【作者】 刘北一；

【导师】 富宁； 姜世勃；

【作者基本信息】 第一军医大学 ， 免疫学， 2004， 博士

【摘要】 结合HIV-1 gP41 NHR的环肽及模拟gP41融合核心结构表位的筛选与鉴定 AIDS为感染HIV-1病毒后所致的继发性免疫缺陷综合症。目前针对HIV感染及AIDS患者的主要治疗药物仍是联合应用逆转录酶抑制剂(PTI)和蛋白酶抑制剂(PI)，即鸡尾酒疗法。但该疗法主要针对HIV感染后期，长期应用有毒、副作用且易诱导病毒突变株产生。 HIV-1跨膜蛋白gp41在病毒和细胞融合中发挥关键作用。当gp120与靶细胞表面的CD4及共受体结合后，gp41胞外段N-末端重复序列(NHR)与C—术端重复序列(CHR)相互作用，形成六螺旋束(6—HB)，即gp41融合活性核心，从而导致病毒融合。源于gp41 CHR的合成肽如C34、T20肽可有效抑制HIV—1介导的细胞融合。因此gp41胞外段已成为研制新型抗HIV的药物靶点。 本研究的目标是：1．筛选针对HIV-1介导细胞融合的肽类先导化合物，对模拟HIV-1 gp41 C螺旋的坏肽抑制病毒与细胞融合作用进行初步鉴定；2．筛选并鉴定HIV-1 gp41核心表位和gp41 NHR结合表位。 本研究包括以下三个部分： 一、HIV-1 gp41 NHR结合序列的筛选与鉴定 1、用源于HIV-1 gp41 N端的肽N36，以PL+S法筛选坏七肽库，得到16个阳性克隆，竞争抑制实验表明阳性克隆No.8与N肽的结合可被C34所阻断。挑选10个阳性克隆测序并推导氨基酸序列，得到8种序列：CQHWWHWYC，CVHWWHWIC，CYWWHRLHC，CTWWRPWHC，CHPHWWRTC，CWYWHAKHC，CFWWHTRHC以及CPAPDQSMC。所有序列富含疏水氨基酸，其中9个克隆均有WW、7个克隆有WWH保守序列，W可模拟C螺旋中的W628及W631与N螺旋中的疏水口袋区相互作用。 2、用固相化N36肤筛选噬菌体环七肤库(P+LS法)，经EL工SA鉴定有n个克隆可特异地与N36肤结合，阳性克隆与N36的结合可被C34和ADS一Jl所抑制。DNA序列分析表明这n个克隆为同一序列，推导的氨基酸序列为CDRHQHKRC。 3、以针对gp41 HR区域的多克隆抗体为靶，筛选环七肤库，13个噬菌体克隆可特异性地与该多抗结合，而不与正常兔工gG结合，ELIsA鉴定表明其中的8个阳性克隆可与N36月太结合。将这8个克隆进行DNA测序并推导氨基酸序列，得到单一序列:CPYGPKLC。提示cPYGPKLC为gp41 NHR结合序列。二、川V--1 gp41 NHR结合肤的合成与鉴定 1、根据游离N36肤筛选所得的阳性克隆No.8所展示的序列CYWWHRLHC，固相合成环肤FJ一08一Ex(序列为SACYWWHRLHCGGGS)。与无关肤相比较，细胞融合抑制实验显示在2小时内，FJ一08一Ex()80瑰/m1)有抑制融膜作用，呈现浓度依赖效应。然而随着时间的延长(超出2小时)，FJ一08一Ex的活性消失，与对照13b月太无明显区别。FJ一08一Ex能抑制HIV介导的膜融合(2小时细胞融合实验)，但不能抑制合胞体的形成(24小时合胞体形成实验)，提示该序列可模拟c多肤与H工V一1 gp41的N螺旋相互作用，抑制HIV一1介导的细胞融合。但因其亲和力较低，导致其抑制作用随时间延长而降低。上述结果提示可以FJ一08一Ex为基础进行改造以获得可抑制HIV融合的、亲和力高的肤类抑制剂。 2、根据固相化N36肤筛选后所得的阳性序列CDRHQHKRC合成环肤NA (SACDRHQHKRCGG)。NA与BSA的交联物可与N36肤特异性结合，竞争EL工SA显示C34月太和游离N36肤可抑制NA一BSA与N36结合，说明NA肤模拟gp41 CHR的抗原性。三、川v--1 gp41核心表位的筛选与鉴定 以针对gp41核心结构的单克隆抗体NC一为靶，筛选噬菌体线性12肤库，得到10个特异地与NC注结合的阳性噬菌体克隆，DNA测序并推导氨基酸，共5种线性表位:HDVHHRWVYLLS，ITVNEWLYTSEQ，HGRSHGMFKPKR，MGPIARPHwHLN，DMYRSPRPKPDT。gp41N肤和C月太所形成的复合物可特异地阻断表达HnVHHRWVYLLS，ITVNEWLYTSEQ和MGP工ARPHWHLN的克隆与NC一的结合。上述结果说明这4种序列为gp41核心表位。更多还原

