尤氏泰勒蟲TuIP、Tu88蛋白特性分析及其應(yīng)用
發(fā)布時間：2019-01-21 20:03
【摘要】：羊泰勒蟲病是由泰勒蟲屬病原經(jīng)硬蜱傳播感染綿羊或山羊而引起的血液原蟲病。尤氏泰勒蟲(Theileria uilenbergi)和呂氏泰勒蟲(T.luwenshuni)是引起我國羊泰勒蟲病的主要病原。在本課題組前期的研究中,從尤氏泰勒蟲裂殖子c DNA文庫中篩選到了Tu IP和Tu88兩個基因,重組表達(dá)的Tu IP和Tu88蛋白都能和尤氏泰勒蟲的陽性血清發(fā)生反應(yīng),表現(xiàn)出了較好的抗原性。本研究進(jìn)一步分析了這兩種蛋白的功能特征,并分別以Tu IP重組蛋白為抗原和以Tu IP基因為靶標(biāo)建立了兩種用于我國羊泰勒蟲檢測的快速診斷技術(shù)。本研究利用間接免疫熒光試驗(IFA)和激光共聚焦顯微鏡技術(shù)研究了Tu IP和Tu88蛋白在尤氏泰勒蟲裂殖子中的表達(dá)和分布情況。結(jié)果表明,制備的抗r Tu IP和抗r Tu88兔血清均能與尤氏泰勒蟲裂殖子中的天然蛋白反應(yīng),同時發(fā)現(xiàn)這兩種蛋白都分布在尤氏泰勒蟲裂殖子胞質(zhì)中。利用Tu IP重組蛋白完成了羊泰勒蟲病免疫膠體金試紙條的研制。在硝酸纖維素膜上分別包被r Tu IP抗原和抗r Tu IP多克隆抗體作為檢測線和質(zhì)控線,膠體金-r Tu IP標(biāo)記物噴涂于玻璃纖維紙上制成膠體金墊。將試紙條各組成成分(樣品墊、膠體金墊、硝酸纖維素膜、吸水墊)按順序組裝在PVC墊上,制成了羊泰勒蟲病免疫膠體金試紙條。該試紙條能夠在15 min內(nèi)完成血清樣品檢測。通過用該試紙條對6只尤氏泰勒蟲感染的實驗羊不同時間點采集的血清進(jìn)行檢測,結(jié)果表明該試紙條能夠檢測到感染后14到85天內(nèi)血清中的抗體,即感染早期和后期病例均能被檢測出。根據(jù)Tu IP基因序列設(shè)計出一對特異性引物,建立了尤氏泰勒蟲實時熒光定量PCR。該方法的檢測靈敏度為102拷貝/μL。與呂氏泰勒蟲、羊無漿體和羊巴貝斯蟲沒有交叉反應(yīng),說明具有較好的特異性。組內(nèi)重復(fù)性和組間重復(fù)性結(jié)果顯示組內(nèi)變異系數(shù)(CV)在0.93%和3.25%范圍內(nèi),小于5%;組間變異系數(shù)(CV)在1.55%和4.32%范圍內(nèi),小于10%;表明該方法具有較好的穩(wěn)定性。結(jié)論,本研究建立的羊泰勒蟲病免疫膠體金試紙條和尤氏泰勒蟲實時熒光定量PCR方法,將為我國羊的泰勒蟲病的診斷和流行病學(xué)的調(diào)查提供快速檢測和定量分析手段。
[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.
【學(xué)位授予單位】：中國農(nóng)業(yè)科學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S852.7
本文編號：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.
【學(xué)位授予單位】：中國農(nóng)業(yè)科學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S852.7
【參考文獻(xiàn)】
相關(guān)期刊論文 前1條
1 李成;;免疫膠體金標(biāo)記技術(shù)[J];中國畜禽傳染病;1987年05期
，本文編號：2412990
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