猫弓形虫抗体ELISA检测方法的建立与评价

发布时间：2018-04-24 18:25

本文选题：致密颗粒蛋白1、7 + 受试者工作特征曲线　；参考：《吉林农业大学》2015年硕士论文

【摘要】：弓形虫病(Toxoplasmosis)由刚地弓形虫(Toxoplasma gondii)感染引起,是一种可导致人及动物流产、畸形或死胎的人兽共患寄生虫病,严重危害公共卫生安全和畜牧业生产。猫是其终末宿主,在弓形虫的传播过程中起重要作用。因此,建立快速、准确的弓形虫检测方法对人类健康具有重大意义。弓形虫致密颗粒蛋白(GRAs)是一类分泌排泄蛋白,其中GRA1与弓形虫速殖子侵染宿主细胞的信号转导有关,而GRA7在弓形虫各个时期均能分泌,是一种潜在的疫苗侯选分子。证明GRA1、GRA7作为诊断抗原具有良好的免疫原性,可作为血清学诊断弓形虫病的基因标志物。本研究利用原核表达的弓形虫致密颗粒蛋白GRA1、GRA7,建立了GRA1-ELISA与GRA7-ELISA检测方法。利用构建原核表达载体pET-28a-GRA1、pET-28a-GRA7,转化大肠杆菌,IPTG诱导表达,经SDS-PAGE验证,重组蛋白GRA1与GRA7分子量分别为25、27 kDa;Western blotting分析表达产物,结果表明,该产物具有较强的免疫反应;应用Ni-NTA对表达蛋白进行纯化,蛋白纯度高达98%;GRA1与GRA7的浓度分别为200μg/mL和600μg/mL。利用纯化重组蛋白GRA1和GRA7分别作为诊断抗原,建立GRA1-ELISA和GRA7-ELISA检测方法。确立了GRA1、GRA7最佳抗原包被浓度5μg/mL、5μg/mL,血清一抗最佳稀释度1:64,HRP标记兔抗猫IgG二抗最佳工作浓度1:20000。试验表明GRA1-ELISA与GRA7-ELISA方法检测灵敏度高,重复性好。应用GRA1-ELISA和GRA7-ELISA方法检测旋毛虫、包虫、猪附红细胞体和猫弓形虫阳性血清,表明建立的GRA1-ELISA与GRA7-ELISA检测方法具有特异性。建立GRA1-ELISA和GRA7-ELISA检测方法以MAT/IFA法检测结果为标准进行比较评价,GRA1-ELISA检测方法假阳性率为10.8%,假阴性率为4.1%,而GRA7-ELISA检测方法假阳性率为7.5%,假阴性率为1.4%,说明GRA7为诊断抗原检测弓形虫病的假阳性率和假阴性率较低。将差异部分通过McNemar卡方检验均无显著性差异(P0.05);将一致部分通过Kappa检验,GRA1:Kappa=0.83,结果一致率为94.6%;GRA7:Kappa=0.92,结果一致率为97.2%;表明GRA7-ELISA检测方法一致率较高。ROC曲线分析出建立的GRA7-ELISA稳定性较好。GRA1-ELISA的cut-off值为0.11时,敏感性为83.6%,特异性为87%;GRA7-ELISA的cut-off值为0.26时,敏感性为89.7,特异性为92.5%。试验表明GRA1作为诊断抗原建立GRA1-ELISA方法检测效果不明显,敏感性与特异性较低、稳定性较弱;GRA7为检测猫弓形虫病的优质诊断抗原,所建立的GRA7-ELISA检测方法具有较高的敏感性与特异性、较强的稳定性。本研究建立的GRA7-ELISA诊断方法,解决了弓形虫病不易准确、快速诊断的难题,对猫弓形虫病的临床诊断、疾病防控具有重要意义,筛选出GRA7优质诊断抗原为我国猫弓形虫病的快速诊断及试剂盒的研发提供依据。
[Abstract]:Toxoplasmosis (Toxoplasmosis) is caused by Toxoplasma gondii (Toxoplasma gondii) infection. It is a kind of human zoonosis which can cause human and animal abortion, deformity or stillbirth. It seriously endangers public health safety and animal husbandry. The cat is its terminal host and plays an important role in the transmission of Toxoplasma gondii. Therefore, it is fast and accurate. The detection method of Toxoplasma gondii is of great significance to human health. Toxoplasma dense granule protein (GRAs) is a class of excretory protein, in which GRA1 and Toxoplasma gondii infect host cell signal transduction, and GRA7 can be secreted at all stages of Toxoplasma gondii. It is a potential candidate for vaccine candidate. It is proved that GRA1 and GRA7 are used as diagnosis. The broken antigen has good immunogenicity and can be used as a gene marker for the diagnosis of toxoplasmosis in serology. In this study, the detection methods of GRA1-ELISA and GRA7-ELISA were established by using the dense granular protein GRA1 and GRA7 of the prokaryotic expression of Toxoplasma gondii. The prokaryotic expression vector pET-28a-GRA1, pET-28a-GRA7, Escherichia coli and IPTG