摘要
目的原核体系诱导表达并纯化人类白细胞抗原-G1(human leukocyte antigen-G,HLA-G1)蛋白,研究HLA-G1分子对人B淋巴细胞内白细胞介素-10(interleukin-10,IL-10)表达水平的影响,进而探究HLA-G1在肾移植受者中B细胞介导的免疫耐受机制。方法通过原核表达体系,构建表达并纯化HLA-G1蛋白,经过蛋白免疫印迹试验(Western Blot)鉴定后,与人外周血单核细胞(peripheral blood mononuclear cell,PBMC)共培养,利用流式细胞仪检测B淋巴细胞内IL-10的表达情况。结果十二烷基硫酸钠-聚丙烯酰氨凝胶电泳(sodium dodecyl sulfate-polyacrylamide gel electropheresis,SDS-PAGE)、Western Blot结果表明,纯化后的蛋白分子量大小约为38.2 kDa,符合HLA-G1蛋白的分子量;人PBMC与HLA-G1蛋白共培养后,随着HLA-G1蛋白浓度的升高(浓度梯度:0、50、100和200 ng/ml),B细胞内IL-10的表达水平明显增加,分别为0.1%、0.36%、0.57%、0.73%。结论通过原核表达系统获得HLA-G1蛋白,纯度达95%以上,HLA-G1蛋白与人PBMC共培养后,可诱导B淋巴细胞表达IL-10,并具有浓度依赖性,提示其对肾移植免疫耐受的建立具有重要作用。
Objective Prokaryotic system was used to induce expression and purification of human leukocyto antigen-G1 （HLA-G1） protein and to investigate the effect of HLA-G1 on expression of interleukin-10 （IL-10） in hunman B lymphocytes, to study the B cell-mediated immune tolerance mechanism of HLA-G1 in kidney transplantation recipients. Methods We used the prokaryotic expression system to express the HLA-G1 protein and purified it. Human peripheral blood mononuclear ceils （PBMCs） were co-cultured with HLA-G1 after Western Blot. Then we detected the expression of IL-10 in B lymphocytes by flow cytometry. Results Results of sodium dodeeyl sulfate-polyaerylamide gel （SDS-PAGE） and Western Blot showed that the molecular weight of the purified protein was about 38.2 kDa which was in accord with that of HLA-G1 protein. The expression of IL-10 in B lymphocytes after co-culture with HLA-G1 significantly increased, and the rate of IL-10 on B lymphocytes were 0.1%, 0.36%, 0.57% and 0.73%, respectively, dependent on the concentration of HLA-G1 （concentration gradient： 0, 50, 100 and 200 ng/ml）. Conclusion We used the prokaryotic expression system to express HLA-G1 protein and purified it. The purity was above 95%. HLA-G1 protein induced the expression of IL-10 in B lymphocytes in a concentration dependent manner after co-culture with human PBMC, which suggested that HLA-G1 might play an important role in the establishment of immune tolerance in kidney transplantation.
出处
《实用器官移植电子杂志》
2017年第2期112-115,共4页
Practical Journal of Organ Transplantation(Electronic Version)
基金
国家自然科学基金面上项目(81571555)
