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恒河猴MHC-I类分子Mamu-A*02与猴免疫缺陷病毒抗原表位复合物的纯化和晶体学分析

来源:中科院微生物研究所 2009-09-05 17:58

生物工程学报 25 July 2009, 25(7):1028~1034

恒河猴MHC-I类分子Mamu-A*02与猴免疫缺陷病毒抗原表位复合物的纯化和晶体学分析

戴连攀1,2, 周斌1, 齐建勋1, 马颖1, 高福1, 杨希才1

1 中国科学院微生物研究所, 北京100101

2 中国科学院研究生院, 北京100039

摘 要: 恒河猴是研究人免疫缺陷型病毒(HIV)感染与获得性免疫缺失综合征(AIDS)疫苗研制的最为重要的动物模型,其组织相容性复合体(MHC)结构的解析将有助于了解HIV 免疫逃逸机制。本研究克隆了1个恒河猴MHC-I类分子Mamu-A*02轻链(β2m)基因并插入到pET21a(+)原核表达载体中, 成功构建了重组表达质粒pET21a(+)-Mamu-β2m。pET21a(+)-Mamu-β2m和之前构建好的恒河猴Mamu-A*02重链(α)重组质粒pET21a(+)-α分别转入BL21(DE3)大肠杆菌表达体系中, IPTG诱导表达。Mamu-A*02重链和猴β2m目的蛋白在BL21(DE3)中以包涵体形式表达。重链、轻链的包涵体蛋白和Mamu-A*02限制的猴免疫缺陷病毒(SIV)Nef表位多肽(YY9)通过稀释法共复性。高纯度的复合物蛋白经分子筛层析和阴离子交换层析纯化后获得。复合物晶体利用悬滴法筛选并优化, 并在0.1 mol/L BIS-TRIS (pH 5.5)、2.0 mol/L (NH4)2SO4 条件下收集一套2.8 .分辨率的X-射线衍射数据。晶体属于正交空间群P212121, 其晶胞参数为a=128.99 ., b=129.01 ., c=129.03 ., α=β=γ=90°。该数据可通过分子置换法解析。

关键词: 猴免疫缺陷病毒(SIV), Mamu-A*02-Nef_YY9复合体, 蛋白表达与纯化, X-射线晶体学

Purification, crystallographic analysis of rhesus MHC-I Mamu-A*02 complexed with simian immunodeficiency virus nonapeptide

Lianpan Dai1,2, Bin Zhou1, Jianxun Qi1, Ying Ma1, George F Gao1, and Xicai Yang1

1 Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China

2 Graduate University of Chinese Academy of Sciences, Beijing 100039, China

Abstract: Rhesus macaque (Macaca mulatta) is the best model to study of human immunodeficiency virus (HIV) infection and to develop acquired immunodeficiency syndrome (AIDS) vaccine. The crystal structure of its major histocompatibility antigen complex (MHC) is helpful to understand the mechanism of HIV immune evasion. In this study, we cloned the light chain (β2m) of MHC class I allele of rhesus macaques, Mamu-A*02, and inserted it into pET21a(+) vector. We transfected the recombinant plasmid pET21a(+)-Mamu-β2m and pET21a(+)-Mamu-β into BL21(DE3). Mamu-A*02 and β2m were expressed in the form of inclusion bodies in BL21 (DE3). We co-refolded the inclusion bodies of Mamu-α and Mamu-β2m with SIV nonapeptide YY9 and obtained the correct refolded protein complex. Then we purified the protein complex by the gel filtration and anion-exchange column. With hanging-drop method, we screened and optimized for the protein crystal. We managed to collect a X-ray diffraction with the resolution to 2.8 A in the condition of 0.1 mol/L BIS-TRIS (pH5.5), 2.0 mol/L(NH4)2SO4 . This crystal belong to perpendicular space group P212121, with unit-cell parameters a=128.99 A, b=129.01 A, c=129.03 A. This data is available for the structure determination.

Keywords: simian immunodeficiency virus (SIV), Mamu-A*02-Nef_YY9 complex, protein expression and purification, X-ray crystallography

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