were used to induce expression. SDS-PAGE showed that the molecular weight of recombinant protein GRA1 and GRA7 was 25,27 kDa, respectively, and Western blotting was used to analyze the expression products. The results showed that the product had strong immune response, and the purity of the protein was 98%, and the concentration of GRA1 and GRA7 was 200 mu g/mL and 600 micron, respectively. GRA1-ELISA and GRA7-ELISA detection methods were established respectively as diagnostic antigens. The optimum antigen inclusion concentration of GRA1, GRA7 was 5 u g/mL, 5 micron g/mL, the best dilution of serum 1:64, HRP labeled Rabbit anti cat IgG two anti optimal working concentration 1:20000. test showed that GRA1-ELISA and GRA7-ELISA methods were highly sensitive and reproducible. GRA7-ELISA method was used to detect Trichinella, echinococcosis, porcine eoeosis and Toxoplasma gondii positive serum, which showed that the method of detecting GRA1-ELISA and GRA7-ELISA was specific. The determination of GRA1-ELISA and GRA7-ELISA was compared with the test results of MAT/IFA, and the false positive rate of GRA1-ELISA detection method was 10.8%, false negative. The rate of sex was 4.1%, while the false positive rate of GRA7-ELISA detection method was 7.5% and the false negative rate was 1.4%. The false positive rate and false negative rate of GRA7 as diagnostic antigen were low. There was no significant difference (P0.05) by the McNemar Chi test. The same part passed the Kappa test, GRA1:Kappa=0.83, the result concordance rate was 94.6. %; GRA7:Kappa=0.92, the result consistent rate was 97.2%, indicating that the GRA7-ELISA detection method with high consistency rate.ROC curve showed that the cut-off value of.GRA1-ELISA with better GRA7-ELISA stability was 0.11, the sensitivity was 83.6%, the specificity was 87%, and the cut-off value of GRA7-ELISA was 0.26, the sensitivity was 89.7, and the specificity of 92.5%. test showed GRA1 as a GRA1. The diagnostic antigen based GRA1-ELISA method has no obvious effect, low sensitivity and specificity and weak stability. GRA7 is a good diagnostic antigen for the detection of toxoplasmosis in cats. The established GRA7-ELISA detection method has high sensitivity, specificity and strong stability. The GRA7-ELISA diagnosis method established in this study solves the Toxoplasma gondii It is very important for the clinical diagnosis of toxoplasmosis and prevention and control of toxoplasmosis. Screening out the GRA7 diagnostic antigen for the rapid diagnosis of toxoplasmosis and the research and development of the kit of toxoplasmosis in China.

【学位授予单位】：吉林农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S858.293

【共引文献】