环形泰勒虫TaSP重组蛋白间接ELISA诊断方法的建立

发布时间：2018-03-15 23:06

本文选题：环形泰勒虫　切入点：TaSP　出处：《河南农业大学》2015年硕士论文　论文类型：学位论文

【摘要】：环形泰勒虫(Theileria annulata)是一种可感染牛且危害严重的血液性原虫,寄生在宿主的巨噬细胞、淋巴细胞和红细胞,易引起被感染者的急性死亡。环形泰勒虫病是一种世界范围内流行的疾病,严重危害各国的养牛业。在中国,感染牛的泰勒虫主要是环形泰勒虫,中华泰勒虫和瑟氏泰勒虫,其中又以环形泰勒虫的危害最大,发病范围最广,在包括新疆、内蒙古等13个省区均有感染环形泰勒虫的病例的报道,给当地的养牛业带来严重影响,并且造成极大的经济损失。在我国,传播环形泰勒虫的媒介主要为璃眼蜱属,其中大部分地区主要是残缘璃眼蜱,新疆南部地区主要是小亚璃眼蜱。本研究以环形泰勒虫的裂殖体膜表面蛋白(Theileria annulata sporozoites surface protein,TaSP)为研究对象,在前期研究的基础上,通过优化重组菌的表达条件,纯化表达的重组TaSP蛋白,并以纯化后的重组蛋白为抗原,建立牛环形泰勒虫TaSP重组蛋白间接ELISA检测方法。研究内容如下:1.对已表达的TaSP(甘肃株)重组蛋白的菌液进行PCR检测,选取琼脂糖凝胶电泳结果条带大小约为400bp的PCR扩增阳性结果的菌液送去测序。分析测序结果发现表达的目的基因大小为396bp,与已上传苏丹株和印度株相应序列比对后发现同源性达97%。通过不同时段的诱导表达,纯化,对重组蛋白的反应原性和特异性进行Western-Blot检测,结果表明重组TaSP蛋白与环形泰勒虫标准阳性血清发生反应,不与环形泰勒虫标准阴性血清发生反应,且不与中华泰勒虫、牛巴贝斯虫、双芽巴贝斯虫、大巴贝斯虫、瑟氏泰勒虫、吕氏泰勒虫、尤氏泰勒虫的标准阳性血清发生交叉反应。2.以纯化的TaSP重组蛋白为抗原,建立牛环形泰勒虫的间接ELISA诊断方法。通过对反应条件的优化,确定了TaSP重组蛋白(即抗原)最适的包被浓度为3μg/ml;血清的最适稀释比例为1:50;最适酶结合底物(原液)最佳稀释比例为1:10000。通过验证,该检测方法能与环形泰勒虫阳性血清发生反应,而与牛巴贝斯虫,双芽巴贝斯虫,瑟氏泰勒虫,中华泰勒虫,吕氏泰勒虫,尤氏泰勒虫阳性血清无交叉反应;对新疆省和甘肃省共140份野外样品进行ELISA和巢式PCR对比检测,符合率为98.5%。因此,该方法特异性达到了100%,敏感性达到了95.7%。
[Abstract]:Theileria annulata is an infectious and seriously harmful blood protozoa that parasitizes macrophages, lymphocytes and red blood cells in the host. It is a worldwide epidemic disease that seriously harms cattle farming in various countries. In China, the main infectious species of Taylor's worms are the ringworm, the Chinese Taylor worm, and the Selenella. Among them, the ringworm is the most harmful and has the most extensive incidence. Cases of the infection have been reported in 13 provinces, including Xinjiang and Inner Mongolia, which have seriously affected the local cattle farming industry. And caused great economic losses. In our country, the main medium for transmission of Taylor's annulus is the genus Tereodes, most of which are mostly residual ticks. In this study, Theileria annulata sporozoites surface protein (TaSP), a merozoite membrane surface protein of Theileria annulata sporozoites surface, was studied in southern Xinjiang, and the expression conditions of recombinant bacteria were optimized on the basis of previous studies. The recombinant TaSP protein was purified and the purified recombinant protein was used as antigen to establish an indirect ELISA detection method for the recombinant TaSP protein of Taylor's cattle. The contents of the study were as follows: 1. The bacterial fluid of the expressed recombinant TaSP protein was detected by PCR. The result of agarose gel electrophoresis (agarose gel electrophoresis) showed that the size of the PCR amplification positive result was about 400bp, and the result of analysis and sequencing showed that the expressed target gene size was 396bp, which was compared with the corresponding sequence of the uploaded Sudan strain and Indian strain. It was found that the homology was 97%. The expression was induced by different periods of time. After purification, the reactivity and specificity of the recombinant protein were detected by Western-Blot. The results showed that the recombinant TaSP protein reacted with the standard positive serum, not with the standard negative serum, and with the Chinese Taylor's. The standard positive serums of bovine Babes, Babes, Babbs, Selenella, Taylor lui and Taylor uvulis were cross-reacted. 2. The purified TaSP recombinant protein was used as antigen. An indirect ELISA diagnostic method for Taylor ringworm was established. The reaction conditions were optimized. The optimal coating concentration of TaSP recombinant protein (antigen) is 3 渭 g / ml; the optimal dilution ratio of serum is 1: 50; the optimal dilution ratio of enzyme binding substrate (substrate) is 1: 10000. This method can react with the positive serum of Taylor's ring, but not with the positive serum of Babes's, Babes, Thessler's, Taylor's, Lui and Taylor's. A total of 140 field samples from Xinjiang and Gansu provinces were detected by ELISA and nested PCR. The coincidence rate was 98.50.Therefore, the specificity of this method was 100 and the sensitivity was 95.7.
【学位授予单位】：河南农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.7

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