Abstract:

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 cells（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 dodecyl sulfate-polyacrylamide 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.

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。