Prokaryotic Expression of Porcine Epidemic Diarrhea Virus S Protein and Preparation of Its Monoclonal Antibodies
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.
国家科技期刊平台
NSTL
万方数据
维普
