裂谷热病毒eGn蛋白的制备及免疫原性验证

Preparation and immunogenicity verification of eGn protein of Rift Valley fever virus

通过PCR扩增裂谷热病毒(RVFV) Gn蛋白胞外区(eGn)基因,连入原核表达载体pET-30a(+),构建重组质粒pET-30a(+)-RVFV-eGn。将重组质粒转化至感受态BL21(DE3),IPTG诱导蛋白表达,经HisTrapTMFF蛋白纯化柱纯化目的蛋白。将目的蛋白免疫BALB/c小鼠,分离小鼠脾细胞与小鼠骨髓瘤细胞融合,筛选分泌特异性抗体的杂交瘤细胞株,制备单克隆抗体,免疫荧光鉴定制备的单克隆抗体特性。结果显示,PCR扩增出1 287 bp的目的基因;构建的重组质粒转化BL21细胞后可有效表达目的蛋白,确定最佳表达条件为30℃,0.8 mmol/L IPTG诱导5 h,目的蛋白以包涵体形式表达;纯化后目的蛋白纯度为94.136%;制备的单克隆抗体可特异性识别哺乳动物细胞表达的Gn蛋白,表明RVFV eGn蛋白具有良好的免疫原性。研究结果为裂谷热新型疫苗和快速诊断试剂的研制奠定基础。

The extracellular domain(eGn) gene of Rift Valley fever virus(RVFV) Gn protein was amplified by PCR and inserted into the prokaryotic expression vector pET-30 a(+) to construct the recombinant plasmid pET-30 a(+)-RVFV-eGn,which was transformed into competent BL21(DE3).Protein expression using IPTG was purified by HisTrapTM FF protein purification column for target protein.Immunized BALB/c mice using the purified protein,and the mouse spleen cells were isolated and fused with myeloma cells.The hybridoma cell lines secreting specific antibodies were screened to prepare monoclonal antibodies,and the characteristics of monoclonal antibodies were determined by immunofluorescence.The results showed that the 1 287 bp target gene was amplified by PCR,the constructed recombinant plasmid was successfully transformed into BL21 cells,and the target protein was effectively expressed.The optimal expression conditions were30℃,0.8 mmol/L IPTG induction for 5 h,and the target protein was in clusion form expression.The purity of the target protein after purification was 94.136%,and the prepared monoclonal antibody specifically recognized the Gn protein expressed by mammalian cells.It has been noted that the eGn protein of Rift Valley fever has good immunogenicity and laid a foundation for the development of new vaccines and rapid diagnostic reagents of Rift Valley fever.