生物工程學(xué)報 25 July 2009, 25(7):1028～1034
恒河猴MHC-I類分子Mamu-A*02與猴免疫缺陷病毒抗原表位復(fù)合物的純化和晶體學(xué)分析
戴連攀1,2, 周斌1, 齊建勛1, 馬穎1, 高福1, 楊希才1
1 中國科學(xué)院微生物研究所, 北京100101
2 中國科學(xué)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)感染與獲得性免疫缺失綜合征(AIDS)疫苗研制的最為重要的動物模型,其組織相容性復(fù)合體(MHC)結(jié)構(gòu)的解析將有助于了解HIV 免疫逃逸機(jī)制,。本研究克隆了1個恒河猴MHC-I類分子Mamu-A*02輕鏈(β2m)基因并插入到pET21a(+)原核表達(dá)載體中, 成功構(gòu)建了重組表達(dá)質(zhì)粒pET21a(+)-Mamu-β2m,。pET21a(+)-Mamu-β2m和之前構(gòu)建好的恒河猴Mamu-A*02重鏈(α)重組質(zhì)粒pET21a(+)-α分別轉(zhuǎn)入BL21(DE3)大腸桿菌表達(dá)體系中, IPTG誘導(dǎo)表達(dá)。Mamu-A*02重鏈和猴β2m目的蛋白在BL21(DE3)中以包涵體形式表達(dá),。重鏈,、輕鏈的包涵體蛋白和Mamu-A*02限制的猴免疫缺陷病毒(SIV)Nef表位多肽(YY9)通過稀釋法共復(fù)性。高純度的復(fù)合物蛋白經(jīng)分子篩層析和陰離子交換層析純化后獲得,。復(fù)合物晶體利用懸滴法篩選并優(yōu)化, 并在0.1 mol/L BIS-TRIS (pH 5.5),、2.0 mol/L (NH4)2SO4 條件下收集一套2.8 .分辨率的X-射線衍射數(shù)據(jù)。晶體屬于正交空間群P212121, 其晶胞參數(shù)為a=128.99 ., b=129.01 ., c=129.03 ., α=β=γ=90°,。該數(shù)據(jù)可通過分子置換法解析,。
關(guān)鍵詞: 猴免疫缺陷病毒(SIV), Mamu-A*02-Nef_YY9復(fù)合體, 蛋白表達(dá)與純化, X-射線晶體學(xué)
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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