【作者】 王金苹；

【导师】 赵俊龙； 姚宝安；

【作者基本信息】 华中农业大学 ， 预防兽医学， 2005， 硕士

【摘要】 弓形虫是细胞内寄生虫,能感染大多数哺乳动物。弓形虫感染畜禽,可使妊娠家畜受到严重威胁,尤其给猪、牛、羊饲养业带来严重的经济损失。由于临床上弓形虫病缺乏特异性临床症状和体征,诊断仍是弓形虫病防治中一个突出的问题。目前,临床上常用、也是目前我国卫生部门推荐的,还是血清学检测方法。但其仍存在一些问题,如出现交叉反应,又多受机体免疫状态以及方法本身敏感性和特异性的影响,容易出现误诊和漏诊。同国外的一些试剂盒相比,目前我国还缺少令人满意的试剂盒。诊断试剂盒中存在的一个主要问题是包被抗原的纯度或抗原活性不够,导致检出的准确率不高,为此,我们探讨利用SAG Ⅰ体外表达产物作为抗原建立弓形虫ELISA诊断试剂盒的可行性,并进行了临床上应用,取得了较好的结果。同时本研究还建立了弓形虫的PCR诊断方法,并初步应用于临床检测。 1.弓形虫聚合酶联反应(PCR)方法的建立和初步应用: 通过对弓形虫各种抗原性的比较,选择SAG Ⅰ基因中比较保守的一段序列设计了一对引物,扩增出长度为270bp的片段,建立了检测弓形虫病的PCR诊断方法,并对该方法的灵敏性和特异性进行了研究:将虫体个数计数后提取模板DNA进行PCR,结果表明当稀释到相当于0.85个虫体的DNA量时结果还是阳性。对临床上几种流行猪病和两种寄生虫病的病料进行检测,结果都没有扩增出目标片段,上述结果表明,该方法敏感性高、特异性强。在此基础上对送检的85份抗凝血样进行了检测,其中检出阳性34份,阳性率为40%。实验结果表明,弓形虫PCR诊断方法是临床检测弓形虫病的一种行之有效的方法。 2.弓形虫SAG Ⅰ基因重组质粒的构建与表达产物抗原性的分析 根据Genebank上公布的弓形虫SAG Ⅰ的序列设计合成引物,PCR扩增和克隆了SAG Ⅰ基因主要表面抗原部分编码区段,并测定了核苷酸序列。克隆的SAG Ⅰ基因长度为978bp。进一步将此片段亚克隆到原核表达载体pGEX-KG,转化大肠杆菌BL21菌株,Western—blot印迹检测获得了分子量为56KD的与谷胱苷肽-S转移酶(GST)相融合的表达蛋白,具有抗原性。这为下一步大规模获取SAG Ⅰ进行疫苗研究和诊断试剂的开发提供了条件。 3.弓形虫主要表面抗原性基因SAG Ⅰ基因表达蛋白ELISA方法的建立和初步应用 利用在大肠杆菌中最佳的表达条件下体外表达了弓形虫主要膜表面抗原SAG Ⅰ基因,提取、纯化表达蛋白形成的包涵体,用提纯的包涵体包被ELISA板来检测抗弓形虫抗体,建立了弓形虫SAG Ⅰ蛋白的ELISA诊断方法。用该方法对猪的几种其它更多还原

【Abstract】 Toxoplasma is an obligate intracellular parasite, and it infects majority mammals. It threatens the pregnant animals, and causes grave loss for industry of livestocks. Because the toxoplasmosis lack peculiar clinical symptom, diagnosis is still an serious problem for it. The serum detection method is common used on clinical and recommended by hygiene department of our country recently. However,there are some problem ,such as cross reaction, false positive and negative. Compared with some kits abroad, the kits developed in our country is still unsatisfied. The main problem is low antigen activation and purity. For this reason, we explore the feasibility of developing ELISA kit by using the expression protein of SAG Ⅰ gene in E.coli. At the same time, the PCR method for toxoplasma gondii was also developed and used on clinical.1. Establishment and application of Toxoplasmosis PCR According to the sequence of SAG I published in Genebank, a pair primers wasdesigned for amplifying the conservative sequence of SAG Ⅰ gene, and the length is 270bp.After defining the suitable condition , we establish the PCR method of Toxoplasma gondii. This method can detecte at least the DNA of 0.85 Toxoplasma gondii. Positive results only found in Toxoplasma gondii samples and didn’t found in other pathogen. 40 positive of 85 samples from clinical sick swine were detected by PCR. Theses results showed that the PCR method had the specificity and high sensitivity.2. The construction and expression of recombinant plasmids of SAG Ⅰ gene of Toxoplasma gondii and analysis the antigenic ofexpression productAccording to the sequence of SAG Ⅰ, we design and synthesize a pair primes .The 978bp fragment of SAG Ⅰ gene was obtained by PCR, its nuclectide sequence was determined by the dideoxy-mediated chain termination method. Then, the DNA fragment of SAG Ⅰ gene was subcloned into prokaryotic expression vector pGEX-KG, the specific fusion protein with GST of molecular weight 56KD was expressed in E.coliBL21 (DE3). Western blotting assay indicated that the polyclonal antibody against Toxoplasma gondii could recognize this protein. This study will give some help on studying epidemiology, vaccine, diagnosis and control of Toxoplasmis.3. Establishment and application of ELISASAG I gene of Toxoplasma gondii was expressed by E.coli BL21(DE3). After the conditions of expression were optimized, the inclusion bodies were purified. The suitable concentration of antigen was determined by square matrix titration, and the specificity, sensitiveness, repetitiveness of this method were determined. 300 serum samples from pig farms were tested by IHA and ELISA at the same time, the coincidence rate is up to 98%,the differencre between the two methods is not remarkable. These Tesults indicate that the ELISAhas high specificity, sensitiveness and good repetitiveness.更多还原