| Two pairs of specific primers was designed according to the relevant sequence of the nucleocapsid (N) gene and GP5-M gene of the porcine reproductive and respiratory syndrome virus (PRRSV) in Genebank,Primers contain restriction enzyme at the 5' end respectively.The nucleocapsid (N) gene and GP5-M gene of PRRSV was amplified with RT-PCR.The result of RT-PCR showed that the length of fragment of PCR was similar to what we expected.The nucleocapsid (N) gene and GP5-M gene was cloned into the prokaryotic expression vector pGEX-4T-1,obtains recombinant particle pGEX-4T-1-N, pGEX-4T-1-GP5.The recombinant plasmid pGEX-4T-1-N was transformed into competent cell E.coli BL31(DE3).which induced with IPTG and expressed,SDS-PAGE showed a unique band with apparent relative molecular mass of 39.866Da.After supersonic stave,which was identified by SDS-PAGE and the protein was soluble protein.which laid the foundation for the later protein's massive purification and the following experiment.The signal peptide sequence (SEC) contain restriction enzyme was synthsised by Shanghai Sangon Biological Engineering Co.,Ltd.The SEC sequence and plasmid pGEX-4T-1-GP5 were digested by two restriction enzymes BamHI and EcoRI,and it was ligased by T4 DNA ligase,the ligased sequence was transformed into competent cell TOP10, obtained recombinant plasmid pGEX-4T-1-GP5-SEC.One pair of specific primers was designed which introduces Kozak sequence according to the multi-clone set of the eukaryotic expression vector pSinRep5.The GP5-SEC sequence was amplified with PCR, the PCR fragment through enzyme was ligased into the eukaryotic expression vector pSinRep5.The recombinant plasmid was extracted,identified and sequenced.The result showed that GP5-SEC sequence was inserted correctly into the vector pSinRep5. |
