Study On Clone Prokaryotic Expression And Biological Activity Of Ictalurus Punctatus (Channel Catfish) Interferon Alpha (IFN-α) Gene

The cDNA sequence which encoding interferon-α(IFN-α) for channel catfish was cloned from the fresh channel catfish's peripheral blood lymphocytotes by means of using the specific primers according to the published sequence in GeneBank(AY267538).The cloned cDNA fragment was inserted into the pMD18-T plasmid,then identified the fragment with enzyme digestion and sequence identification.The results showed that the cloned cDNA fragment is channel catfish's IFN-α.The length of the cloned cDNA fragment was 492bp with an ORF,and deduced it encoding 164 amino acids.After nucleotide blasting,it displayed that there were high homology of 96.5%and the identity of the deduced amino acids was 92.6%between the imported channel catfish's IFN-αsequence and the published channel catfish's IFN-αsequence.The GenBank accession number of IFN-αgene sequence of channel catfish is EU783919.In order to construct the recombinant expression vector pET-32(a)+-CC- IFN-α,the cloned IFN-αcDNA fragment was inserted between the restriction sites EcoR I and Xho I of Prokaryotic Expression plasmids pET-32(a)+ vector.Transformed the correct recombinant plasmid which had been identified by enzyme digestion into the E.coli BL21.The recombinant plasmid was induced to express IFN-αprotein with different temperatures and times.The outcomes of the SDS-PAGE and Western Blotting showed that the expression products had the molecular weight about 39KD.It also showed the expression products reached their peaks in 5 hours with the tempreture of 30℃,and the expression quantity of fusion protein was 48.4%in the total thallin protein.At the same time,the E.coli BL21 without inducing express no IFN-αprotein at all.The non-inclusion bodies which express IFN-αwere washed,then test their biological activity by Cytopathic Effect(CPE).The results indicated that the recombinant IFN-αprotein have the ability to protect the CCO cells against the VSV,that means the protein do have biological activity.The Chinese channel catfish's IFN-αwas cloned,then the IFN-αproteins were obtained from prokaryotic expression in this experiment.And we introduced international standered to test channel catfish's IFN for the first time.The expressed protein can supply material base for further research in channel catfish's IFN-αand can also take precaution against and control channel catfish's virus diseases.Meanwhile,this can be a guid to future domestic aquacultural drugs's productiong,making them no poluution,no remains and safe.