重组hcmvIL-10的表达纯化及免疫抑制活性研究

摘要

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 PCR ARRAY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-e2X-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 recombi — nant 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 Th 1-Th2-Th3 PCR ARRAY. Results:Both SDS-PAGE and Western blot analysis indicated that the recombinant hcmvIL 40 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-a 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 -a in other treated groups were obviously lower than the control group rn(P <0.01). Different concentrations of hcmvIL-10 and hIL-10 were added in THP-1 cells for 48 h respectively. The relative expression level of HLA4DR mRNA in THP4 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 (P <0.01). 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 (P <0.01). Totally ,84 differentiation related genes were detected by the functional classified Th 1 -Th2-Th3 PCR ARRAY. There were 8 differentially expressed genes in hcmvIL 40 treated group, in which IL42RB2,NFATC2,SOCS2,TYK2,GFI1 and CEBPB were up -regulation, and CD80,CTLA4 were down-regulation. There were 5 differentially expressed genes in hIL 40 treated group, in which S0CS2 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 THP4 cells. This function is similar to that of hIL40. Recombinant hcmvIL40 could influence differentiation-related gene expression of THP4 cells. Differentially expressed genes of it are different with that of hIL 40.