确诊断具有一定实际意义；本研究发现弓形虫 ROP18 对小白 鼠具有一定的抗弓形虫免疫保护力，为今后研制弓形虫疫苗提供了理论依据。 关键词：弓形虫，LAMP，ROP18，表达，免疫保护力 PAGE PAGE IV Abstract Toxoplasmosis is a zoonotic protozoal disease in humans and other warm-blood animals, caused by Toxoplasma gondii which is an obligate intracellular parasite. It is widely popular all over the world. Not only is it caused a serious threat to human health, but also has a great economic losses of animal husbandry. The diagnostic method of toxoplasmosis is mainly etiology and immunological methods, with the poor sensitivity and specificity. Therefore, for control toxoplasmosis, it is a great significance to establish a simple, rapid, sensitivity and specificity diagnostic method of Toxoplasmosis. Unfortunately, there has not been safe and effective drug developed to prevent or cure toxoplasmosis yet. Researching the protective immunity of parasite protein and developing an effective vaccine are new means against T. gondii infection. ROP18 involved in the invasion of host cell, and mediated immune evasion of T.gondii, has been shown to be an important virulence factor. Some researchers have already cloned and expressed ROP18, and analyzed the reactionogenicity of it. But there are only few reports about its immune protection. Firstly, the conserved sequence was obtained by analysising the clone sequence of 529bp PCR product which is from swine in AnHui province. Secondly, a pair of LAMP amplification primers was designed by Primer Explorer 4.0, a program which can design LAMP sepecific primers onli