负载SIV抗原肽的中国恒河猴Mamu-B*1703可溶性单体及其四聚体的制备和鉴定  

Preparation and identification of soluble Chinese rhesus macaques Mamu-B* 1703 monomer and tetramer loaded with a SIV peptide

作　　者：王笑迎[1] 欧阳东云[1] 何贤辉[1] 徐丽慧[2] 施焕敬[1] 高琦[1] 郭贺[1] 

机构地区：[1]暨南大学生命科学技术学院,组织移植与免疫实验中心,广东广州510632 [2]暨南大学生命科学技术学院,生物工程研究所,广东广州510632

出　　处：《暨南大学学报（自然科学与医学版）》2009年第4期349-354,共6页Journal of Jinan University(Natural Science & Medicine Edition)

基　　金：国家自然科学基金项目(30572199,30230350);广东省自然和学基金项目(8451063201000340);生物化学与分子生物学广东省重点学科资助项目

摘　　要：目的:制备负载SIV抗原肽的中国恒河猴Mamu-B*1703可溶性单体及其四聚体。方法:以含Mamu-B*1703重链cDNA序列的pMD19-T克隆为模板,通过PCR的方法克隆Mamu-B*1703重链基因,进而构建羧基端融合生物素化酶BirA底物肽(BSP)的Mamu-B*1703重链胞外域融合蛋白的表达载体,并在大肠杆菌中获得表达。β微球蛋白、SIV抗原肽共存时,通过稀释法复性可溶性Mamu-B*1703单体,经生物素化并纯化后与荧光素标记的链亲和素按4∶1的比例混合形成四聚体。结果:ELISA检测显示获得具有正确构象的负载SIV抗原肽的Mamu-B*1703四聚体。结论:印度恒河猴Mamu-B*1701特异性抗原肽IW9,与中国恒河猴的Mamu-B*1703相结合形成可溶性Mamu-B*1703/IW9单体和四聚体。Aim:To prepare soluble Chinese rhesus macaques Mamu-B*1703 monomer and tetramer loaded with a SIV peptide. Methods: Mamu-B*1703 sequence cloned in pMD19-T vector was used as an initial template for amplication of the fragment encoding the extracellular domain of Mamu-B*1703 heavy chain,then the DNA fragment encoding the ectodomain of Mamu-B*1703 heavy chain fused at its carboxyl-terminal a BirA substrate peptide(BSP).The recombinant protein was expressed in E.coli.It was then refolded in the presence of β2-microglobulin and SIV peptide to form a soluble Mamu-B*1703 monomer.After purification and biotinylation,the tetramer was formed by incubation with streptavidin-PE at a ratio of 4∶1.Results: The spatial conformation of the monomer and tetramer was correct as confirmed by an ELISA-based assay.Conclusion:It was shown that IW9 peptide,identified to be Mamu-B*1701-restricted in Indian-origin macaques,could combined with the Mamu-B*1703 to form soluble Mamu-B*1703/IW9 monomer and tetramer.

关 键 词：恒河猴 Mamu-B*1703 猴免疫缺陷病毒 四聚体 

分 类 号：R392.3[医药卫生—免疫学]