摘要
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PEDVS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EcoR Ⅰ/Sal Ⅰ双酶切测序鉴定后,转化BL21菌,经IPTG诱导,成功表达了约74kDa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∶100 000.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PEDVS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.
Abstract
The hydrophilic region of porcine epidemic diarrhea virus(PEDV)S gene was amplified from the whole genome by RT-PCR,cloned into pMAl-c2E prokaryotic expression vector.The resulting recombinant plasmid pMAl-c2E-PEDV-S was identified by EcoRⅠ/SalⅠ 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 purified by affinity 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∶100 000.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.
基金项目
江苏省基础研究计划(自然科学基金)(BK20210807)
江苏省高等学校重点学科建设项目()
国家科技期刊平台
NSTL
维普
万方数据