新型抗HIV-1蛋白GRFT的构建、表达及活性研究

根据已知的新型抗HIV-1蛋白GRFT基因氨基酸序列,推测其DNA编码序列,密码子优化及修饰后进行全基因化学合成,连接到原核表达载体pET28a（+）中,转化大肠杆菌BL21(DE3),IPTG诱导表达,获得目的蛋白. SDS-PAGE、Western Blot分析结果表明,目的蛋白得到良好表达并具有抗原活性;对表达条件进行优化,在最佳表达条件下,目的蛋白的表达量可占菌体总蛋白的55.84%;利用Ni2+-NTA柱亲和层析法进行目的蛋白的复性和纯化,凝胶成像系统扫描分析表明,纯度可达94.06%;运用Dot-ELISA法进行复性蛋白的抗原结合活性检测,结果显示,目的蛋白能够与HIV-1膜蛋白抗原特异性结合,初步证明所表达的重组蛋白具有良好的体外结合活性;基于制备的能够表达HIV-1 gp120基因的靶细胞模型,运用IFA法开展目的蛋白的细胞结合活性研究,结果显示,目的蛋白能够与靶细胞发生特异性反应,表明成功制备并获得了具有生物活性的新型抗HIV-1蛋白GRFT,为进一步研制新型抗HIV-1基因工程药物及其靶向治疗制剂奠定了坚实基础.     

新型抗HIV-1重组导向制剂SL41在毕赤酵母中的表达及活性实验

以酵母分泌型表达载体pPIC9k为基础, 通过一段柔性连接肽Linker构建含有人源化抗HIV-1 gp41单链抗体(ScFv41)和免疫诱导因子葡萄球菌肠毒素A(staphylococcal enterotoxin A, SEA)的融合表达质粒pPIC9k-SL41, 线性化后, 采用电转化法整合入巴斯德菌毕赤酵母GS115中, 经His+MutS表型鉴定、PCR分析以及G418筛选获得高拷贝重组转化子. 摇瓶培养、甲醇诱导表达、SDS-PAGE和Western Blot分析结果表明, 目的蛋白得到良好表达, 表达量最高可达到47.9 mg/L. 目的蛋白经初步纯化后, 用于制备的HIV-1感染靶细胞复制模型进行抗体亲和力测定、细胞结合活性测定和细胞杀伤活性研究, 结果显示, 目的蛋白能够很好地与靶细胞模型中的HIV-1外膜蛋白gp160发生结合反应, 并可介导特异的CTL反应, 对靶细胞具有明显的杀伤活性, 表明获得了具有生物活性的抗HIV-1重组导向制剂.     

临床肝素类药物酶解分析二糖组成

肝素是一类结构复杂的高分子糖类,以N-硫酸、6-O硫酸氨基葡萄糖和2-O硫酸艾杜糖醛酸为主要成分组成二糖。常见的肝素注射液是以未分级肝素为原料纯化灭菌制备而来,在临床上使用更为广泛的是将肝素降解得到的低分子片段,低分子肝素保留了肝素糖链基本结构,但是不同制备工艺导致其具有不同的还原及非还原端。本研究以肝素类药物为研究对象,使用肝素裂解酶玉,域及芋将肝素注射液、低分子肝素注射液(达肝素、那屈肝素、依诺肝素)、化学合成的类肝素(磺达肝素)进行酶催化降解产生二糖,结合强阴离子交换色谱( Strong anion exchange high performance liquid chromatography,SAX-HPLC)以及紫外检测器在线分析,使用市售肝素二糖标准品确定各种二糖结构。此外,使用反向离子对色谱和电喷雾质谱联用分析磺达肝素中的甲基二糖以及达肝素和那屈肝素中的脱氨二糖结构,为肝素以及类肝素药物的质量控制提供更为精确的结构信息。     

用作HIV-1融合抑制剂的多肽及其类似物

HIV-1通过其包膜糖蛋白跨膜亚基gp41介导的病毒-细胞膜融合进入和感染靶细胞.HIV-1融合抑制剂以gp41为靶点,通过阻断病毒与宿主细胞膜的融合,在感染的初始环节切断HIV-1的复制周期.2003年,首个多肽类融合抑制剂T-20获美国食品药物管理局（FDA）批准上市,但其易被体内蛋白酶降解、临床剂量大、耐受性差,且耐药性HIV-1毒株也很快出现.针对这些缺点,近年来在融合抑制剂的作用机制研究和新融合抑制剂的研发等方面取得了重要进展.以gp41不同功能区为靶点,具有高活性和更好代谢性质的多肽及多肽类似物候选分子不断被发现,成为抗HIV药物研究领域的热点之一.本文综述了多肽和类肽类融合抑制剂的研究进展,为相关的药物开发和基础研究提供参考.     

