Construction and identification of the eukaryotic coexpression plasmid p-IRES-P1-3CD

Objective To amplify P1 and 3CD gene from patient infected with EV71 and construct the eukaryotic co-expression plasmid encoding P1 and 3CD.Methods The internal ribosomal entry site(IRES) from encephalomyocarditis virus(EMCV) was amplified and inserted into eukaryotic vector pcDNA3.0,named pc-IRES.Then the cDNA encoding P1 and 3CD were obtained by RT-PCR amplification from the fecal of patient infected with EV71 and cloned into pc-IRES vector.The constructed plasmid p-IRES-P1-3CD was confirmed by restriction enzymolysis and PCR.Results A 2.5 kb and 1.9 kb fragment were obtained from amplification,the sequences were confirmed to be EV71 P1 and 3CD gene by sequence analysis.PCR and double endonucleases results confirmed that the eukaryotic coexpression plasmid p-IRES-P1-3CD was successfully constructed.Conclusion The successful construction of eukaryotic expression plasmid p-IRES-P1-3CD establishes an experimental basis for virus like particle vaccine of EV71.