兔感染新孢子虫间接ELISA方法的建立与初步应用

发布时间：2018-06-22 03:51

本文选题：新孢子虫 + Nc-43-p　；参考：《吉林农业大学》2017年硕士论文

【摘要】：新孢子虫病(Neosporosis)由犬新孢子虫(Neospora caninum)感染引起的一种原虫病[1]。新孢子虫病是严重危害奶牛和犬的一种原虫病,临床报道发生新孢子虫病的动物包括绵羊、山羊、鹿、犀牛和马,除此之外,在水牛、红狐、灰狐、狼、骆驼以及猫科动物中也检测到了新孢子虫抗体[2]。甚至在人的血清中也检测到新孢子虫的抗体,目前还没有在人体内发现新孢子虫体的报导,但是新孢子虫可能存在人兽共患的风险。新孢子虫可感染兔,为了减少新孢子虫病给养兔业带来的经济损失,做好该病的诊断与防控非常重要。目前没有针对兔感染新孢子虫的诊断方法。因此,本研究选取新孢子虫的主要表面抗原基因Np-43基因,克隆到pET-32a,构建pET-Nc-p43重组质粒,在BL21中进行Ncp43P重组蛋白表达,利用镍柱进行表达蛋白的纯化,作为包被抗原,建立检测兔感染新孢子虫的间接ELISA抗体检测方法。利用表达的Ncp43P蛋白建立检测兔血清中新孢子虫抗体的间接ELISA方法。包被抗原浓度为5μg/mL。分别选择了小鼠血清、胎牛血清、明胶作为封闭剂,并对一抗、二抗的稀释浓度进行了优化,结果确定采用10%的小鼠血清作为封闭剂,血清按照1:100稀释,酶标二抗按照1:1000稀释效果最好。建立的方法通过同一板内检测不同的血清,和同一样品在不同板间进行检测,分析建立的方法批间、批内稳定性。通过实验测得批间差为1.6%-5.0%,批内差为0.4%-2.3%,均小于10%,说明建立的方法具有很好的稳定性。因为新孢子虫与弓形虫最为接近,所以利用该方法对弓形虫抗体阳性血清进行检测,结果显示该方法与弓形虫阳性血清无交叉反应,具有较好的特异性。Cut off值的确定,根据Cut off值=阴性血清OD值平均值+3SD,计算得Cut off值为0.37。为减少假阳性与假阴性,在Cut off值的基础上加减一个标准差作为判断阴阳性血清的标准,即OD值0.4为阳性,OD值0.3为阴性,OD值在两者之间为疑似。该方法与VMRD的ELISA试剂盒进行比较符合率达97.5%。该方法可用于兔血清中新孢子虫抗体的检测,利用该方法对吉林地区采集的468份兔血清样品进行检测,结果检测为阳性血清为149份,阳性率达31.8%。
[Abstract]:Neosporosis A protozoa disease caused by Neospora caninum infection. Neosporidiosis is a protozoan disease that seriously harms cows and dogs. Clinical reports of neosporidiosis include sheep, goats, deer, rhinoceros and horses, in addition to buffalo, red fox, gray fox, wolf, etc. Antibodies to neosporidium were also detected in camels and cats [2]. Antibodies to neosporidium have been detected even in human serum. There are no reports of neosporidium in human body, but neosporidium may be at risk of zoonosis. Neosporidium can infect rabbits. In order to reduce the economic loss caused by neosporidiosis, it is very important to make a good diagnosis, prevention and control of the disease. There is no diagnostic method for neosporidium infection in rabbits. Therefore, the main surface antigen gene of neosporidium, Np-43 gene, was cloned into pET-32a, and the recombinant plasmid pET-Nc-p43 was constructed. Ncp43P recombinant protein was expressed in BL21 and purified by nickel column. An indirect Elisa method for detection of neosporidium infection in rabbits was established. An indirect Elisa method for detection of neosporidium antibodies in rabbit serum was established by using the expressed Ncp43P protein. The concentration of coated antigen was 5 渭 g / mL. Mouse serum, fetal bovine serum and gelatin were selected as sealants, and the dilution concentration of first antibody and second antibody was optimized. The results showed that 10% mouse serum was used as sealant and the serum was diluted according to 1: 100. The enzyme labeled second antibody was best diluted according to 1: 1000. The established method was used to detect different serum in the same plate, and the same sample was detected in different plates. The stability of the method between batches and batches was analyzed. The results showed that the difference between batches was 1.6-5.0, and the difference within batches was 0.4-2.3, which was less than 100.The results showed that the established method had good stability. Because neosporidium and Toxoplasma gondii are the closest, the method is used to detect Toxoplasma antibody positive serum. The results show that this method has no cross reaction with Toxoplasma gondii positive serum, and has good specificity. According to the mean value of cut off = negative serum OD value 3 SD, the cut off value was calculated to be 0. 37. In order to reduce false positive and false negative, a standard deviation was added and subtracted on the basis of cut off value as the criterion for judging yin-yang serum, that is, OD value 0.4 was positive, OD value 0.3 was negative, OD value was suspected between them. Compared with the Elisa kit of VMRD, the coincidence rate of this method was 97.5. The method can be used for the detection of neosporidium antibody in rabbit serum. 468 rabbit serum samples collected in Jilin area were detected by this method. 149 positive sera were detected, and the positive rate was 31.8%.
【学位授予单位】：吉林农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S858.291

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