Cloning And Expression Of Porcine IFN-γ Gene And Its Use As Vaccine Adjuvant

IFN- is a dimeric glycoprotein predominantly secreted by T cells and NK cells, mitogen or antigen activated T cell clone can also secret IFN-γ. It plays a very important role in resistance to viruses, intracellular bacterial and parasitical infections, anti-tumor proliferation and immunomodulatory activities. It has proved that IFN-y played a very important role in virus therapy and is predicted to be used as a new adjuvant in vaccine. In this study we co-stimulated pig peripheral lymphocytes cells with PHA and LPS, then extracted total RNA from the cells and used it as a template to amplify the IFN-γ gene using RT-PCR. The amplified fragment was subsequently ligated into the pGEM-T easy vector for sequencing. The results revealed that the sequence had 100% homology with the sequence of IFN-y gene obtained from GeneBank. We then designed primers according to the ORF sequence of the IFN-γ gene and used the pGEM-T-easy/pIFN-γ recombinant plasmid as template in the PCR reaction. After double digestion with EcoR1 and NotI, the amplified products and the pGEX-4T-1 vector were ligated together and transformed into E.coli cells, this construct was also sequenced to ensure fidelity. Expression of the recombinant plasmid was induced in E.coli using IPTG resulting in a high level of protein expression. The expressed products were analyzed by SDS-PAGE and Western-blot, showing that the 40 kDa IFN-γ GST fusion was expressed successfully. The fusion protein was also recognized by anti-human IFN-γ serum, with the expressed protein making up 30-50% of total E. coli proteins. We also inserted the IFN-y gene into the pcDNA3.1 vector and named pcDNA3.1/pIFN-γ, then used it as an adjuvant of Cysticercus cellulosae vaccine to immunize mice. Antibodies were subsequently detected, showing that the pcDNA3.1/pIFN-γ recombinant plasmid has a promising role as an adjuvant to Cysticercus cellulosae vaccine and much more better than aluminum and 206 adjuvant.