我国中山病血清流行病学调查及VP7抗原间接ELISA方法的建立与应用

发布时间：2018-04-08 14:42

本文选题：中山病　切入点：VP7蛋白　出处：《黑龙江八一农垦大学》2017年硕士论文

【摘要】：中山病是由中山病病毒(Chuzan virus,CHUV)引起的一种虫媒性传染病,感染牛羊等家畜,临床症状以流产、早产、死产、产生先天性异常犊牛为主要特征。中山病来源于日本,国内外相关研究较少。为了解中山病在我国的流行状况以控制本病在我国的发生和发展,本研究开展了我国中山病流行病学调查及间接ELISA抗体检测方法的研究。对我国黑龙江、内蒙古、新疆、山东、江苏、湖北、广西、云南和海南9个省区的543份牛羊血清,用经典的病毒微量中和试验方法进行中山病的流行病学调查。结果表明:海南68份牛羊血清样品中阳性样品39份,阳性率高达57.35%;黑龙江的80份样品检测结果均为阴性;其他各省的血清阳性率介于6%-48.65%之间,气候温暖地区感染率较高;对新疆、湖北、广西、云南4个省区牛和羊的易感性进行比较,除新疆(羊阳性率比牛高)外,其他地区牛的阳性率均比羊高,牛血清阳性率在55.56%-58.6%,羊在32.73%-33.33%之间;对新疆、湖北、广西3个省区不同季节中山病的感染率进行比较,秋季(9-11月)感染率(16.67%-43.33%)最高,明显高于春季和夏季。为建立一种简便快速的检测中山病抗体的间接ELISA方法,本研究将携带群特异性抗原决定簇的CHUV VP7蛋白作为检测抗原,参考CHUV广西分离株GX871毒株的S7基因序列设计引物,经RT-PCR扩增得到VP7基因,克隆至p ET-28a(+)表达载体中,经IPTG诱导表达、SDS-PAGE和Western blot鉴定,获得重组VP7蛋白。用纯化的VP7蛋白作为包被抗原,用方阵滴定法确定包被抗原的最佳使用浓度,经过对ELISA术式进行优化和完善,初步建立了检测中山病抗体的间接ELISA方法。其临界值分别为0.384-0.347,即OD450nm大于0.384为阳性,小于0.347为阴性,二者之间为可疑;对阿卡斑病毒(Akabane virus,AKAV)、牛轮状病毒(Rotaviruses,RV)、蓝舌病病毒(Bluetongue virus,BTV)、口蹄疫病毒(Foot and mouth virus,FMDV)、牛病毒性腹泻-粘膜病(Bovine viral diarrhoea virus,BVDV)、牛传染性鼻气管炎(Infectious bovine rhinotracheitis virus,IBRV)、牛副流感3型(Parainfluenza virus type3,PIV3)阳性血清检测结果均为阴性;批内和批间重复试验的变异系数分别小于7.1%和13.2%;与中和试验符合率为83.05%。用所建立的间接ELISA检测方法对我国黑龙江、新疆、广西和云南四省区121份血清样品进行了检测,其阳性率分别为0%、13.33%、39.39%和64.28%,与微量中和试验检测结果一致。综上所述,中山病在我国被调查的8个省区均有不同程度的流行,气候温暖地区感染率较高,秋季阳性率较高,牛比羊更易感,符合虫媒病的流行规律。本研究建立的以重组VP7蛋白为抗原的间接ELISA方法,具有良好的特异性和敏感性,且成本较低,可用于中山病血清的高通量检测。
[Abstract]:Zhongshan disease is caused by Zhongshan disease virus (Chuzan virus, CHUV) caused by an insect borne infectious diseases, infection of cattle and other livestock, clinical symptoms of abortion, premature birth, stillbirth and congenital anomalies were characterized. Zhongshan disease originated from Japan, less relevant research at home and abroad. In order to understand the disease epidemic situation in Zhongshan our country to control the disease in the occurrence and development of our country, this paper carried out research on detection method of investigation of Zhongshan disease epidemiology in China and the indirect ELISA antibody in China. Heilongjiang, Inner Mongolia, Xinjiang, Shandong, Jiangsu, Hubei, Guangxi, Yunnan and Hainan 9 provinces 543 cattle and sheep serum, were epidemiological investigation of Zhongshan disease virus neutralization test using classical methods. The results show that 68 samples of Hainan cattle and sheep serum samples positive in 39 samples, the positive rate is as high as 57.35% in Heilongjiang; 80 samples test results were negative for each other; Between the serum positive rate between 6%-48.65%, a higher rate of warm climate in the region of Xinjiang, Hubei, infection; Guangxi, Yunnan 4 provinces and the susceptibility of cattle and sheep were compared, in addition to Xinjiang (the positive rate of sheep than cattle, cattle in other high) positive rates than sheep, bovine serum positive rate in 55.56%-58.6% sheep, 32.73%-33.33%; in Xinjiang, Hubei, Guangxi 3 provinces were compared in different seasons in Zhongshan disease infection rate in autumn (9-11 months) (16.67%-43.33%) the highest infection rate was significantly higher than that in spring and summer. For the indirect ELISA method to establish a simple and rapid detection of Zhongshan disease antibody, the research group will carry the specific epitope of CHUV VP7 protein as antigen, primers S7 gene sequences of reference CHUV Guangxi isolate GX871 strain, VP7 gene was amplified by RT-PCR, cloned into P (+) ET-28a expression vector, expression of SDS induced by IPTG. -PAGE and Western blot identification, obtain the recombinant VP7 protein. The purified VP7 protein as antigen to determine the optimal concentration of coating antigen by matrix titration method, through the optimization and improvement of the ELISA operation, the initial establishment of the indirect ELISA method for detection of Zhongshan disease antibody. Its critical value was 0.384-0.347, i.e. OD450nm was more than 0.384 less than 0.347 positive, negative, between the two as suspicious of Akabane virus (Akabane; virus, AKAV), bovine rotavirus (Rotaviruses, RV), bluetongue virus (Bluetongue virus, BTV of foot-and-mouth disease virus (Foot), and mouth virus, FMDV), bovine viral diarrhea (Bovine viral diarrhoea virus, BVDV), infectious bovine rhinotracheitis (Infectious bovine rhinotracheitis virus, IBRV), bovine parainfluenza virus type 3 (Parainfluenza virus, type3, PIV3) detection results of positive serum were negative; intra and inter repeated test The coefficient of variation of the test were less than 7.1% and 13.2%; and with indirect ELISA neutralization test rate of 83.05%. by using the proposed detection method on China's Heilongjiang, Xinjiang, Guangxi and Yunnan four provinces of 121 serum samples were detected, the positive rates were 0%, 13.33%, 39.39% and 64.28%, and the micro neutralization test results consistent. To sum up, Zhongshan disease was investigated in China's 8 provinces have different levels of popularity, a higher rate of infection is higher in autumn warm climates, the positive rate of cattle sheep are more susceptible, meet the epidemic regularity of insect borne diseases. This study established the recombinant VP7 protein as antigen by indirect ELISA method, with good specificity and sensitivity, and low cost, can be used for high-throughput serum Zhongshan disease detection.

【学位授予单位】：黑龙江八一农垦大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S855.3

【相似文献】