猪流行性腹泻病毒S蛋白原核表达及其单克隆抗体制备

Prokaryotic Expression of Porcine Epidemic Diarrhea Virus S Protein and Preparation of Its Monoclonal Antibodies

本研究以猪流行性腹泻病毒(PEDV)全基因组为模板,利用RT-PCR方法扩增PEDV S基因的亲水区,将其克隆至pMAl-c2E原核表达载体,获得重组质粒pMAl-c2E-PEDV-S。经EcoRⅠ/SalⅠ双酶切测序鉴定后,转化BL21菌,经IPTG诱导,成功表达了约74 kDa的重组蛋白MBP-PEDV-S,亲和层析法纯化重组蛋白。将重组蛋白免疫3只健康的6~8周龄雌性BALB/c小鼠,将小鼠脾脏B淋巴细胞与SP2/0瘤细胞融合,采用间接ELISA方法筛选阳性杂交瘤细胞株,获得了4株能稳定分泌抗PEDV S蛋白抗体的杂交瘤细胞株1B11、1B10、1C8、3L3,其腹水效价都高于1∶100000。获得的4株单克隆抗体与PEDV经IFA和Western blot检测,结果显示均具有良好的反应性。而且3株单克隆抗体重链为IgG2a,1株重链为IgG2b;3株轻链为Kappa,1株轻链为Lambda。本研究表达了PEDV S蛋白并制备了单克隆抗体,为研究S蛋白的结构和功能提供了条件,为揭示PEDV致病机制奠定了基础。

The hydrophilic region of porcine epidemic diarrhea virus(PEDV)S gene was amplifi ed from the whole genome by RTPCR,cloned into pMAl-c2E prokaryotic expression vector.The resulting recombinant plasmid pMAl-c2E-PEDV-S was identifi ed by Eco RI/Sal I double enzyme digestion and sequencing and then transformed into BL21 competent cells for protein expression with IPTG induction.The recombinant protein of molecular mass at 74 kDa was further purifi ed by affi nity chromatography.Three healthy female BALB/c mice at age of 6-8 weeks were immunized with the recombinant protein.The spleen B lymphocytes of mice were fused with SP2/0 myeloma cells.The hybridoma cells were screened by indirect ELISA.Four hybridoma cell lines 1B11,1B10,1C8 and 3L3 were found to stably secret monoclonal antibodies(MAbs).The ascites of these monoclonal cell lines was prepared and their ELISA titers were higher than 1:100000.These MAbs also had good reactivity with PEDV in IFA and Western blot.Isotyping analysis found that 3 MAbs belonged to heavy chain IgG2a and one Mab to IgG2b.Additionally,3 MAbs had light chain Kappa and one MAb had light chain Lambda.The prokaryotic expression of PEDV S protein and preparation of MAbs provided tools for further study of the structure and function of S protein and laid a foundation for revealing the pathogenic mechanism of PEDV.