【作者】 张剑；

【导师】 马卫明；

【作者基本信息】 山东农业大学 ， 临床兽医学， 2013， 硕士

【摘要】 布鲁氏菌病(Brucellosis)是由布鲁氏菌(Brucella)引起的一种人兽共患性传染病，它以流产和发热为主要特征，极大阻碍了畜牧业的发展，严重危害了人类的健康。布鲁氏菌病是世界范围内影响公共卫生的一个难题，发病后如果转入慢性就无法根治。目前，我国布鲁氏菌病的流行状况仍然令人堪忧，严峻的疫情形势为该病的预防和检测提出了更高的要求。本研究拟选取牛布鲁氏菌毒力基因Virb8作为研究对象，构建其原核表达载体，获得高纯度蛋白后，利用此蛋白作为包被抗原构建检测奶牛布鲁氏菌血清的iELISA方法，并与传统的虎红平板凝集试验进行对比，探讨所建立的iELISA检测方法用于生产实践的可行性。通过查阅牛布鲁氏菌毒力基因Virb8序列（Gen Bank:AF226278.1），设计一对特异性引物，在特异性引物的上游和下游分别引入BamHI酶切位点和HindIII酶切位点。提取布鲁氏菌A19疫苗株的基因组DNA后，将其作为模板扩增出Virb8的基因片段，片段大小为720bp。将目的基因连接到pEASY-T3上，然后把产物转入到大肠杆菌感受态细胞DH5α中，构建完成重组表达质粒pET-T3-Virb8。随后利用限制性内切酶BamHI和HindIII对pET-T3-Virb8和pET-32a(+)质粒同时进行双酶切鉴定，证明载体已构建成功。利用T4DNA连接酶将试验所需的目的基因同表达载体进行连接，转化入DH5α，构建出重组表达质粒pET-32a-Virb8，然后将pET-32a-Virb8转入E.coli BL21(DE3)中，用1mM/L的IPTG对其进行诱导表达，经SDS-PAGE分析和Western-blot分析鉴定后，采用Ni-NTASpin Kit对蛋白进行纯化。将获得的目的蛋白作为包被抗原确定iELISA方法的最佳包被浓度、最佳包被条件、最佳二抗稀释度及阴阳性临界值，同时进行敏感性试验和重复性试验，用所建立的iELISA方法对235个临床奶牛血清样本进行检测，并与虎红平板凝集试验进行比较。通过实验，成功的构建起了布鲁氏菌毒力Virb8的克隆表达载体，并且获得了大量的目的蛋白，所建立的iELISA方法具有稳定、灵敏的特点，与虎红平板凝集试验的符合率可达97.02%，有效地证明了该iELISA方法可以应用于牛布鲁氏菌病血清学诊断，为生产实践中布鲁氏菌病的预防和检测提供了便利。更多还原

【Abstract】 Brucellosis is a zoonotic disease which is caused by Brucella. It obstacls livestockdevelopoment and is harmful to the human health. The disease is characterized by abortionand fever. Brucellosis is a worldwide public health problem, it is hard to cure completelywhen infected. The monitoring results showed that the situation of disease had been bad inrecent years, so prevention and detection must be strengthened.In order to explore the possibility of Virb8iELISA for practice,Brucella Virb8wasselected to construct a prokaryotic expression cloning vectors. When the recombinant proteinwas purified, an iELISA which used the purified recombinant protein as coating antigen wasestablished for detecting antibodies to Brucella abortus. Serum samples were also tested bythe RBPT as reference.According to the sequence of Brucella Virb8gene(Gen Bank:AF226278.1), one pair ofspecific primer was designed. BamHI restriction site in the upstream was introduced inprimers and also HindⅢ restriction site in the downstream. Using the method of boiling,Brucella abortus vaccine strain S19genomic DNA was extracted. The Virb8gene fragmentwas amplified using the total genomic DNA as template, and the size is720bp. The amplifiedgene fragment was cloned into pEASY-T3, then transformed into DH5α to construct thecloned carrier pEASY-T3-Virb8. The recombinant plasmid and the pET-32a were doubledigested with BamHI and HindⅢ. The target gene fragment was ligated into the expressionvector by using T4ligase, then transformed into DH5a, and constructed the recombinantexpression plasmid pET-32a-Virb8. After sequencing, the restructuring expression plasmidwas transformed into E.coli BL21(DE3), then expressed by1mM/L IPTG, identified bySDS-PAGE and Western-blot. The recombinant protein was purified by the Ni-NTA Spin Kit.When the recombinant protein was purified, an iELISA which used the purified recombinantprotein as coating antigen was established for detecting235serum samples. During thedevelopoment, sunch as the optimal coating condition of recombinant protein and the optimal dilution of peroxidise were determined. Serum samples were also tested by the RBPT asreference.In this sdudy, Brucella Virb8was successfully cloned and high purified protein wasobtained. The iELSIA established was stable and sensitive.The coincidence of iELISA withRBPT was97.02%. The iELISA could be used as a method for serological diagnosis ofbrucellosis and could be convenient to practice.更多还原