牛病毒性腹泻病毒宁夏地方分离株的分离及其E0基因原核表达载体的构建

发布时间：2018-08-20 12:03

【摘要】：牛病毒性腹泻／黏膜病(Bovine viral diarrhea-mucosal disease, BVD-MD)是由牛病毒性腹泻／粘膜病病毒(BVDV)引发的牛的一种传染病。该病主要发生于牛,犊牛更易感,还可感染其它动物,主要引起牛腹泻,急、慢性粘膜病,持续性感染与免疫耐受,母畜流产和死胎等,给养牛业的经济造成严重的损失。为了调查宁夏地区牛病毒性腹泻/粘膜病的流行病学及分子流行病学情况,掌握该病在宁夏地区的流行状况,本试验运用ELISA方法对宁夏BVDV的感染及流行情况进行调查。并在此基础上对血清学阳性牛进行分子生物学跟踪检测,以获得BVDV地方分离株。本研究对送到实验室检测的326份血清样品和278份耳组织样品,运用BVDV抗体ELISA检测试剂盒对血清样品进行抗体检测,对耳组织样品使用BVDV抗原ELISA检测试剂盒进行抗原检测。对送检样品BVDV血清学的研究。检测结果显示,326份血清样品的BVDV抗体阳性率为90.2%,278份耳组织样品的BVDV抗原阳性率为0.3%。检测结果表明宁夏地区送检的样品中BVDV抗体阳性率很高,可能是因为送检的都是具有腹泻、流产等症状的样品有关。利用细胞培养技术对诊断出的抗原阳牛进行病毒分离,并顺利获得牛病毒性腹泻病毒分离株,命名为BVDV-NX。该分离株在MDBK细胞系上进行增殖培养,出现空斑、拉网和脱落等一系列典型的细胞病变(CPE)。随后收集病毒液,提取病毒总RNA并经反转录获得BVDV-NX毒株cDNA.利用PCR方法对5'-UTR基因片段进行扩增和测序,应用MEGA6分析软件,选择N-J(Neighbor-Joining)方法绘制遗传进化树,这株BVDV-NX属于BVDV基因1b型。根据NCBI上已发表的BVDV-EO基因序列设计一对特异性引物,利用PCR方法扩增出710bp的E0基因,进行测序及序列分析。将PCR扩增的E0基因克隆入原核表达载体pET-32a,转化到大肠杆菌BL21(DE3)进行双酶切鉴定,构建原核表达载体pET-32a-E0,为进一步的试验奠定基础。
[Abstract]:Bovine viral diarrhea / mucosal disease (BVD-MD) is an infectious disease caused by bovine viral diarrhea / mucosal disease virus (BVDV). The disease mainly occurs in cattle, calves are more susceptible to infection, but also can infect other animals, mainly causes bovine diarrhea, acute, chronic mucosal disease, persistent infection and immune tolerance, female abortion and stillbirth, and so on, causing serious economic losses to the cattle industry. In order to investigate the epidemiology and molecular epidemiology of bovine viral diarrhea / mucosal disease in Ningxia and to know the epidemic situation of the disease in Ningxia, the ELISA method was used to investigate the infection and prevalence of BVDV in Ningxia. On the basis of this, the seropositive cattle were tracked by molecular biology to obtain the local isolates of BVDV. In this study, 326 serum samples and 278 ear tissue samples sent to the laboratory were detected by BVDV antibody ELISA assay kit, and BVDV antigen ELISA kit was used to detect the antigen of ear tissue samples. Study on BVDV serology of sample. The results showed that the positive rate of BVDV antibody in 326 serum samples was 90.2and the positive rate of BVDV antigen in 278 ear tissue samples was 0.3%. The results showed that the positive rate of BVDV antibody was very high in the samples from Ningxia area, which may be related to the samples with diarrhea, abortion and other symptoms. Using cell culture technique, the virus was isolated from the diagnosed antigen, and the isolated strain of bovine viral diarrhea virus was successfully obtained, which was named BVDV-NX. The isolate was cultured on MDBK cell lines and showed a series of typical cytopathic (CPE)., such as plaque, mesh drawing and shedding. Then the virus solution was collected, the total RNA of the virus was extracted and the BVDV-NX strain cDNA was obtained by reverse transcription. The 5'-UTR gene fragment was amplified and sequenced by PCR method. N-J (Neighbor-Joining) method was selected to map the genetic evolution tree using MEGA6 analysis software. The BVDV-NX belongs to BVDV gene 1b type. According to the published sequence of BVDV-EO gene in NCBI, a pair of specific primers were designed. The E0 gene of 710bp was amplified by PCR method and sequenced. The E0 gene amplified by PCR was cloned into the prokaryotic expression vector pET-32a and transformed into Escherichia coli BL21 (DE3) for double digestion to construct the prokaryotic expression vector pET-32a-E0, which laid a foundation for further experiments.
【学位授予单位】：宁夏大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S852.65

【相似文献】