HIV-1病毒DNA与整合酶结合后的构象变化

用分子动力学(MD)模拟方法优化了HIV-1病毒DNA与整合酶(IN)二聚体(IN2)复合物模型结构, 并分析了HIV-1病毒DNA结合IN2后的构象变化. 结果表明, 按照HIV-1病毒DNA与IN2结合能力的强度, 病毒DNA可分为五个区域: 非结合区、强结合区1、弱结合区、强结合区2和反应区, 并用结合自由能计算验证了该分区的合理性. 与未结合IN2的病毒DNA相比, 复合物模型中病毒DNA除了非结合区碱基外, 其它四个区域的碱基构象变化较大. 复合物模型中病毒DNA主链较大程度地偏离标准B型DNA以及结合部位的小沟变宽都是识别IN的结构基础. 模拟结果与实验数据吻合较好, 为基于HIV-1 IN的药物分子设计提供了一定的结构信息.     

HIV-1嵌合抗原的纯化及免疫原性分析

为获得高效的HIV诊断试剂,选定HIV-1的外膜蛋白env中469-511aa,538-674aa和700-734aa3处包含较多抗原位点的区域作为免疫抗原,用PCR的方法从HIV-1全基因扩增编码这3处片段的基因序列,将它们克隆到原核表达载体中,利用大肠杆菌表达系统表达嵌合蛋白.结果发现,3段嵌合基因能在大肠杆菌BL21(Star)中表达,通过Ni-sepharose4B金属Ni螯合层析柱分离纯化目的产物,酶联免疫检测结果表明,纯化抗原有较强的抗原特异性.     

异肽键交联的N肽类HIV-1融合抑制剂的设计、合成及活性评价

报道了一种通过在HIV-1 gp41 NHR区域的多肽(N肽)之间引入异肽键稳定N3螺旋的新方法;以天然N肽序列N38为模板,采用此方法设计合成了一系列异肽键交联的N38三股α螺旋结构.该结构具有极强的热稳定性和纳摩尔水平抑制HIV-1包膜糖蛋白(Env)介导的细胞融合活性.     

含N-乙酰化肝素寡糖的制备及序列分析

建立了含N-乙酰化肝素寡糖的分离提纯及其序列结构分析方法.首先应用肝素酶Ⅰ深度酶解低分子量肝素来富集含N-乙酰化结构寡糖,通过Bio-Gel P10凝胶色谱法分离制备了包括二糖至十四糖的系列肝素寡糖粗样品,ProPac PA-1强阴离子高效液相色谱(SAX-HPLC)等方法对粗样品进一步分离,提纯得到4种六糖和3种八糖片段.其次应用肝素酶Ⅰ,Ⅱ和Ⅲ复合酶解与HPLC法分析各纯化寡糖的二糖组分,并结合肝素酶Ⅰ底物特异性,初步推断4种六糖和3种八糖的序列结构.在寡糖的糖链两端均含有N-硫酸化二糖,而N-乙酰化二糖分布在糖链当中.应用电喷雾离子阱-飞行时间质谱(ESI-IT-TOF-MS)在负离子模式下进一步表征寡糖并分析其裂解规律.结果表明,各寡糖中均出现大量因SO32-丢失形成的碎片离子峰,六糖中主要有双电荷和三电荷碎片离子峰;在八糖中出现了一系列从双电荷至五电荷的离子峰.各寡糖的双电荷离子峰质荷比进一步确定了上述寡糖的序列结构.六糖的裂解规律表明,裂解主要存在于糖苷键,N-乙酰葡糖胺和糖醛酸上的裂解方式分别为0,2X和0,2Z.本研究提供了切实有效的分离、分析未知结构肝素寡糖序列的新方法.     

