导　　师： 黄会岭;吴清民

学科专业： I0603

授予学位： 硕士

作　　者： ;

机构地区： 河北农业大学

摘　　要： 布鲁氏菌病(brucellosis)是由细胞内寄生的革兰氏阴性布鲁氏菌引起的一种人畜共患病。临床上以发热，流产为主要症状，严重威胁着人畜健康。据世界卫生组织统计表明，全世界每年出现50万例人布鲁氏菌病，每年因布鲁氏菌病造成的经济损失近30亿美元。 在布鲁氏菌病控制过程中，检疫淘汰是一项重要措施。检疫淘汰的前提就是准确的诊断，但目前常用的布鲁氏菌病血清学诊断方法不能区分是疫苗免疫还是自然感染后引起的血清学反应。寻找合适的布鲁氏菌诊断性抗原和研制具有标记性的疫苗一直是人们追求的目标。 布鲁氏菌外膜蛋白位于细菌细胞膜的最外层，因其具有免疫原性和免疫保护作用一直备受注目。本试验从genbank中下载编码布鲁氏菌外膜蛋白的基因，用blast分析后选取与布鲁氏菌猪、牛、羊这三种生物型基因序列的同源性高又与其他革兰氏阴性细菌没有交叉反应的基因；同时对目的基因编码的蛋白用tmhmm和conpredⅡ软件进行跨膜结构和亲水性的分析后，选取没有跨膜结构且亲水性较好的蛋白进行研究。本试验依据以上条件选取了羊布鲁氏菌16m的外膜蛋白omp39和ompl4进行初步研究。 通过premier 5.0设计引物用于扩增选定的两个目的基因，以灭活的16m布鲁氏茵基因组为模板用降落pcr扩增目的片段。将限制性内切酶bamhi、ecori双酶切的目的片段直接与同样双酶切的表达载体pgex-4t-2连接，之后将重组表达载体转化到大肠杆菌bl2l。提取的重组质粒经菌液pcr鉴定、重组载体双酶切鉴定和测序分析确定目的基因成功插入到了表达载体中。然后将含有重组表达载体的菌株在不同温度、不同时间、不同iptg浓度下诱导目的蛋白表达，sds-page电泳检测结果显示目的蛋白在大肠杆菌中获得了大量表达。表达蛋白通过western-blotting分析发现能够与布鲁氏菌病豚鼠阳性动物血清发生较强烈的反应。之后用电洗脱的方法纯化了目的蛋白，并将其作为包被抗原，用牛布鲁氏菌阳性血清为一抗，兔抗牛igg为二抗优化elisa检测条件，试图进一步探索目的蛋白作为诊断性蛋白的可能性。本试验为寻找布鲁氏菌病的诊断性蛋白和布鲁氏菌基因缺失标记苗的研制奠定了一定基础。 Brucellosis is a zoonotic disease caused by a small intracellular gram-negative bacterium which is pathogenic for humans as well as for many species of animals. This infection induces abortions, undulant fever and osteomyelitis in humans as well as livestock animals leading to severe economic losses. Every year Brucella caused about 500,000 cases of Brucellosis in mankind in the world. According to the World Health Organization statistical data, Brucellosis caused economic losses of nearly 3 billion dollars every year. Accurate diagnosis was premise for effective control and eradication of the brucellosis, but the common brucellosis serological diagnostic methods can not distinguish between the vaccine or natural infection. Development of a marker of the vaccine and find a suitable brucella diagnostic antigen has been the people's goals. Brucella outer membrane protein as the immunogenic and protective effect antigen has been well known to the world. The gene of Brucella outer membrane protein was download from GenBank. The gene was selected by BLAST analysis that showed high gene homologous of the three Brucella species/(B. melitensis, B.abortus, B.suis/) and the gene did not high cross-reaction with other Gram- negative bacteria, while the gene of outer membrane protein was analysised of the signal peptide and transmembrane structure by TMHMM and ConPred II Software. This test select the Omp39 and 0mpl4 of B.melitensis 16M for being the preliminary study. First, the primer of the two selected outer membrane protein genes were designed by the Premier 5.0 software. The genome of inactivated B.melitensis 16M was used as amplification templates. The amplified fragments digested with the restriction endonuclease BamH I, EcoR I were ligaed directly to the expression vector-pGEX-4T-2 which were digested with BamH I, EcoR I. The recombinant vectors were respectively transformed into Escherichia coli BL21. By colony PCR identification, recombinant vector digested identification and sequencing analysis, it showed that the recombinant expression vectors were successfully constructed. Next the two recombinant vectors were induced to express the fusion proteins at the different temperatures, different time points, different concentrations of IPTG. After expressed, the recombinant fusion proteins were checked by SDS-PAGE and Western-blotting analysis. The result showed that the immunized sera from guinea pig immunized with Brucella melitensis could specifically recognize the recombinant fusion proteins. The recombinant fusion proteins purified by electrodialysis method were coated as antigen for ELISA assay. Although the results didn't support the recombinant proteins as the possibility of Diagnosis protein, this study may provide a solid foundation for the further study on the function of proteins and brucellosis diagnostic antigens.

关 键 词： 布鲁氏菌 原核表达 免疫原性 布鲁氏菌病 蛋白 蛋白

分 类 号： [S855.12]

领　　域： [农业科学] [农业科学] [农业科学]