Cloning and protein expression of spike gene of porcine epidemic diarrhea virus isolated in Korea
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Porcine epidemic diarrhea (PED) is a coronavirus infection that has devastated piglets in many countries. The spike (S) protein of the porcine epidemic diarrhea virus (PEDV) plays an important role in inducing neutralizing antibodies during infection in pigs. In the present study, to establish a genetic resource for the development of recombinant DNA and subunit protein vaccines against PED, the S gene of a PEDV strain previously isolated in Korea was cloned. Furthermore, attempts were made to express the S gene in the eukaryotic expression system. S gene of about 4.2 kb (4,152 bases) in full length was cloned and featured having one open reading frame for amino acid production with initiation codon at 5’ end and stop codon at 3’ end. After recombination of the cloned S gene with pFastBacHT plasmid, a eukaryotic expression vector, it was constructed and named pFBspike. The recombined S gene in pFBspike was transposed into a bacmid at its expression cassette, by means of transformation of the pFBspike DNA into competent E. coli DH10Bac which harbors the bacmid and a helper plasmid supporting transposition of foreign gene into the bacmid. The recombinant bacmid was then transfected into the Spodoptera frugiperda (Sf) cells, which are eukaryotic, where expression of the S protein was confirmed. In a fluorescent antibody (FA) test using monoclonal antibodies against the PEDV S protein, the expressed S protein showed a positive reaction. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and radio-immunoprecipitation (RI) tests, the protein expressed from the recombinant S gene in bacmid featured a molecular weight of about 200 kDa, which suggests that the recombinant S gene produced the same protein as in natural PEDV. After further establishment of the methods for mass production and analysis for antigenicity of the protein, it would be feasible to develop genetically engineered vaccines from recombinant S gene of the PEDV.
Abstract
Porcine epidemic diarrhea (PED) is a coronavirus infection that has devastated piglets in many countries. The spike (S) protein of the porcine epidemic diarrhea virus (PEDV) plays an important role in inducing neutralizing antibodies during infection in pigs. In the present study, to establish a genetic resource for the development of recombinant DNA and subunit protein vaccines against PED, the S gene of a PEDV strain previously isolated in Korea was cloned. Furthermore, attempts were made to express the S gene in the eukaryotic expression system. S gene of about 4.2 kb (4,152 bases) in full length was cloned and featured having one open reading frame for amino acid production with initiation codon at 5’ end and stop codon at 3’ end. After recombination of the cloned S gene with pFastBacHT plasmid, a eukaryotic expression vector, it was constructed and named pFBspike. The recombined S gene in pFBspike was transposed into a bacmid at its expression cassette, by means of transformation of the pFBspike DNA into competent E. coli DH10Bac which harbors the bacmid and a helper plasmid supporting transposition of foreign gene into the bacmid. The recombinant bacmid was then transfected into the Spodoptera frugiperda (Sf) cells, which are eukaryotic, where expression of the S protein was confirmed. In a fluorescent antibody (FA) test using monoclonal antibodies against the PEDV S protein, the expressed S protein showed a positive reaction. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and radio-immunoprecipitation (RI) tests, the protein expressed from the recombinant S gene in bacmid featured a molecular weight of about 200 kDa, which suggests that the recombinant S gene produced the same protein as in natural PEDV. After further establishment of the methods for mass production and analysis for antigenicity of the protein, it would be feasible to develop genetically engineered vaccines from recombinant S gene of the PEDV.