利用糖芯片研究半乳寡糖与蓖麻凝集素和鸡冠刺桐凝集素间的相互作用

建立了用糖芯片技术研究寡糖与凝集素相互作用的方法.以新乳糖-N-四糖(LNnT)、乳糖-N-四糖(LNT)、λ-卡拉胶四糖(L4)和琼胶五糖(A5)为原料,经还原胺化法分别将其与1,2-十六烷基磷脂酰乙醇胺偶联得到拟糖脂,再将其与卵磷脂和胆固醇按4∶2∶5比例混合制备成脂质体后,用全自动芯片点样仪将其点印在硝酸纤维素膜包被的玻片上制成糖芯片,并进行寡糖与凝集素的结合实验.结果表明,蓖麻凝集素(Rieinus communis agglutinin 120,RCA120)特异性识别非还原端糖残基为Galβ(1 →4)的寡糖,而鸡冠刺桐凝集素(Erythrina cristagalli lectin,ECL)特异性识别非还原端糖残基为Galβ(1→4)GlcNAc的LNnT.采用接触式芯片点样仪制备糖芯片,并用荧光扫描仪对糖与凝集素结合信号进行检测,增加了灵敏度和准确性.本方法不仅适合于糖与蛋白相互作用的研究,也有助于加快糖类药物的发现.     

电喷雾电离质谱法研究环肽对HIV-1调控区DNA的识别及其相关碎裂机理和稳定性

利用电喷雾电离质谱(ESI-MS)和二级质谱(MS/MS)研究了六种结构不同的环五肽, 环七肽以及环十肽与HIV-1调控区DNA的非共价键相互作用. 在研究中比较了不同识别分子与靶序列DNA结合的强弱, 发现环七肽CP5对靶点DNA具有高亲合性的结合. 用MS/MS法研究了环肽与DNA复合物的碎裂机理; 用升温实验研究了其热稳定性, 发现与CP5结合后能提高HIV-1双螺旋DNA的热稳定性.     

Theoretical studies on the interactions and interferences of HIV-1 glycoprotein gp120 and its coreceptor CCR5

The interaction between the HIV gp120 protein and coreceptor CCR5 or CXCR4 of the host cell is critical in mediating the HIV entry process. A model for the CCR5-gp120 complex has been developed. In the model, the N-terminus of CCR5 binds to three discontinuous domains of gp120, including the fourth conserved (C4) region, β19/β20 connecting loop, and V3 loop. The second extra-cellular loop (ECL2) of CCR5 also interacts with the crown part of the gp120 V3 loop. The bindings of the three CCR5 antagonists, maraviroc, aplaviroc, and vicriviroc, to the trans-membrane domain of CCR5 have been modeled. The bindings are found to affect the conformation of the ECL2 domain, which in turn drives the N-terminus of CCR5 to an altered state. Aplaviroc is more hydrophilic than maraviroc and vicriviroc, and its binding is more interfered by solvent, resulting in a quite different effect to the structure of CCR5 compared with those of the other two molecules. The above results are in accord with experimental observations and provide a structural basis for further design of CCR5 antagonists.     

Interaction of human immunodeficiency virus (HIV) glycans with lectins of the human immune system

Approximately half of the molecular mass of gp120, the receptor-binding envelope protein of human immunodeficiency virus (HIV), consists of N-linked glycans. Nearly half of these glycans are of the high mannose type. These high mannose glycans furnish a rich forest of mannose residues on the virus surface making HIV a prime target for interaction with mannose-specific lectins of the immune system. This review focuses on the known interactions between gp120 and immune system lectins some of which HIV appears to exploit. The effect of variation in glycosylation of gp120, especially with respect to clades of HIV, on binding of immune system lectins is highlighted.     

Intricate Recognition of Glycolipid-Like Compounds by HIV-1 Envelope Proteins Evaluated with Surface Plasmon Resonance Imaging

Fusion of human immunodeficiency virus (HIV) to the cell membrane occurs by the specific binding of an envelope protein of HIV-1 (gp120 and gp160) and a glycosphingolipid of the cell membrane. In this study, quantitative and array-based affinity evaluation of gp120 and gp160 was performed by surface plasmon resonance (SPR) and the SPR imaging technique using a substrate immobilized with glycolipid-like compounds (Gb3, GM3, and Lac). Quantitative affinity evaluation showed that gp160 specifically bound to Gb3 and Lac compared with GM3, whereas gp120 showed lower binding affinity and specificity. Array-based evaluation showed that gp160 binds to Gb3 more favorably than Lac and GM3.     

