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

Prokaryotic expression,purification and identification of the E2 gene from Venezuelan equine encephalitis virus

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。

To express and purify the E2 extracellular domain of Venezuelan equine encephalitis virus （VEEV）, produce the polyclonal antibody of mouse anti-VEEV E2 protein and identify the polyclonal antibody. Truncated E2 gene was amplified by PCR and inserted into prokaryotic ex- pression vector pET-30a（ ＋ ）. The constructed plasmid pgT-30a（ ＋ ）-VEEV-E2 was transformed into BL21（DE3） to express under induction of IPTG. The induction conditions were optimized to express a maximum of target protein. BABL/c mice were immunized with the purification target protein to produce polyclonal antibody. Antibody combining ability is detected by indirect immunofluorescence method. The E2 extracellular domain gene of VEEV was successfully amplified by PCR to yield a product 1 023 bp. PCR analysis and sequencing proved that the recombinant plasmid pET-30a（＋）-VEEV E2 was successfully constructed. The target protein was expressed in the form of inclusion body. The optimal conditions of protein expression were 37℃,with the final concentration of 0.6 mmol/L IPTG for 3h. BABL/c mice were immunized three times with the purified recombinant protein to obtain polyclonal antibody. The result of indirect immunofluorescence showed that the sera could specifically recognize the prokaryotically expressed E2 protein. The E2 extracellular domain gene of VEEV was stably expressed in E. coli. The recombinant protein E2 has expected immunogenicity,whieh may lay a foundation of establishment of a method for rapid diagnosis of VEEV and study on novel vaccines.