Generation And Identification Of Stable Cell Line Expressing Bovine Interferon-γ

Interferon-gamma is a kind of cytokine mainly produced by CD4+Th1cells、CD8+CTL cells and NK cells in response to antigenic or mitogenic stimulus, which has anti-viral activity and anti-tumor activity, and plays many critical roles in the modulation of immune responses. The IFN-y can be used as adjuvant to improve immune effects. The level of IFN-y in body can reflect the state of cellular immunity. The special monoclonal antibody against BoIFN-y can be developed to establish some immunological method, which can provide useful diagnostic reagent for bovine diseases, such as tuberculosis. In this study, a stable cell line expressing BoIFN-y was generated, and then a sandwish ELISA was established based on specific monoclonal antibodies for the detection of different BoIFN-y products.Bovine interferon-y (BoIFN-y) gene was amplified from plasmid pVAX1-preBoIFN-y, and then cloned into pCR2.1vector. The identified recombinant plasmid pCR2.1-preBoIFN-y was digested with Kpn I and Xba I to obtain BoIFN-y fragment, and then inserted into pcDNA3.1-FLAG to construct recombinant expression plasmid pcDNA3.1-preBoIFN-y-FLAG. The BoIFN-y-FLAG fragment was obtained by digesting plasmid pcDNA3.1-preBoIFN-y-FLAG with Xba I and Apa I, and was inserted into pcDNA5/FRT to construct recombinant expression plasmid pcDNA5/FRT-preBoIFN-y-FLAG.COS-1cells were transfected with recombinant expression plasmid pcDNA3.1-preBoIFN-y-FLAG,48h later after transfection, transient expression of BoIFN-y was detected by indirect immunofluorescent assay, the results indicated that the eukaryotic expression plasmid was constructed correctly and expressed successfully. Flp-In293cells were cotransfected with Flp recombinase expression plasmid pOG44and pcDNA5/FRT-preBoIFN-y-FLAG using lipofectamine reagent,48h later after transfection, the cells were cultured with selective medium containing120μg/ml Hygromycin B,3-4weeks later the Hygromycin-resistant foci were formed on dishes. Foci were then picked, expanded and subcloned. The positive cell line named B1was identified by ELISA, FACS, Western-blot assay, the results confirmed that BoIFN-y was expressed successfully and stablely in this cells.The antiviral activities of His-BoIFN-y, GST-BoIFN-y, Bac-BoIFN-y and FLAG-BoIFN-y were up to8.389×107U/mg,6.554×105U/mg,4.069×104U/ml,1.024×104U/ml and1.28×103U/ml respectively, which were analyzed in MDBK/VSV system.Antiviral assay is a useful biological method earliest established to detect the activity of interferon. At present, all the bioassays of interferon have the defects of time-consuming, trival, low specificity and low sensitivity, which need to be improved urgently. In this study, a sandwish ELISA was established using monoclonal antibodies5G4and3E6against BoIFN-γ, which can detect the activity of all kinds of recombinant BoIFN-y and provide a useful method for the clinical practice and research of BoIFN-y. The method can be used to detect the antiviral activities of recombinant bovine interferon-gamma in quantity, which provide a good foundation for the further study of BoIFN-y.