Clone And Prokaryotic Expression Of Porcine Interferon-α1 Gene

In our country, we have the largest Pig-farming industry in the whole world. At present, there are many infections diseases that cumber the thriving and the updating of this industry, and cause huge economic losses. Interferons are cytokines that functions as antiviral factors and immune regulators, which are naturally produced by the immune system of mammal animals immediately following viral Infection or viral vaccination. Interferon alpha is famous as the potentant antiviral factors. It is of great significance on control porcine virus disease to obtain active recombinant porcineα-interferon by genetic engineering technology.Accordding to the complete gene sequence of porcineα1 interferon (poIFN-α1) published in GenBank(registed number: EU364896), two pairs primers (P1-P2 and eP1-eP2) were designed and synthesized. Primers were used for amplification poIFN-α1 gene.The gene of porcine interferon-alpha was amplified by RT-PCR from the total RNA in the lymphocytes stimulated wih 15μg / mL of lipopolysaccharide from the peripheral blood of pig. The gene fragment was cloned into the pGEM-T vector and recombinant plasmid pGEM-T-poIFN-α1 was constructed. It showed that the cloned sequence includes the purpose of IFN-α1 gene of swine and pGEM-T-poIFNα1 plasmid had been successfully constructed.PoIFN-α1 with restriction Enzyme digestion site was amplified by polymerase chain reaction (PCR) from cloning vector pGEM-T-poIFN-α1, subcloned into pGEX-6P-1 vector, and recombinant expression pGEX-6P-poIFNα1 plasmid was constructed. Moreover, the recombinant plasmids were transformed into the E.Coli DH5α. The positive clones were selected and incubated. And analyze the best expression protein conditions. Sequencing result showed that cloned Porcine IFN-αlong 546 bp, encoding 181 amino acids with a signal peptide of 23 amino acids, without glycosylation sites. The protein expression magnitude is most at 9 h, 37℃, IPTG concentration 1.0 mmol/L. SDS-PAGE and Western-blot analysis showed that a 46 kDa fusion proteinwas expressed in the form of inclusion body. This study paved the way for further study of its antiviral activity and utilization.