羊布鲁杆菌脂蛋白OMP19单克隆抗体制备与鉴定  

Production and identification of monoclonal antibodies against Brucella melitensis U-lipoprotein OMP19

作　　者：廖卿宇 胡飞环[1] 吴静波[2] 罗佩芳[1] 王文敬[1] 黎诚耀[1] 

机构地区：[1]南方医科大学生物技术学院输血医学系,广州510515 [2]韶关学院英东生命科学学院,韶关512005

出　　处：《中国人兽共患病学报》2015年第10期899-902,913,共5页Chinese Journal of Zoonoses

基　　金：国家自然科学基金项目(31372443;31100657);广东省自然基金项目(2011B090400407)~~

摘　　要：目的制备与鉴定羊布鲁杆菌脂蛋白OMP19单克隆抗体,用于布菌感染免疫机制研究。方法将OMP19基因连接入pET-30a(+)表达载体中,构建pET-30a(+)/OMP19质粒,转化入大肠埃希氏菌BL21(E3),以不同浓度异丙基-β-D硫代半乳糖苷(IPTG)进行诱导表达,采用镍金属螯合亲和层析(NI-NTA)纯化;以布菌阳性血清检测重组蛋白免疫反应性,并利用杂交瘤技术制备单克隆抗体,以天然OMP19蛋白及布菌外膜蛋白提取物(NMP)对制备的单克隆抗体进行Western Blot及酶免疫染色试验(IEST)鉴定。结果表达了OMP19蛋白,分子量约19kDa,通过纯化纯度可达95%,Western Blot分析显示蛋白具有良好的抗原性,并制备了OMP19抗原23株鼠源性单克隆抗体,鉴定结果显示22(95.65%)株能与天然OMP19蛋白反应,18(78.26%)株能与NMP反应,其中IgG1(k)亚型占91.30%;4株能与羊种布鲁氏菌菌涂片反应。结论成功制备具有良好抗原性的重组OMP19蛋白,筛选出识别天然蛋白的单克隆抗体,已初步应用于布菌的检测,为OMP19抗原B细胞表位的筛选奠定基础。We produced and identified the monoclonal antibodies against Brucella rnelitensis U lipoprotein OMP19. A DNA fragment coding ompl9 of Brucella melitensis was amplified by PCR, and inserted into the vector of pET-30a（＋）, the result- ant recombinant plasmid, which we designated as pET-30a（＋）/omp19. We then transformed the plasmid into BL21 （DE3） competent cells for the expression of the OMP19 protein. After induction with different concentrations of IPTG, the colleted cells were analyzed by SDS-PAGE, and then OMP19 monoclonal antibodies were prepared through hybridoma technology. These mAbs were tested to reactivity to rOMP19 and nature membrance proteins （NMP） of Brucella melitensis by Western blot and lEST. We successfully constructed an expression vector of pET30a（＋）/omp19. An IPTG-induced expression of the OMP19 protein （19 kDa in molecular weight） was demonstrated by SDS-PAGE. The fusion protein existed in the form of solu- ble, and the OMP19 protein of high purity could be obtained by Ni-NTA. Western blot assay showed that the refolded protein could be recognized by the anti-serum against Brucella melitensis. Twenty-three mAbs to OMP19 was produced in which 91.30M were IgG1, twenty-two （95.65%） mAbs could recognize nature OMP19 protein, and eighteen （78.26%） mAbs could recognize NMP, four mAbs could react with Brucella melitensis. The protein maintained good immunogenicity and twenty- three mAbs were obtained, which we believe provides a good protein candidate for the immunological research.

关 键 词：布鲁杆菌 OMP19 蛋白表达 单克隆抗体 

分 类 号：R378.5[医药卫生—病原生物学]