CAJ | 학술논문

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 0.036 nm,Fc 段与单体 Fc 的主链碳原子间 RMSD 值为 0.064 nm. 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36 kD,与理论预测值 34 kD 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 BLvS 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.
B세포성숙항원(BCMA)시B림파세포자격인자(BLyS)적수체지일.타적포외구여인IgG1Fc적융합단백eBCMA-Fc,우칭위유이수체,구유길항 BLyS 적활성.위료설계신적길항태,기우BCMA 화 Fc 적정체결구,통과계산궤도형학기술、분자모의방법,건립료 eBCMA-Fc 융합단백적삼유이론결구.이용균방근위이 (root mean square distance,RMSD) 대 eBCMA-Fc 융합단백여단체eBCMA、Fc 구상차이진행분석.융합단백 eBCMA-Fc 중적 eBCMA 단여단체 eBCMA 적주련탄원자간RMSD 치위 0.036 nm,Fc 단여단체 Fc 적주련탄원자간 RMSD 치위 0.064 nm. 결과표명,대비단체,융합단백 eBCMA-Fc 병미인eBCMA 여 Fc직접련접이발생구상적변화.분자대접방법현시,융합단백 eBCMA-Fc 중적 BCMA 여 BLyS 작용,이 Fc 분연착은정BCMA구상적지가작용.위진일보험증상술이론분석,구건 eBCMA-Fc 융합기인,병장재유eBCMA-Fc융합기인적원핵표체질립전화 BL21(DE3) 균、재세균중표체.목적단백경단백 A 친화주순화대약위36 kD,여이론예측치 34 kD 상근.면역인적표명항인 IgG 항체능구식별 eBCMA-Fc 융합단백.ELISA 증실,eBCMA-Fc 융합단백능구결합BLyS.수착 eBCMA-Fc 융합단백증가,결합 BLyS 적융합단백야상응증가.이대조인 IgG,즉사재고농도조건하,야불결합 BLyS.차외,eBCMA-Fc 융합단백능구억제 BLvS 대 B 세포종류 Daudi 세포적작용.저사연구위하일보설계화사선 BLyS 길항태제공료실험기출.
B cell maturation antigen (BCMA) is a receptor of B lymphocyte stimulator (BLyS). Human IgG1Fc fusion proteins with the extracellular domain of BCMA(eBCMA), also called decoy receptors, have beenused as a potential BLyS antagonists to block BLyS activities. In order to design novel BLyS antagonistpeptides, computer-aided homologue modeling was used to construct an eBCMA-Fc fusion protein based on thecrystal structures of BCMA and Fc fragmant. To ensure the activity of eBCMA not to be interfered by Fcfusion, the root mean square distance (RMSD) for eBCMA and Fc were calculated to be 0.036 nm and 0.064nm, respectively, based on molecular docking modeling. An eBCMA-Fc fusion gene was constructed andintroduced into E. coli for expression. As expected, the purified 36 kD eBCMA-Fc fusion protein was able tobind BLyS in vitro at a dosage-dependent manner and demonstrated an anti-proliferative activity induced byBLyS in Daudi cells. The results have provided useful information on the evaluation of computer modeling andthe in vitro biological activity for the design of potential BLyS antagonist peptides.