| Interferons(IFNs)are very important cytokines of human and other animals,which are generated by specific cells induced by inducers.IFNs are glycoprotein that possess antiviral activity,anti-tumor activity and immunoregulation function.IFNs include three types:I,II and III.Among them,IFN-γ belongs to type II interferon,and type II interferon consists of only IFN-gamma.JFNs are secreted by lymphocytes cells,including CD4+ Th1 cells,CD8+ cells and NK cells,when they are stimulated by specific or aspecific antigen.The native form of the IFNs is a N-glycosylated homodimer,and the IFNs possess certain species-specific.IFN-γ not only has anti-viral activity,but also participate in immune regulation and MHC(major histocompatibility complex)expression of the antigen,as well as controlling cells growth.Genetically engineered interferon not only bear similarity in biological activity to natural interferon,but also has many benefits such as high purity,high yield,good quality,stability and being suitable for large-scale production.More importantly,large-scale production of genetically engineered interferon could significantly reduce the cost of manufacturing enterprise and it could be wlidely used in livestock and poultry breeding industry.The aim of the present study is to develop the recombinant rabbit interferon gamma with technique of genetic engineering and study the antiviral activity and to prepare monoclonal antibodies of the recombinant protein.The study laid the foundation for engineering production and clinical application of the recombinant interferon.The DNA sequence of rabbit IFN-y gene was amplified by RT-PCR and obtained 504bp fragment from total RNA of rabbit peripheral blood lymphocytes which have been previously treated with concanavalin A(ConA).The IFN-y gene was then cloned into pET-28a(+)to generate the recombinant pET28a-IFN-γ plasmid,which was transformed into Escherichia coli BL21(DE3).The recombinant IFN-y protein was induced by IPTG and purified with HisTrap affinity chromatography.Finally,the protein was obtained with a size of 18KDa.The recombinant rabbit IFN-γ protein was abundantly expressed and renaturation to obtain active protein.The antiviral activity of the recombinant rabbit IFN-γ protein was detected using the micro-cytopathic-effect-inhibition method.The results are as follows:(1)The anti-VSV activity of the recombinant rabbit IFN-y protein was about 2.51×103U/mL measured using VERO cells,protein concentration was 0.556mg/mL,specific activity was 4.51×103U/mg.(2)The anti-VSV activity of the recombinant rabbit IFN-γ protein was about 3.98×103U/mL measured using MDCK cells,protein concentration was 0.556mg/mL,specific activity was 7.16×10 U/mg.The results suggested that the recombinant rabbit IFN-y protein was effective on resisting the toxicity of VSV to cells in vitro.Four hybridoma cell lines secreting monoclonal antibodies(McAbs)against IFN-y were prepared by fusing mouse myeloma cells(SP2/0)with spleen cells from BALB/c mice immunized with the purified recombinant protein.These McAbs were named as 3H8,4G4,4F10 and 6C12,belonging to IgG1-κ,IgG2b-κ,IgGl-κ,IgGl-κ,respectively.The ELISA titers of the McAbs were 10-4 to 10-5.The western blot experiments showed that all of the four mAbs had good specificities.The addition ELISA revealed that 3H8 and 4F10 identified one antigen epitope,and 4G4 and 6C12 identified another antigen epitope.The preparation of rabbit anti-IFN-y monoclonal antibodies provided an important tool for the research and detection of IFN-y.This research laid the foundation for the study on rabbit’s immune mechanisms and immune function. |
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