尤氏泰勒虫TuIP、Tu88蛋白特性分析及其应用
发布时间：2019-01-21 20:03
【摘要】：羊泰勒虫病是由泰勒虫属病原经硬蜱传播感染绵羊或山羊而引起的血液原虫病。尤氏泰勒虫(Theileria uilenbergi)和吕氏泰勒虫(T.luwenshuni)是引起我国羊泰勒虫病的主要病原。在本课题组前期的研究中,从尤氏泰勒虫裂殖子c DNA文库中筛选到了Tu IP和Tu88两个基因,重组表达的Tu IP和Tu88蛋白都能和尤氏泰勒虫的阳性血清发生反应,表现出了较好的抗原性。本研究进一步分析了这两种蛋白的功能特征,并分别以Tu IP重组蛋白为抗原和以Tu IP基因为靶标建立了两种用于我国羊泰勒虫检测的快速诊断技术。本研究利用间接免疫荧光试验(IFA)和激光共聚焦显微镜技术研究了Tu IP和Tu88蛋白在尤氏泰勒虫裂殖子中的表达和分布情况。结果表明,制备的抗r Tu IP和抗r Tu88兔血清均能与尤氏泰勒虫裂殖子中的天然蛋白反应,同时发现这两种蛋白都分布在尤氏泰勒虫裂殖子胞质中。利用Tu IP重组蛋白完成了羊泰勒虫病免疫胶体金试纸条的研制。在硝酸纤维素膜上分别包被r Tu IP抗原和抗r Tu IP多克隆抗体作为检测线和质控线,胶体金-r Tu IP标记物喷涂于玻璃纤维纸上制成胶体金垫。将试纸条各组成成分(样品垫、胶体金垫、硝酸纤维素膜、吸水垫)按顺序组装在PVC垫上,制成了羊泰勒虫病免疫胶体金试纸条。该试纸条能够在15 min内完成血清样品检测。通过用该试纸条对6只尤氏泰勒虫感染的实验羊不同时间点采集的血清进行检测,结果表明该试纸条能够检测到感染后14到85天内血清中的抗体,即感染早期和后期病例均能被检测出。根据Tu IP基因序列设计出一对特异性引物,建立了尤氏泰勒虫实时荧光定量PCR。该方法的检测灵敏度为102拷贝/μL。与吕氏泰勒虫、羊无浆体和羊巴贝斯虫没有交叉反应,说明具有较好的特异性。组内重复性和组间重复性结果显示组内变异系数(CV)在0.93%和3.25%范围内,小于5%;组间变异系数(CV)在1.55%和4.32%范围内,小于10%;表明该方法具有较好的稳定性。结论,本研究建立的羊泰勒虫病免疫胶体金试纸条和尤氏泰勒虫实时荧光定量PCR方法,将为我国羊的泰勒虫病的诊断和流行病学的调查提供快速检测和定量分析手段。
[Abstract]:Taylor's disease of sheep is caused by the infection of sheep or goat with the pathogen of Taylor's genus by ticks. (Theileria uilenbergi) and T.luwenshuni are the main pathogens of Taylor's disease in sheep in China. Two genes, Tu IP and Tu88, were screened from c DNA library of Taylor's parasite, and the expressed Tu IP and Tu88 proteins could react with the positive serum of Taylor eurei. It showed good antigenicity. In this study, the functional characteristics of the two proteins were further analyzed, and two rapid diagnostic techniques for the detection of Taylor's sheep were established using Tu IP recombinant protein as antigen and Tu IP as target, respectively. In this study, the expression and distribution of Tu IP and Tu88 proteins in the merozoites of Taylor eutectus were studied by indirect immunofluorescence assay (IFA) and laser confocal microscopy. The results showed that both anti-r Tu IP and anti-r Tu88 rabbit sera could react with the natural proteins in the merozoites of Taylor's parasite, and both proteins were found to be distributed in the cytoplasm of the merozoites of Taylor's parasite. Tu IP recombinant protein was used to prepare the gold strip of immune colloid for sheep Taylor's disease. R Tu IP antigen and anti r Tu IP polyclonal antibody were coated on the nitrocellulose membrane as detection line and quality control line respectively. Colloidal gold-r Tu IP label was sprayed on glass fiber paper to make colloidal gold pad. The composition of the test strip (sample pad, colloidal gold pad, nitrocellulose film, water absorbent pad) was assembled on the PVC pad in order to make the immune colloidal gold strip of sheep Taylor's disease. The test strip can be used to detect serum samples within 15 min. The test strip was used to detect the serum samples collected at different time points from 6 sheep infected with Taylor's Eurex. The results showed that the test strip could detect the antibody in serum within 14 to 85 days after infection. Early and late cases of infection can be detected. According to the sequence of Tu IP gene, a pair of specific primers were designed, and a real-time fluorescent quantitative PCR. of Taylor Eurex was established. The detection sensitivity of this method is 102 copies / 渭 L. There was no cross reaction with Taylor lui, sheep without serous body and sheep Babes, which indicated that it had better specificity. The results of intra-group reproducibility and inter-group reproducibility showed that the coefficient of variation (CV) was less than 5% in the range of 0.93% and 3.25%, and the coefficient of variation (CV) was less than 10% in the range of 1.55% and 4.32%. It shows that the method has good stability. Conclusion the gold strip of immune colloid and the real-time fluorescent quantitative PCR method of Taylor's disease in sheep developed in this study will provide a rapid detection and quantitative analysis method for the diagnosis and epidemiological investigation of Taylor's disease in sheep in China.
