Expression, Purification Of Porcine Interferon-gamma And Its Biological Activity Study

Interferon-gamma (IFN-γ) secreted by T lymphocytes and natural killer (NK) cells is an anti-viral and anti-tumor proliferation and immunoregulatory cytokine. It has powerful regulatory effects on immune system, and it is a necessary element in playing immune functions, scavenging pathogens in vivo.Therefore, IFN-γplays more important roles in diagnosis, treatment of clinical diseases and in detecting immune efficacy of vaccines. It is one of the research focuses in the modern molecular biology , immunology and clinical medicine. In our research, a pair of primers were designed according to the sequence of pIFN-γwithout the sequence encoding signal peptide, then the gene was cloned into prokaryotic expression vector pET-30a and the recombinant expression vector pET-30a-pIFN-γwas constructed. The recombinant plasmid was transformed into E.coli BL21 (DE3) followd by identification by PCR, double enzyme digestiion and sequencing, and induced by IPTG. Culture condition of expression system was optimized, and the result indicated that the recombinant pIFN-γwas expressed correctly and the optimal expression was induced by 0.1mmol/L IPTG in LB mediumt at 28℃. Besides, we also found that induced expression of the recombinant pIFN-γwas done under any working condition, inclusion body was more than soluble protein, and the soluble protein increased evidently after reducing temperature of induction. The soluble protein was purified through Nickel-affinity chromatography and further purified by Sephadex-G100. After washed by DOC, the inclusion bodies dissolved in SKL and subsequently renatured by dialysis. The purified protein was analyzed by SDS-PAGE and Western blot and the results indicated that the highly purified pET-30a-IFN-γwas harvested. Study on a serial of biological activities of purified recombinant pIFN-γwas carried out:(1)antiviral effect of the purified product was tested by cytopathogenic effect inhibition assay. The test result indicated that the purified product had a higher activity of interfering virus replication. The activity of IFN-γagainst PRV in PK15 cell line was about 2.0×103 U·mg-1, and the activity against standard O type FMDV was about 2.56×105U·mg-1;(2)the passage of PK15 cells was induced by purified recombinant pIFN-γ. The results showed that high concentration pIFN-γcould induce PK15 cells apoptosis;(3)abdominal macrophage cells and PK15 cells were irritated by different dose irradiation of rpIFN-γ, followed by the anti-toxoplasma gondii challege infection, increasing dose of rpIFN-γ, the anti-toxoplasma gondii effect of macrophage was augmented. rpIFN-γdid not affect the invading of toxoplasma gondii tachyzoites to PK cells. This research is the basis for the development of pIFN-γ-related genetically modified bioproducts.