Molecular Cloning, Expression And Purification Of Recombinant Canine Interferon-β Using A Novel Prokaryotic Expression System

With the development of pet industry in China, more and more dogs are raised as companion animals, which has become a part of elder's life. However, viral diseases such as canine parvovinis disease, canine distemper and canine adenovirus infection remain to be the main threat for domestic dogs. So development of safe and efficient anti-viral preparations is needed. Interferon-β(IFN-β) is produced in vertebrate cells with antiviral and immune regulatory activities. E.coli-expressed recombinant canine interferons have been used to treat viral diseases, but the purification procedure is complex and thus the production cost is high. In this study we explore the feasibility of a novel prokaryotic expression system for expression of recombinant canine IFN-β(Ca IFN-β).1. Genomic DNA was extracted from dog's ear skin tissue and used as the DNA template for Ca IFN-βgene amplification b y PCR. The PCR product was cloned into pMD 18 T Simple Vector for sequencing. Sequence alghnment showed that the PCR-amplified Ca IFN-βgene was identical to the published sequence in GeneBank. 2. The Ca IFN-βgene without signal peptide coding sequence was subcloned into expression vector PET-MIE, which contained an Mxe intein and elastin-like polypeptide coding sequence. After induction at low temperature with IPTG, a fusion protein of expected size was detected in the cell lysate of the recombinant vector-transformed E.coli by SDS-PAGE.3. By using the property of temperature-sensitive reverse phase change of ELP, the fusion protin was purified by repeated centrifugations at different temperatures. By using autocleavage of intein, recombinant Ca IFN-βwas obtained after incubation at low temperature in a buffer containing DTT. SDS-PAGE showed that the released protein had an expected molecular weight of 18kDa.4. By using the VSV-MDCK cell system, a high antiviral effect of the recombinant CaIFN-βwas detected with an antiviral titer of 106 U/ml.T...