Co-expression Of Chicken Interferon α/γ In Insect Cells

Interferon (Interferon, IFN) is one of multifunctional cytokine, a member of highly reactive multifunctional glycoprotein. With the rapid development of molecular biology and recombinant DNA technology, it has been applied to human anti- tumor, anti-virus with genetic engineering technology to produce a large number of highly effective interferon. In this study, chicken interferonαandγgenes were cloned and then subcloned into the insect cell / baculovirus expression vector, its expression in the insect cells was studied. The main contents include:1. Primers were designed according to chicken interferonαandγgene sequences published in GenBank. The chicken interferonαandγcDNA were amplified by RT -PCR from total RNA extracted from spleen lymphocytes, Then the amplified cDNA was cloned into pGEM-T Easy vector. By identification of plasmid by PCR and enzyme digestion, positive recombinant strains were sequenced. DNA sequencing result showed that the sequence of chicken interferonαandγwere correct. Compare to the interferonαandγsequence of other species reported in NCBI, it was also proved to be chicken interferonαandγgene correctly.2. To express chicken interferonαandγin baculovirus expression vector, primers were designed according to chicken interferonαandγmature protein genes. Chicken interferonαandγwere subcloned into the baculovirus transfer vector of pFastBacDual under the control of pH and p10 promoters respectively. To achieve the chicken interferonαandγsecreted protein expression, the authentic signal sequences of chicken interferonαandγwere substituted with the honeybee melittin signal sequence. To facilitate the detection of recombinant protein and application of nickel affinity chromatography purification, 6×His tag was fused in the C terminal.3. Recombinant plasmid was transformed into DH10Bac which contains a shuttle vector of Bacmid and named rDH10-CG. After co-transfected the recombinant plasmid into insect cells, the 18-20kDa expressed protein of chicken interferonαandγwere detected by SDS-PAGE, mRNA level of expressed protein was certificated by real time PCR. The results showed that chicken interferonαandγgenes were successfully expressed in insect cells. In summary, in this experiment, the chicken interferonαandγwere cloned and expressed in the insect cell / baculovirus expression system. This study paved the way for the development of chicken interferonαandγprotein as a vaccine adjuvant to promote the vaccine immunity, and as a therapeutic agent in the treatment of viral diseases...