委内瑞拉马脑炎病毒E2蛋白的原核表达、纯化及多克隆抗体的制备

发布时间：2018-05-21 15:39

  本文选题：委内瑞拉马脑炎病毒 + 原核表达　； 参考：《中国兽医学报》2017年11期

【摘要】：原核表达并纯化委内瑞拉马脑炎病毒(Venezuelan equine encephalitis virus,VEEV)E2蛋白胞外区,并以此为抗原免疫动物制备多克隆抗体。利用PCR扩增VEEV E2蛋白胞外区基因并将其插入到原核表达载体pET-30a(+),构建重组质粒pET-30a(+)-VEEV-E2,转化至大肠杆菌BL21(DE3),IPTG诱导目的蛋白表达。优化诱导条件并对目的蛋白进行亲和纯化,用纯化后的蛋白免疫BALB/c小鼠制备多克隆抗体,通过间接免疫荧光方法检测抗体的结合能力。通过PCR扩增出约为1 023bp的VEEV E2基因胞外区,成功构建重组表达质粒pET-30a(+)-VEEV-E2,在37℃,0.6mmol/L IPTG诱导3h条件下目的蛋白表达量最高且主要以包涵体形式存在;将纯化的目的蛋白免疫BALB/C小鼠成功制备了抗VEEV E2蛋白胞外区鼠源多克隆抗体,经间接免疫荧光鉴定制备的抗体能够特异性识别真核表达的VEEV E2蛋白。VEEV E2基因胞外区在大肠杆菌中获得稳定表达,且表达的目的蛋白具有良好的免疫原性,为VEEV快速诊断方法及新型疫苗的研究奠定了基础。
[Abstract]:The extracellular domain of Venezuela equine encephalitis virus (Venezuelan equine encephalitis virus) VEEV E 2 protein was expressed and purified in prokaryotic cells and used as antigen to immunize animals to prepare polyclonal antibodies. The extracellular domain gene of VEEV E2 protein was amplified by PCR and inserted into the prokaryotic expression vector pET-30a.Recombinant plasmid pET-30a (-VEEV-E2) was constructed and transformed into E. coli BL21DE3 / IPTG to induce the expression of the target protein. The BALB/c mice were immunized with the purified protein to prepare polyclonal antibody and the binding ability of the antibody was detected by indirect immunofluorescence method. The extracellular region of VEEV E2 gene was amplified by PCR, and the recombinant expression plasmid pET-30a (-VEEV-E2) was successfully constructed. The expression of the target protein was the highest at 37 鈩，

本文编号：1919794