重组hcmvIL-10的表达纯化及免疫抑制活性研究

Study on the expression,purification and immunosuppression function of recombinant hcmvIL-10

目的:探讨重组人巨细胞病毒白细胞介素10(hcmvIL-10)对单核细胞的免疫抑制活性及分化相关基因表达的影响。方法:将重组的原核表达质粒pMal-c2X-hcmvIL-10和pMal-c2X-hIL-10转化受体菌E.coli BL21(DE3)进行诱导表达,采用Amylose-resin亲和层析纯化重组蛋白,并用葡聚糖凝胶(Sephadex G-75)进行二次纯化。纯化的重组蛋白hcmvIL-10和人白细胞介素10(hIL-10)分别与THP-1细胞作用后,采用酶联免疫吸附试验(ELISA)检测培养细胞上清液TNF-α水平;实时荧光定量PCR与流式细胞术分别检测THP-1细胞HLA-DR mRNA及HLA-DR分子的表达;采用功能分类Th1-Th2-Th3 PCRARRAY检测分化相关基因表达谱。结果:重组蛋白hcmvIL-10和hIL-10经SDS-PAGE及Western blot鉴定与目的相符;不同浓度(2、10和50 ng/ml)重组hcmvIL-10和hIL-10分别与经PHA诱导的THP-1细胞作用48小时后,培养上清液中TNF-α的浓度分别为:(3.8±0.96)pg/ml、(1.95±0.51)pg/ml、(1.6±0.45)pg/ml和(2.49±0.43)pg/ml、(1.77±0.38)pg/ml、(0.98±0.16)pg/ml,除2 ng/ml重组hcmvIL-10处理组外,其它处理组与对照组比较均明显降低(P<0.01);不同浓度重组hcmvIL-10和hIL-10分别与THP-1细胞作用48小时后,HLA-DR mRNA相对表达量分别为:0.86±0.025、0.76±0.023、0.75±0.017和0.73±0.017、0.61±0.017、0.55±0.015,HLA-DR分子的表达分别为:(11.5±0.44)%、(10.1±0.30)%、(9.1±0.38)%和(10.9±0.10)%、(9.7±0.50)%、(7.5±0.32)%,除2 ng/ml重组hcmvIL-10处理组外,其它处理组与对照组比较均明显降低(P<0.01);功能分类Th1-Th2-Th3 PCR ARRAY共检测84个分化相关基因,hcmvIL-10处理组共有8个基因差异表达,上调基因为IL-12RB2、NFATC2、SOCS2、TYK2、GFI1和CEBPB,下调基因为CD80和CTLA4;hIL-10处理组共有5个基因差异表达,上调基因为SOCS2,下调基因为CD80、CD28、CTLA4和IL-6。结论:重组蛋白hcmvIL-10可抑制THP-1细胞分泌TNF-α,下调THP-1细胞HLA-DR分子的表达,类似hIL-10的免疫抑制功能;重组蛋白hcmvIL-10可影响THP-1细胞相关基因的表达,其差异表达基因与hIL-10不同。

Objective：To investigate the effects of the recombinant human cytomegalovirus interleukin 10（hcmvIL-10） on immunosuppression function and differentiation-related gene expression of monocyte.Methods：The recombinant expression vector pMal-c2X-hcmvIL-10 and pMal-c2X-hIL-10 were transformed into E.coli strain BL21（DE3） and expressed.The recombined protein was purified by affinity chromatography using Amylose-resin,and further purified by Sephadex G-75.After stimulation by purified recombinant protein hcmvIL-10 and hIL-10 in THP-1 cell,the level of TNF-α was detected by enzyme-linked immunoabsorbent assay（ELISA）;The expression of HLA-DR mRNA was detected by real-time quantitative PCR.The expression of HLA-DR on the surface of THP-1 cells was detected by Flow cytometry.Differentiation-related genes were detected by the functional classified Th1-Th2-Th3 PCR ARRAY.Results：Both SDS-PAGE and Western blot analysis indicated that the recombinant hcmvIL-10 and hIL-10 were consistent with the purpose.Different concentrations of hcmvIL-10 and hIL-10（2,10 and 50 ng/ml） were added into THP-1 cells for 48 h respectively,which were stimulated by PHA.The concentrations of TNF-α in the cell supernatant were（3.8±0.96） pg/ml,（1.95±0.51） pg/ml,（1.6±0.45） pg/ml and（2.49±0.43） pg/ml,（1.77±0.38） pg/ml,（0.98±0.16） pg/ml respectively.Except the 2 ng/ml hcmvIL-10 treated group,the concentration of TNF-α in other treated groups were obviously lower than the control group（P0.01）.Different concentrations of hcmvIL-10 and hIL-10 were added in THP-1 cells for 48 h respectively.The relative expression level of HLA-DR mRNA in THP-1 cells were 0.86±0.025,0.76±0.023,0.75±0.017 and 0.73±0.017,0.61±0.017,0.55±0.015 respectively.The level of HIA-DR mRNA in treated groups were obviously lower than the control group（P0.01）.The relative expression level of HLA-DR on the surface of THP-1 cells were（11.5±0.44）%,（10.1±0.30）%,（9.1±0.38）% and（10.9±0.10）%,（9.7±0.50）%,（7.5±0.32）% respectively.Except the 2 ng/ml cmvIL-10 treated group,the level of HLA-DR in other treated groups were obviously lower than the control group（P0.01）.Totally,84 differentiation-related genes were detected by the functional classified Th1-Th2-Th3 PCR ARRAY.There were 8 differentially expressed genes in hcmvIL-10 treated group,in which IL-12RB2,NFATC2,SOCS2,TYK2,GFI1 and CEBPB were up-regulation,and CD80,CTLA4 were down-regulation.There were 5 differentially expressed genes in hIL-10 treated group,in which SOCS2 was up-regulation,and CD80,CD28,CTLA4 and IL-6 were down-regulation.Conclusion：The recombinant protein hcmvIL-10 can inhibit TNF-α secretion and reduce the expression of HLA-DR of THP-1 cells.This function is similar to that of hIL-10.Recombinant hcmvIL-10 could influence differentiation-related gene expression of THP-1 cells.Differentially expressed genes of it are different with that of hIL-10.