Synthesis of the 2',3'-dideoxynucleoside derivatives of the specific binding peptide part of CD4.

The 2',3'-dideoxynucleoside derivatives of the specific binding peptide part of CD4 to HIV envelope protein gp120 were synthesized.     

Multisite and multivalent binding between cyanovirin-N and branched oligomannosides: calorimetric and NMR characterization

Binding of the protein cyanovirin-N to oligomannose-8 and oligomannose-9 of gp120 is crucially involved in its potent virucidal activity against the human immunodeficiency virus (HIV). The interaction between cyanovirin-N and these oligosaccharides has not been thoroughly characterized due to aggregation of the oligosaccharide-protein complexes. Here, cyanovirin-N's interaction with a nonamannoside, a structural analog of oligomannose-9, has been studied by nuclear magnetic resonance and isothermal titration calorimetry. The nonamannoside interacts with cyanovirin-N in a multivalent fashion, resulting in tight complexes with an average 1:1 stoichiometry. Like the nonamannoside, an alpha1-->2-linked trimannoside substructure interacts with cyanovirin-N at two distinct protein subsites. The chitobiose and internal core trimannoside substructures of oligomannose-9 are not recognized by cyanovirin-N, and binding of the core hexamannoside occurs at only one of the sites on the protein. This is the first detailed analysis of a biologically relevant interaction between cyanovirin-N and high-mannose oligosaccharides of HIV-1 gp120.     

Characterization of a discontinuous epitope of the HIV envelope protein gp120 recognized by a human monoclonal antibody using chemical modification and mass spectrometric analysis

A subset of the neutralizing anti-HIV antibodies recognize epitopes on the envelope protein gp120 of the human immunodeficiency virus. These epitopes are exposed during conformational changes when gp120 binds to its primary receptor CD4. Based on chemical modification of lysine and arginine residues followed by mass spectrometric analysis, we determined the epitope on gp120 recognized by the human monoclonal antibody 559/64-D, which was previously found to be specific for the CD4 binding domain. Twenty-four lysine and arginine residues in recombinant full-length glycosylated gp120 were characterized; the relative reactivities of two lysine residues and five arginine residues were affected by the binding of 559/64-D. The data show that the epitope is discontinuous and is located in the proximity of the CD4-binding site. Additionally, the reactivities of a residue that is located in the secondary receptor binding region and several residues distant from the CD4 binding site were also altered by Ab binding. These data suggest that binding of 559/64-D induced conformational changes which result in altered surface exposure of specific amino acids distant from the CD4-binding site. Consequently, binding of 559/64-D to gp120 affects not only the CD4-binding site, which is recognized as the epitope, but appears to have a global effect on surface exposed residues of the full-length glycosylated gp120.     

Synthetic peptides for study of human immunodeficiency virus infection

The formation of a complex among gp120, CD4, and CCR5/CXCR4 represents a key step in human immunodeficiency virus (HIV) infection. The use of synthetic peptides reproducing sequences of these surface proteins has increased knowledge about the interactions that determine the penetration of HIV viruses into target cells. The final aim of such investigations is the design of molecules able to inhibit the initial step of infection and the development of high-sensitivity in vitro assays for detection of HIV. In particular, the studies presented herein concern the role of the gp120 V3 loop in the CD4 binding, the importance of the N-terminal sequence of HIV-coreceptor CCR5, the sequences patterned on CXCR4 natural ligand (stromal-derived factor 1 [SDF-1]) as inhibitory peptides, and the importance of substrate secondary structure in determining the enzymatic processing of gp120 precursor (gp160).     

Oligosaccharide and glycoprotein microarrays as tools in HIV glycobiology; glycan-dependent gp120/protein interactions.

Defining HIV envelope glycoprotein interactions with host factors or binding partners advances our understanding of the infectious process and provides a basis for the design of vaccines and agents that interfere with HIV entry. Here we employ carbohydrate and glycoprotein microarrays to analyze glycan-dependent gp120-protein interactions. In concert with new linking chemistries and synthetic methods, the carbohydrate arrays combine the advantages of microarray technology with the flexibility and precision afforded by organic synthesis. With these microarrays, we individually and competitively determined the binding profiles of five gp120 binding proteins, established the carbohydrate structural requirements for these interactions, and identified a potential strategy for HIV vaccine development.     