【学位授予单位】：中国农业科学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.7
本文编号：2412990
[Abstract]:Taylor's disease of sheep is caused by the infection of sheep or goat with the pathogen of Taylor's genus by ticks. (Theileria uilenbergi) and T.luwenshuni are the main pathogens of Taylor's disease in sheep in China. Two genes, Tu IP and Tu88, were screened from c DNA library of Taylor's parasite, and the expressed Tu IP and Tu88 proteins could react with the positive serum of Taylor eurei. It showed good antigenicity. In this study, the functional characteristics of the two proteins were further analyzed, and two rapid diagnostic techniques for the detection of Taylor's sheep were established using Tu IP recombinant protein as antigen and Tu IP as target, respectively. In this study, the expression and distribution of Tu IP and Tu88 proteins in the merozoites of Taylor eutectus were studied by indirect immunofluorescence assay (IFA) and laser confocal microscopy. The results showed that both anti-r Tu IP and anti-r Tu88 rabbit sera could react with the natural proteins in the merozoites of Taylor's parasite, and both proteins were found to be distributed in the cytoplasm of the merozoites of Taylor's parasite. Tu IP recombinant protein was used to prepare the gold strip of immune colloid for sheep Taylor's disease. R Tu IP antigen and anti r Tu IP polyclonal antibody were coated on the nitrocellulose membrane as detection line and quality control line respectively. Colloidal gold-r Tu IP label was sprayed on glass fiber paper to make colloidal gold pad. The composition of the test strip (sample pad, colloidal gold pad, nitrocellulose film, water absorbent pad) was assembled on the PVC pad in order to make the immune colloidal gold strip of sheep Taylor's disease. The test strip can be used to detect serum samples within 15 min. The test strip was used to detect the serum samples collected at different time points from 6 sheep infected with Taylor's Eurex. The results showed that the test strip could detect the antibody in serum within 14 to 85 days after infection. Early and late cases of infection can be detected. According to the sequence of Tu IP gene, a pair of specific primers were designed, and a real-time fluorescent quantitative PCR. of Taylor Eurex was established. The detection sensitivity of this method is 102 copies / 渭 L. There was no cross reaction with Taylor lui, sheep without serous body and sheep Babes, which indicated that it had better specificity. The results of intra-group reproducibility and inter-group reproducibility showed that the coefficient of variation (CV) was less than 5% in the range of 0.93% and 3.25%, and the coefficient of variation (CV) was less than 10% in the range of 1.55% and 4.32%. It shows that the method has good stability. Conclusion the gold strip of immune colloid and the real-time fluorescent quantitative PCR method of Taylor's disease in sheep developed in this study will provide a rapid detection and quantitative analysis method for the diagnosis and epidemiological investigation of Taylor's disease in sheep in China.
【学位授予单位】：中国农业科学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.7
【参考文献】
相关期刊论文 前1条
1 李成;;免疫胶体金标记技术[J];中国畜禽传染病;1987年05期
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