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口疮病毒GIF蛋白的原核表达及对小鼠免疫细胞的影响

Prokaryotic expression of GIF protein of orf virus and its effects on mouse immune cells
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摘要 探究口疮病毒GIF蛋白对小鼠骨髓源树突状细胞成熟及Naive CD4^+T细胞极化作用的影响。原核表达口疮病毒GIF蛋白,用纯化后的蛋白刺激小鼠树突状细胞,利用流式细胞术检测细胞表面分子的表达情况,ELISA方法检测细胞培养上清中细胞因子IL-12P40、TNF-α、IL-10、IL-1β的分泌情况。将Naive CD4^+T细胞与经不同质量浓度的GIF蛋白刺激后的树突状细胞共培养,用ELISA检测该树突状细胞对Naive CD4^+T细胞的免疫刺激能力,并进一步检测培养上清中IFN-γ和IL-5的分泌水平。结果显示,成功克隆了ORFV117基因,表达了羊口疮病毒GIF蛋白。经不同质量浓度蛋白刺激后,树突状细胞的表面共刺激分子CD86、CD40的表达水平均明显升高,其中5μg/mL蛋白刺激后,MHCⅡ表达水平显著升高,IL-12P40、TNF-α、IL-1β、IL-10释放水平显著升高。经不同质量浓度的GIF重组蛋白刺激后的树突状细胞对Naive CD4^+T细胞的刺激能力增强,淋巴细胞共培养上清中IFN-γ分泌量显著增加。这表明一定质量浓度的口疮病毒GIF蛋白能促进小鼠骨髓源树突状细胞的成熟,能够激活抗原递呈作用,并且具有刺激T细胞分化的能力。 This study was designed to investigate the effect of orf virus(ORFV)GIF protein on the mature and fuction of mouse bone marrow-derived dendritic cells(DC).The ORFV GIF protein was expressed in prokaryotic and purified,and used to stimulate the DC.Then flow cytometry was used to detect the expression level of surface costimulatory molecules on the dendritic cells,while ELISA was used to test the secretion levels of IL-12 P40,TNF-α,IL-10 and IL-1β in culture supernatant.In addition,Naive CD4^+ T cell was co-cultured with the dendritic cells stimulated with different mass concentrations of GIF protein.And the immunostimulatory capacity of DCs and the secretion level of IFN-γ and IL-5 in the supernatant were tested by ELISA.The results showed that the ORFV117 gene was cloned and protein was expressed.After stimulation with different concentrations of GIF protein,the expression levels of co-stimulatory molecules CD86 and CD40 in dendritic cells increased significantly.However,the expression of MHCⅡ on DC was increased after stimulation with 5μg/mL ORFV GIF protein.Moreover, ELISA showed that the secretion of IL-12 P40,TNF-α,IL-10 and IL-1β enhanced obviously after ORFV GIF protein stimulation.DC stimulated with different mass concentrations of GIF protein enhanced Naive CD4^+ T stimulation, and IFN-γ secretion increased significantly in lymphocyte co-culture supernatant.In conclusion,a certain mass concentration of ORFV GIF protein can promote the maturation of mouse bone marrow-derived dendritic cells,activate antigen presentation,and has the ability to stimulate T cell differentiation.
作者 冯倩 吴锦艳 李玲霞 杜国玉 尚佑军 刘湘涛 刘永生 FENG Qian;WU Jin-yan;LI Ling-xia;DU Guo-yu;SHANG You-jun;LIU Xiang-tao;LIU Yong-sheng(State Key Laboratory of Veterinary Etiological Biology/Key Laboratory of A nimal Virology, Ministry of A griculture/Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences , Lanzhou 730046, China)
出处 《中国兽医科学》 CAS CSCD 北大核心 2019年第10期1222-1232,共11页 Chinese Veterinary Science
基金 国家重点研发计划项目(2017YFD0500903) 国家现代肉羊产业技术体系项目(CARS-38-04B) 内蒙古科技专项
关键词 口疮病毒 GIF蛋白 纯化 树突状细胞 orf virus GIF protein purification dendritic cells
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