猪脑心肌炎病毒抗体ELISA方法的建立和应用
发布时间：2019-04-29 20:16
【摘要】：脑心肌炎病毒(Encephalomyocarditis virus,EMCV)是一种自然疫源性人畜共患病。可以感染多种动物和人,引起以脑炎、心肌炎或心肌周围炎为主的一类高度传染病。猪是EMCV的易感宿主,引起哺乳仔猪的高死亡率和妊娠母猪的繁殖障碍。给养猪业带来了极大的经济损失。多数猪场虽然未用EMCV的疫苗进行免疫,但血清学调查表明,我国规模化猪场的猪的血清阳性率达50%以上。表明猪群中EMCV的野毒感染较为普遍,因此需要对EMCV的流行进行检测和监控。为了对EMCV的流行病学进行调查和分析,我们参考GenBank中的序列,人工合成一段含有VP1抗原的DNA序列,转化到BL21感受态细胞中,诱导表达蛋白并纯化。将纯化的VP1蛋白包被ELISA板,建立检测猪脑心肌炎病毒抗体的间接ELISA的诊断方法,并对ELISA检测条件进行优化。最佳包被缓冲液为PH9.6的碳酸盐缓冲液;最佳包被蛋白的浓度为用1mg/ml;4℃包被过夜;封闭条件是5%milk作为封闭液,37℃,封闭1h;待检血清的最佳稀释倍数为1:100倍稀释,待检血清的孵育条件为37℃,0.5h;HRP标记二抗抗体的最佳稀释倍数为1:1000倍稀释,反应条件为37℃,孵育1.5h;TMB显色时间为室温下20min,在OD450进行读数,并对数据进行分析。通过对2106份猪血清进行检测,用TG-ROC软件对检测的数据进行分析统计,确定本方法的判定标准是:OD值大于0.5的为阳性;OD值小于0.4的为阴性;界于两者之间的为可疑。应用该方法,检测了山东省区域内猪的血清2106份,OD值大于0.5的数值有984份,阳性率为46.72%;OD值小于0.4的数值有985份,阴性率为46.77%;界于两者之间的数值有137份,为可疑值,可疑率为6.51%。结果证明山东省内的猪群,大部分存在EMCV抗体,EMCV感染非常普遍。本实验为山东省内的EMCV的感染的流行病学监测及诊断提供了有效手段,并可以大概了解山东省内猪群的EMCV的感染状况。
[Abstract]:Cerebral myocarditis virus (Encephalomyocarditis virus,EMCV) is a natural zoonotic disease. Can infect a variety of animals and people, causing encephalitis, myocarditis, or perimyocardial inflammation is a high-level infectious disease. Pig is a susceptible host of EMCV, which causes high mortality of suckling piglets and reproductive disturbance of pregnant sows. It has brought great economic losses to the pig industry. Although most pig farms were not immunized with EMCV vaccine, serological investigation showed that the seropositive rate of pigs in large-scale pig farms in China was more than 50%. The results showed that the wild virus infection of EMCV was common in pigs, so it was necessary to detect and monitor the prevalence of EMCV. In order to investigate and analyze the epidemiology of EMCV, we synthesized a sequence of DNA containing VP1 antigen and transformed it into BL21 competent cells, induced the expression of protein and purified it. The purified VP1 protein was coated with Elisa board to establish an indirect ELISA diagnostic method for detection of antibodies against porcine encephalomyocarditis virus. The detection conditions of ELISA were optimized. The optimal coating buffer was carbonate buffer of PH9.6, the optimal concentration of coated protein was 1 mg / ml 路ml ~ (- 1) for the night at 4 鈩，
本文编号：2468501
[Abstract]:Cerebral myocarditis virus (Encephalomyocarditis virus,EMCV) is a natural zoonotic disease. Can infect a variety of animals and people, causing encephalitis, myocarditis, or perimyocardial inflammation is a high-level infectious disease. Pig is a susceptible host of EMCV, which causes high mortality of suckling piglets and reproductive disturbance of pregnant sows. It has brought great economic losses to the pig industry. Although most pig farms were not immunized with EMCV vaccine, serological investigation showed that the seropositive rate of pigs in large-scale pig farms in China was more than 50%. The results showed that the wild virus infection of EMCV was common in pigs, so it was necessary to detect and monitor the prevalence of EMCV. In order to investigate and analyze the epidemiology of EMCV, we synthesized a sequence of DNA containing VP1 antigen and transformed it into BL21 competent cells, induced the expression of protein and purified it. The purified VP1 protein was coated with Elisa board to establish an indirect ELISA diagnostic method for detection of antibodies against porcine encephalomyocarditis virus. The detection conditions of ELISA were optimized. The optimal coating buffer was carbonate buffer of PH9.6, the optimal concentration of coated protein was 1 mg / ml 路ml ~ (- 1) for the night at 4 鈩，
本文编号：2468501
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