B细胞成熟抗原(BCMA)是B淋巴细胞刺激因子(BLyS)的受体之一.它的胞外区与人IgG1Fc的融合蛋白eBCMA-Fc,又称为诱饵受体,具有拮抗BLyS的活性.为了设计新的拮抗肽,基于BCMA和Fc的晶体结构,通过计算机图形学技术、分子模拟方法,建立了eBCMA...B细胞成熟抗原(BCMA)是B淋巴细胞刺激因子(BLyS)的受体之一.它的胞外区与人IgG1Fc的融合蛋白eBCMA-Fc,又称为诱饵受体,具有拮抗BLyS的活性.为了设计新的拮抗肽,基于BCMA和Fc的晶体结构,通过计算机图形学技术、分子模拟方法,建立了eBCMA-Fc融合蛋白的三维理论结构.利用均方根位移(root mean square distance,RMSD)对eBCMA-Fc融合蛋白与单体eBCMA、Fc构象差异进行分析.融合蛋白eBCMA-Fc中的eBCMA段与单体eBCMA的主链碳原子间RMSD值为0.036nm,Fc段与单体Fc的主链碳原子间RMSD值为0.064nm.结果表明,对比单体,融合蛋白eBCMA-Fc并未因eBCMA与Fc直接连接而发生构象的变化.分子对接方法显示,融合蛋白eBCMA-Fc中的BCMA与BLyS作用,而Fc扮演着稳定BCMA构象的支架作用.为进一步验证上述理论分析,构建eBCMA-Fc融合基因,并将载有eBCMA-Fc融合基因的原核表达质粒转化BL21(DE3)菌、在细菌中表达.目的蛋白经蛋白A亲和柱纯化大约为36kD,与理论预测值34kD相近.免疫印迹表明抗人IgG抗体能够识别eBCMA-Fc融合蛋白.ELISA证实,eBCMA-Fc融合蛋白能够结合BLyS.随着eBCMA-Fc融合蛋白增加,结合BLyS的融合蛋白也相应增加.而对照人IgG,即使在高浓度条件下,也不结合BLyS.此外,eBCMA-Fc融合蛋白能够抑制BLyS对B细胞肿瘤Daudi细胞的作用.这些研究为下一步设计和筛选BLyS拮抗肽提供了实验基础.展开更多
Objective： To measure the expression levels of BLyS and its receptors mRNA in peripheral blood ronnonuclear cells （PBMC） using real-time fluorescence quantitative polymerase chain reaction（RFQ-PCR） method and to ...Objective： To measure the expression levels of BLyS and its receptors mRNA in peripheral blood ronnonuclear cells （PBMC） using real-time fluorescence quantitative polymerase chain reaction（RFQ-PCR） method and to investigate the relationship between BLyS and its receptors mRNA expression and systemic lupus erythematosus （SLE）. Methods： Specific primers and TaqMan probe were designed, and RFQ-PCR was perfomed. According to the standard curve of plasmid DNA, the level of BLyS and its receptors mRNA expression in 23 patients with SLE and 23 healthy subjects were detemined. The ratio of the copy number of BLyS mRNA to that of β2-microgluobulin （β2M） mRNA and the ratio of the copy number of BLyS receptors mRNA to that of β2M mRNA were regarded as indicator for the levels of BLyS and BLyS mRNA expression. Results： The concentration of RFQ-PCR was in the range of 10 - 109 pg/ml, and the coefficient of variation values for both intra-experimental and inter-experimental reproducibility ranged from 2.40% to 10.12% and from 4,.26% to 12.29%, respectively. In 23 SLE patients, the level of BLyS and its receptors（BCMA, TACI, BAFF-R） mRNA were in the ranges of 1.27 ± 1.4,9, 0.64 ± 0.77, 0.83 ± 1.05 and 0.9±3 -1.37, respectively. The mean values were 1.38 ± 0.07, 0.70 ± 0.04, 0.91 ±0.06 and 1.15±0.12, respectively. In 23 healthy donors, the levels of BLyS and its receptors（BCMA, TACI, BAFF-R） mRNA were： 0.60±1.0, 0.55±0.80, 0.54±0.74 and 0.54± 0.77, respectively. The mean values were 0.83 ±0.13, 0.68±0.08, 0.65± 0.07 and 0.68 ± 0.06, respectively. Conclusion： This results suggest that BLyS, TACI and BAFF-R might be involved in the pathogenesis of SLE and the mRNA expression levels might be used as new markers for the diagnosis of SLE.展开更多
