Gene Clonging, Prokaryotic Expression And Research Of Antiviral Activity Of Porcine Interferon-α

Pork Livers were collected of different varieties, including DanishLandrace, French Landrace, English Large-white and French Yorkshine, from certain species nourish field of Henan province. Their genome DNA were extracted separately and were used to amplificate porcine Interferon-alpha gene directly with primers which was designed according to existent gene sequence of porcine Interferon-alpha. Specific fragments were connected to pGEM-T Easy vectors, then introduced into competence cells of JM109. Screened of doubtful strains by idenfication of blue-white spot screening, plasmid PCR and enzyme digestion, suspected strains were send to Takara for sequencing. And sequencing results display we obtain Interferon-alpha gene of four strains.The results of homology analysis with DNAstar reveal their nucleotide homology are all over 97.2 % and their amino acid homology are all over 92.8 %.Amplifying the gene of IFN-α gene of crossbred growing and finishing pig and then cloning PoIFN-α gene into cloning vector of pGEX-4T-1, we obtain a recombinant plasmid named pGEM-T-IFN- α Z. After subcloned ,PoIFN-α gene was inserted into pGEX-4T-1 of eukaryotic expression vector.The recombinant expression vector can express fusion proteins of recombinant PoIFN-α in BL21 of E.coli. The fusion protein'N-end has GST. There is a strip occurring at the location labled 45.0 kDa when expression products were identifited by SDS-PAGE.The strip is specific,because it's molecular weight is just the same as the one of GST adding up the one of matural PoIFN-α .Using mouse anti porcine interferon alpha-1 as first antibody and goat anti mouse IgG(H & L) peroxidase conjugated affinity purified antibody as second antibody, the result of Western blotting proved that there is a specific stripe appearing on NC-Membran.Aanalysised with thin slice scan,the result manifest that the express products occupy 18.6 % in the whole thallus proteins. The expressed products exist at the form of inclusion body.After being degenerated and then renaturated,the activity of the inclusion bodies was detected by inhibiting the cytopathic effect. As a result, the activity of recombinant PoIFN-α against VSV at vero cell is 2.24×10~3U/mg.A pairs of new primers were resynthesised. PoIFN-α gene was subcloned from the recombinant cloning vector pGEM-T-IFN- α Z and then cloned to pET-43.1a-c(+) to get a prokaryotic expression plasmid pETIFN α which can successful express recombinant PoIFN-α.The expression products are fusion proteins.The fusion protein' N-end has a fusion tag. There is a strip occurring at the location labled 85.0 kDa when...