Expression And Bioactivity Of Yak Recombinant Interferon

IFN-αand IFN-βare produced by leucocyte and fibral cell respectively.They are the main members of interferon type I ( IFN-? ), involved in broad-spectrum antivirus and immunomodulatory activities.IFN-γis a dimeric glycoprotein predominantly secreted by T cells and NK cells, mitogen or antigen activated T cell, belongs to interferon type II ( IFN- II ). IFN-γplays a very important role in resistance to viruses, intracellular bacterial and parasitical infection, anti-tumor proliferation immunomodulatory activities. Therefore, IFN plays more important roles in diagnosis and treatment of diseases, detecting on immune efficacy of vaccine and soon. It is one of the research focuses in the modern molecular biology , immunology and clinical medicine.In this study, yak-IFN-γcDNA was amplified by Reverse Transcription Polymerase Chain Reaction (RT-PCR). Then the gene was sub cloned into pET-30a vector, and the recombinant plasmid named pET-30a-yakIFN-γwas constructed. Compared with the data from GenBank,the nucleotide homology gene was 99%。The result indicated that the recombinant yakIFN-γwas expressed correctly, and the optimal condition of expression was the induction with 0.5 mmol/L IPTG in LB medium at 28℃.Besides, we also found that inducing expression of the recombinant yak IFN-γwas conducted under any working condition, and the soluble target protein increased evidently after reducing temperature. The soluble expressed product was purified through nickel-affinity chromatography column.The protein in inclusion bodies was washed by DOC, then dissolved with SKL and subsequently renatured by dialysis, and the purifiel protein was analyzed by SDS-PAGE and Western-blotting.The results confirmed that the highly purified pET-IFN-γwas harvested.The yakIFN-? was cloned into the express plasmid vector pPIC9K and transformed into E.coli. Positive recombinant pPIC9K-yakIFN-? were named selected, sequenced .They were linearized by SalⅠ,then transfored into Pichia Pastoris GS115 by electroporation. The expression vector pPIC9K-yakIFN-? integrated into GS115 via homologous recombination between the transformed DNA and the P.Pastoris the genome. Multicopy recombinant strains were screened by G418. PCR showed that the interest yakIFN-? gene was integrated within the genome, and the multicopy recombinants were named GS115/pPIC9K-yakIFN-?. The expression of recombinant fusion protein was induced by methanol, then was tested by SDS-PAGE . Experimental results showed the fusion protein of yakIFN-? secreted by GS115 reached at expression peak of 180mg/L at 72h.Researches on biological activities purified of yakIFN-γand yakIFN-I showed that the recombined yakIFN was capable of inducing lymphcyte proliferation. yakIFN-γhad more powerful ability than yakIFN-I to induce lymphcyte proliferation. Results of yakIFN-γand yakIFN-I anti-Pseudorabies virus ( PRV ) showed that yakIFN-γhad more powerful ability than yakIFN-I to resist virus. Combination of yakIFN-α-A and yakIFN-βwas twice more active than the individual, and was four times active than yakIFN-γ. Hence, expressed yakIFN-γand yakIFN-Ⅰhad powerful and broad biological activities. Our research provides basic theory for development of biological products of yakIFN gene for the therapy and prevention of related diseases.