HIV-1 gp120 V3 loop for structure-based drug design

HIV-1 cell entry is mediated by sequential interactions of the envelope protein gp120 with the receptor CD4 and a coreceptor, usually CCR5 or CXCR4, depending on the individual virion. Considerable efforts on exploiting the HIV coreceptors as drug targets have led to the new class of coreceptor antagonists. While these antiretroviral drugs aim at preventing virus/coreceptor interaction by binding to host proteins, neutralizing antibodies directed against the coreceptor-binding sites on gp120 have attracted attention as possible vaccine candidates. However, both approaches are complicated by the multiple protective mechanisms of gp120 which allow for rapid escape from selective pressures exerted by drugs or antibodies. Thus, advances in rational drug and vaccine design rely heavily on improved insights into the relation between genotype and phenotype, the evolution of coreceptor usage, and, ultimately the structural biology of coreceptor usage and inhibition. The third variable (V3) loop of gp120, crucially involved in all these aspects, will be a major focus of this review.     

The potent anti-HIV protein cyanovirin-N contains two novel carbohydrate binding sites that selectively bind to Man(8) D1D3 and Man(9) with nanomolar affinity: implications for binding to the HIV envelope protein gp120.

Cyanovirin-N (CVN) is a monomeric 11 kDa cyanobacterial protein that potently inactivates diverse strains of human immunodeficiency virus (HIV) at the level of cell fusion by virtue of high affinity interactions with the surface envelope glycoprotein gp120. Several lines of evidence have suggested that CVN-gp120 interactions are in part mediated by N-linked complex carbohydrates present on gp120, but experimental evidence has been lacking. To this end we screened a comprehensive panel of carbohydrates which represent structurally the N-linked carbohydrates found on gp120 for their ability to inhibit the fusion-blocking activity of CVN in a quantitative HIV-1 envelope-mediated cell fusion assay. Our results show that CVN specifically recognizes with nanomolar affinity Man(9)GlcNAc(2) and the D1D3 isomer of Man(8)GlcNAc(2). Nonlinear least squares best fitting of titration data generated using the cell fusion assay show that CVN binds to gp120 with an equilibrium association constant (K(a)) of 2.4 (+/- 0.1) x 10(7) M(-1) and an apparent stoichiometry of 2 equiv of CVN per gp120, Man(8)GlcNAc(2) D1D3 acts as a divalent ligand (2 CVN:1 Man(8)) with a K(a) of 5.4 (+/- 0.5) x 10(7) M(-1), and Man(9)GlcNAc(2) functions as a trivalent ligand (3 CVN:1 Man(9)) with a K(a) of 1.3 (+/- 0.3) x 10(8) M(-1). Isothermal titration calorimetry experiments of CVN binding to Man(9)GlcNAc(2) at micromolar concentrations confirmed the nanomolar affinity (K(a) = 1.5 (+/- 0.9) x 10(8) M(-1)), and the fitted data indicated a stoichiometry equal to approximately one (1 Man(9):1 CVN). The 1:1 stoichiometry at micromolar concentrations suggested that CVN has not only a high affinity binding site-relevant to the studies at nM concentrations-but a lower affinity site as well that facilitates cross-linking of CVN-oligomannose at micromolar concentrations or higher. The specificity of CVN for Man(8) D1D3 and Man(9) over the D1D2 isomer of Man(8) indicated that the minimum structure required for high affinity binding comprises Manalpha1 --> 2Manalpha. By following the (1)H-(15)N correlation spectrum of (15)N-labeled CVN upon titration with this disaccharide, we unambiguously demonstrate that CVN recognizes and binds to the disaccharide Manalpha1 --> 2Manalpha via two distinct binding sites of differing affinities located on opposite ends of the protein. The high affinity site has a K(a) of 7.2 (+/- 4) x 10(6) M(-1) and the low affinity site a K(a) of 6.8 (+/- 4) x 10(5) M(-1) as determined by isothermal titration calorimetry. Mapped surfaces of the carbohydrate binding sites are presented, and implications for binding to gp120 are discussed.