弓形虫特异性抗原的筛选及ELISA检测方法的建立

发布时间：2018-05-04 19:10

本文选题：弓形虫 + 重组蛋白质　；参考：《沈阳农业大学》2017年硕士论文

【摘要】：弓形虫病是由刚地弓形虫(Toxoplasma gondii)引起的一种人畜共患寄生虫病,广泛的分布于世界各地。弓形虫对宿主无严格的选择性,在众多温血动物及人群中广泛存在。猫科动物为弓形虫的终末宿主也是重要的传染源,哺乳类、鸟类都是其中间宿主。弓形虫作为一种细胞内寄生的机会性致病原虫,对免疫功能不全者及妊娠动物的危害严重。猪感染了弓形虫病后造成大量死亡,造成怀孕母猪流产、早产、死胎、畸形胎等,对猪场危害严重。本研究利用生物信息学进行分析,对猪弓形虫诊断抗原进行了筛选,目的是为了寻找和确定弓形虫高免疫原性的蛋白质,建立一种快速灵敏的弓形虫病诊断方法。本实验在确定弓形虫分泌蛋白质的基础上,将筛选出的基因克隆到表达载体,表达出2个重组蛋白质。通过亲和层析法纯化得到His标签重组蛋白质和GST标签重组蛋白质。利用酶联免疫吸附试验(ELISA)和免疫印迹法(Western-blot)筛选出了 TGME49__223140基因重组蛋白质的特异性单克隆抗体,通过间接免疫荧光(IFA)确定抗原在虫体上的表达部位。最后,以该重组蛋白质作为抗原,利用ELISA方法对54份疑似感染弓形虫的猪血清进行了检测,检出率为98.15%,结果与标准检测方法具有非常好的吻合。高抗原的确定对建立弓形虫病临床检测/诊断方法具有重要意义。
[Abstract]:Toxoplasmosis is a zoonotic parasitic disease caused by Toxoplasma gondii. It is widely distributed all over the world. Toxoplasma gondii has no strict selectivity to the host and is widely found in many warm-blooded animals and people. Cat is the terminal host of Toxoplasma gondii, and mammals and birds are the intermediate hosts of Toxoplasma gondii. Toxoplasma gondii is a kind of opportunistic protozoa parasitic in cells, which is harmful to the immune insufficiency and pregnant animals. Pigs infected with Toxoplasma gondii caused a large number of deaths, resulting in pregnant sows miscarriage, premature delivery, stillbirth, abnormal fetus, and so on, which caused serious harm to pig farms. In this study, the diagnostic antigens of Toxoplasma gondii were screened by bioinformatics analysis. The aim of this study was to find and identify the proteins with high immunogenicity of Toxoplasma gondii and to establish a rapid and sensitive diagnostic method for toxoplasmosis. On the basis of determining the secreted protein of Toxoplasma gondii, the selected gene was cloned into the expression vector and two recombinant proteins were expressed. His tag recombinant protein and GST label recombinant protein were purified by affinity chromatography. The specific monoclonal antibody of recombinant protein of TGME49__223140 gene was screened by Elisa and Western-blot, and the expression site of the antigen was determined by indirect immunofluorescence assay (IFA). Finally, the recombinant protein was used as antigen to detect 54 sera of pigs suspected to be infected with Toxoplasma gondii by ELISA method. The detection rate was 98.15. The results were in good agreement with the standard detection method. The determination of high antigen is of great significance for the establishment of clinical detection / diagnosis of toxoplasmosis.
【学位授予单位】：沈阳农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.7

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