文摘B细胞成熟抗原(BCMA)是B淋巴细胞刺激因子(BLyS)的受体之一.它的胞外区与人IgG1Fc的融合蛋白eBCMA-Fc,又称为诱饵受体,具有拮抗BLyS的活性.为了设计新的拮抗肽,基于BCMA和Fc的晶体结构,通过计算机图形学技术、分子模拟方法,建立了eBCMA-Fc融合蛋白的三维理论结构.利用均方根位移(root mean square distance,RMSD)对eBCMA-Fc融合蛋白与单体eBCMA、Fc构象差异进行分析.融合蛋白eBCMA-Fc中的eBCMA段与单体eBCMA的主链碳原子间RMSD值为0.036nm,Fc段与单体Fc的主链碳原子间RMSD值为0.064nm.结果表明,对比单体,融合蛋白eBCMA-Fc并未因eBCMA与Fc直接连接而发生构象的变化.分子对接方法显示,融合蛋白eBCMA-Fc中的BCMA与BLyS作用,而Fc扮演着稳定BCMA构象的支架作用.为进一步验证上述理论分析,构建eBCMA-Fc融合基因,并将载有eBCMA-Fc融合基因的原核表达质粒转化BL21(DE3)菌、在细菌中表达.目的蛋白经蛋白A亲和柱纯化大约为36kD,与理论预测值34kD相近.免疫印迹表明抗人IgG抗体能够识别eBCMA-Fc融合蛋白.ELISA证实,eBCMA-Fc融合蛋白能够结合BLyS.随着eBCMA-Fc融合蛋白增加,结合BLyS的融合蛋白也相应增加.而对照人IgG,即使在高浓度条件下,也不结合BLyS.此外,eBCMA-Fc融合蛋白能够抑制BLyS对B细胞肿瘤Daudi细胞的作用.这些研究为下一步设计和筛选BLyS拮抗肽提供了实验基础.
文摘Objective： To measure the expression levels of BLyS and its receptors mRNA in peripheral blood ronnonuclear cells （PBMC） using real-time fluorescence quantitative polymerase chain reaction（RFQ-PCR） method and to investigate the relationship between BLyS and its receptors mRNA expression and systemic lupus erythematosus （SLE）. Methods： Specific primers and TaqMan probe were designed, and RFQ-PCR was perfomed. According to the standard curve of plasmid DNA, the level of BLyS and its receptors mRNA expression in 23 patients with SLE and 23 healthy subjects were detemined. The ratio of the copy number of BLyS mRNA to that of β2-microgluobulin （β2M） mRNA and the ratio of the copy number of BLyS receptors mRNA to that of β2M mRNA were regarded as indicator for the levels of BLyS and BLyS mRNA expression. Results： The concentration of RFQ-PCR was in the range of 10 - 109 pg/ml, and the coefficient of variation values for both intra-experimental and inter-experimental reproducibility ranged from 2.40% to 10.12% and from 4,.26% to 12.29%, respectively. In 23 SLE patients, the level of BLyS and its receptors（BCMA, TACI, BAFF-R） mRNA were in the ranges of 1.27 ± 1.4,9, 0.64 ± 0.77, 0.83 ± 1.05 and 0.9±3 -1.37, respectively. The mean values were 1.38 ± 0.07, 0.70 ± 0.04, 0.91 ±0.06 and 1.15±0.12, respectively. In 23 healthy donors, the levels of BLyS and its receptors（BCMA, TACI, BAFF-R） mRNA were： 0.60±1.0, 0.55±0.80, 0.54±0.74 and 0.54± 0.77, respectively. The mean values were 0.83 ±0.13, 0.68±0.08, 0.65± 0.07 and 0.68 ± 0.06, respectively. Conclusion： This results suggest that BLyS, TACI and BAFF-R might be involved in the pathogenesis of SLE and the mRNA expression levels might be used as new markers for the diagnosis of SLE.
