激活鼠FcγRⅡb对天然免疫因子产生的影响

Effect of Activated Murine FcγRIIb on the Production of Innate Immune Factors

目的:了解FcγRIIb对小鼠巨噬细胞天然免疫及抗病毒免疫作用的影响。方法:构建鼠源FcγRIIb原核表达质粒pET28a-FcγRIIb,将其转化至大肠杆菌BL21感受态细胞中进行诱导表达,采用镍柱亲和层析法对重组蛋白进行纯化。以纯化后的重组蛋白作为抗原,免疫兔制备多克隆抗体,采用间接ELISA法检测血清效价,利用Western blotting技术鉴定抗体特异性,高免血清经饱和硫酸铵溶液粗纯和ProteinG Sapharose精纯获得兔抗鼠FcγRIIb多克隆抗体。以该抗体激活小鼠腹腔巨噬细胞表面的FcγRIIb,利用荧光定量PCR技术检测MHV感染上述细胞后相关细胞因子的mRNA转录水平。结果:单独激活FcγRIIb 12~48 h时巨噬细胞中TLR3、IRF3、IFN-α、IFN-β、IFN-γ、TNF-α和IL-1β的mRNA转录水平上调,免疫抑制因子TGF-β的mRNA转录水平下调。此外,在MHV感染小鼠腹腔巨噬细胞12~48 h时,与未激活组相比,FcγRIIb激活后可上调细胞中TLR3、IRF3、IFN-α、IFN-β、IFN-γ、TNF-α和IL-1β的mRNA转录水平,下调TGF-β的mRNA转录水平,且其病毒拷贝数及病毒滴度低于未激活组。结论:研究证明FcγRIIb的活化能促进小鼠腹腔巨噬细胞天然免疫因子产生,同时具有一定抗病毒作用,为进一步探究FcγRIIb在MHV感染过程中的作用奠定了基础。

Objective:To study the effect of FcγRIIb on innate and antiviral immunity of macrophages in mice.Methods:The recombinant vector pET28a-FcγRIIb was constructed and transformed into BL21 competent cells for expression induction.The recombinant protein was purified by Nickel affinity chromatography.The purified protein was then used as an antigen to immunize rabbits for polyclonal antibody production.The serum titers were detected by indirect ELISA,and the specificity was determined by Western blotting.Then,the rabbit polyclonal antibody against mouse FcγRIIb was obtained with saturated ammonium sulfate solution and protein G sapharose.Thus,the antibody was used to activate the FcγRIIb on mouse peritoneal macrophages,and the mRNA transcript levels of cytokines in MHV-infected cells were detected by qPCR.Results:The activated FcγRIIb could up-regulate the mRNA transcript levels of TLR3,IRF3,IFN-α,IFN-β,IFN-γ,TNF-αand IL-1βin macrophages at 12-48 hours,and down-regulate the transcript level of TGF-βmRNA.In addition,after MHV infection with FcγRIIb activated cells for 12-48 hours,the mRNA transcript levels of TLR3,IRF3,IFN-α,IFN-β,IFN-γ,TNF-αand IL-1βwere up-regulated and the mRNA transcription levels of TGF-βwas down-regulated in the FcγRIIb activated group,compared with the non-activated group.In addition,the viral copy numbers and viral titers of MHV in the FcγRIIb activated group were much lower than those in the non-activated group.Conclusion:This study demonstrates that activation of murine FcγRIIb can effectively enhance the production of the innate immunity related cytokines in macrophages and inhibit virus replication.It provides a basis for further investigation of the role of FcγRIIb in MHV infection.