计算机辅助设计可溶性BLyS受体,eBCMA-Fc融合蛋白及其在大肠杆菌中的表达(英文)

Prokaryotic Expression of eBCMA-Fc Fusion Protein,a Soluble BLyS Receptor,with Computer-aided Molecular Design in E. coli

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.

B cell maturation antigen （BCMA） is a receptor of B lymphocyte stimulator （BLyS）. Human IgG1 Fc fusion proteins with the extracellular domain of BCMA（eBCMA）, also called decoy receptors, have been used as a potential BLyS antagonists to block BLyS activities. In order to design novel BLyS antagonist peptides, computer-aided homologue modeling was used to construct an eBCMA-Fc fusion protein based on the crystal structures of BCMA and Fc fragmant. To ensure the activity of eBCMA not to be interfered by Fc fusion, the root mean square distance （RMSD） for eBCMA and Fc were calculated to be 0.036 nm and 0.064 nm, respectively, based on molecular docking modeling. An eBCMA-Fc fusion gene was constructed and introduced into E. coli for expression. As expected, the purified 36 kD eBCMA-Fc fusion protein was able to bind BLyS in vitro at a dosage-dependent manner and demonstrated an anti-proliferative activity induced by BLyS in Daudi cells. The results have provided useful information on the evaluation of computer modeling and the in vitro biological activity for the design of potential BLyS antagonist peptides.