MERS-CoV S1蛋白的表达及鉴定

Expression,purification and identification of MERS-CoV S1 protein

目的真核表达具有天然构象的中东呼吸综合征冠状病毒(MERS-CoV)S1蛋白,并进行免疫反应性鉴定。方法以pcDNA3.1-S重组质粒为模板,PCR扩增S1基因,连接至pFastBac1质粒,构建重组质粒pFB1-S1。将pFB1-S1转化至DH10Bac感受态中,构建重组杆粒BpFB1-S1,然后转染sf9细胞获得重组杆状病毒rpFB1-S1并连续传代。将第四代rpFB1-S1接种sf9细胞后悬浮培养96h,取上清,用PEG8000浓缩并进行免疫亲和层析纯化,采用SDS-PAGE和Western blot鉴定其分子质量及免疫反应性;另取感染rpFB1-S1 48h的sf9细胞进行IFA鉴定。结果 S1基因PCR扩增产物大小为2 212bp,与预期相符。重组质粒pFB1-S1经酶切和测序分析鉴定构建正确。重组杆粒BpFB1-S1经PCR鉴定构建正确。IFA试验可见感染rpFB1-S1的sf9细胞出现特异性绿色荧光,即有目的蛋白表达。SDSPAGE显示纯化的重组S1为单一100×103(Mr)蛋白条带,Western blot检测该蛋白能被鼠抗S-RBD蛋白单克隆抗体识别。结论成功表达了具有免疫反应性的MERS-CoV S1蛋白,为MERS-CoV快速诊断方法的建立及候选疫苗的构建奠定了基础。

Objectives To express the MERS-CoV S1 protein with natural conformations using a baculovirus/insect cell system and to determine its immunoreactivity. Methods The plasmid pcDNA3.1-S was used as a template to amplify the S1 gene using PCR.After double-enzyme digestion,the S1 gene and pFastBac1 plasmid were ligated to construct the recombinant plasmid pFB1-S1.The pFB1-S1 plasmid was transformed into DH10 Bac competent cells to construct the recombinant bacmid BpFB1-S1.The recombinant baculovirus rpFB1-S1 was rescued by transfecting the BpFB1-S1 bacmid into sf9 cells and was passaged in succession.A suspension of sf9 cells was cultured for 96 hafter infection with the fourth passage of rpFB1-S1.The supernatant was concentrated with PEG8000 and purified using immunoaffinity chromatography.The molecular mass of the purified protein was determined with SDS-PAGE and its immunoreactivity was determined with Western blotting.sf9 cells infected with rpFB1-S1 for 48 hwere identified using an IFA. Results The PCR product was 2 212 bp in length.The pFB1-S1 plasmid was verified as correct using restriction analysis and DNA sequencing.The BpFB1-S1 bacmid was verified using PCR.In an IFA,specific green fluorescence was observed in sf9 cells infected with rpFB1-S1,i.e.the protein of interest was expressed.SDS-PAGE indicated that the purified S1 protein was a single 100×10^3（Mr）protein band.Western blotting indicated that the purified protein was recognized by mouse anti-S-RBD monoclonal antibody. Conclusion Immunoreactive MERS-CoV S1 protein was successfully expressed.This has laid the foundation for the development of methods to rapidly diagnose MERS-CoV and to create potential vaccines.