Expression Of Pig Interferon A And Construction Of Recombinant Bacmid

The lymphocytes were extracted from venous blood which came from healthy certain species nourished in the field of Henan province. The lymphocytes were cultured and the interferon alpha was induced by ConA.Their RNA were extracted and were used to amplificate porcine Interferon-alpha gene by RT-PCR with a pair of primer which was designed according to existent gene sequence of porcine Interferon-alpha. The specific fragments were connected to pGEM-T Easy vectors,then they were transformed into competence cells of JM109. The doubtful strains were idenfied by blue-white spot screening, plasmid PCR and enzyme digestion.The suspected strains were sent to shanghai sangon biological engineering technology and services co ltd for sequencing. And the sequencing results display:our obtained gene contained 498bp which encoded 166 amino.The results of homology analysis with DNAstar revealed their nucleotide homology are all over 97.0% .The pGEM-T-pIFNαwere digested by EcoRⅠand HindⅢ,and IFN-αgene was inserted into pET32a of procaryon expression vector.The recombinant expression vectors were idenfied by PCR and enzyme digestion and sequencing . The fusion protein were expressed after the recombinant expression vectors were transformed in BL21 of E.coli. There is a strip occurring at the location labled 40.2 kD when expression products were identifited by SDS-PAGE.The strip is specific,because it's molecular weight is just the same as the tag in the pET32a of procaryon expression vector and the protein deduced according to porcine IFN-αgene .Optimizeing to the concentration of IPTG and the time of IPTG induceing, the produce of IFN-αincreased .The proteins were purityed by NTA and had only one strip by SDS-PAGE. The machophages of lung of pig were washed and cultured in the 24 plate. The proteins of IFNαwere incubating on the machophages for 24 hours,and prrsv was enchaged. When we watched the result of Indirect lmmunofluorescent Antibody Technique to test prrsv, the machophages enchanged by prrsv had flavo-green fluorescent,the machophages protected by IFN-αhad no fluorescent,the machophages had no fluorescent . therefore ,the grown of the prrsv was exhibited.The amplificateed gene of IFN-αwas connected to pFastBac Dual,then the recombinant pFastBac Dual was succeessfully constructed by analysising to the result of PCR and enzyme digestion and sequencing . The recombinant pFastBac Dual was transformed into DH10 Bac ,and DH10 Bac was growned and screened on the LB with X-gal and IPTG and threee antibiotics.The recombinant Bacmid was succeessfully constructed when the plasmid was amplificateed by PCR and There is a strip occurring at the location labled 3000bp.