【Abstract】 Acquired immunodeficiency syndrome (AIDS), one of the serious emerging infectious diseases worldwide, is caused by the human immunodeficiency virus (HIV).The HIV envelope glycoprotein (Env) plays an important role in HIV entry into the target cell. The Env surface subunit is responsible for virus binding and the transmembrane subunit gp41 mediates fusion between the viral and target cell membranes. HIV-1 infection is initiated by gp120 binding to CD4 and a co-receptor (i.e., CXCR4 or CCR5) on target cell, which triggers a conformational change in gp41. The interaction between the N- and C-terminal heptad repeat (NHR and CHR, respectively) regions of the gp41 extracellular domain leads to the formation of a six-helix bundle (6-HB), representing the fusion-active gp41 core. Peptides derived from the CHR region, named C-peptides, e.g., C34 and T-20, inhibit HIV fusion by interacting with the viral NHR region and blocking the fusogenic core formation. Therefore, gp41 NHR region can serve as an attractive target for developing HIV fusion inhibitors, a new class of anti-HIV drugs and the 6-HB formed by peptides derived from the NHR and CHR regions can be used as a model of the gp41 core to study fusogenic mechanism of HIV and the mechanism of action of the anti-HIV peptides.Accordingly, the main aims of this proposal are: (1) To screen short peptides with inhibitory activity on HIV-1 entry from phage display libraries as leads for developing peptidic HIV fusion inhibitors;and (2)To identify the epitopes on gp41 core structure and the epitope which can bind gp41 NHR.1.Screening the C7C phage library using soluble form of biotinylated peptide N36 derived from gp41 NHR region (PL+S method). In this assay, the positive clones binding to biotin-N36 in solution were captured by streptavidin-coated on the solid phase. After three cycles of biopanning, we identified 16 clones which may be the mimotopes of peptide C34 since the binding of the positive clone No.8 to the peptide N36 could be blocked by peptide C34 in a competitive ELISA.Amino acid sequences of the displayed peptides in 10 phage clones were deduced from DNA sequences.They are CQHWWHWYC, CVHWWHWIC, CYWWHRLHC, CTWWRPWHC, CHPHWWRTC, CWYWHAKHC, CFWWHTRHC and CPAPDQSMC. Every peptide sequence contains at least two hydrophobic residues. 9 of the 10 clones have a conservative sequence WW, which may mimic the critical conserved hydrophobic residues W628 and W631 in the CHR cavity-binding sequence to interact with the NHR cavity-forming sequence.2. Screening of the C7C phage display peptide library using the peptide N36 coated on the solid phase (P+LS method). In this assay, the positive clones were captured by the biotin-N36 bound to the streptavidin-coated wells. After three cycles of biopanning, we identified 11 positive phage clones. The binding of the positive clones to N36 could be inhibited by C34 and ADS-J1, a small molecule HIV fusion inhibitor targeting gp41 NHR region. All the positive clones showed a unique amino acid sequence: CDRHQHKRC.3. Screening C7C phage display peptide library using polyclonal antibodies against the gp41 heptad repeat regions. Thirteen positive phage clones were identified and 8 of 13 clones could bind to N36 peptide. All these 8 positive clones shared a common sequence: CPYGPKLC, suggesting that CPYGPKLC may be a gp41 NHR binding sequence.1. The peptide FJ-08-Ex corresponding to the sequence of the positive clone No.8 (SACYWWHRLHCGGGS) identified from the PL+S screening method was synthesized. The natural sequences SAC and CGGGS were added to the N- and C-termini, respectively, of the core sequence YWWHRLH in order to keep a stable cyclic conformation, which may be required for its interaction with the gp41 NHR region. Compared with unrelated control peptide 13b, FJ-08-Ex significantly inhibited HIV-1 mediated cell-cell fusion during the first 2 hrs of coculture of HIV-1 infected cells and the target cells in a dose-dependent manner. However, the inhibitory activity disappeared 24 hrs later.更多还原