磺胺类药物单链抗体的制备及其与磺胺噻唑相互作用研究

抗体与对应的小分子待测物之间的相互作用模式决定了免疫分析的特性。本研究以磺胺类药物杂交瘤细胞株4C7为起点,应用基因工程技术制备出单链抗体scFv4C7,采用间接竞争ELISA法对比其与母本单克隆抗体的识别特性,同时采用同源建模构建scFv4C7的三维立体结构,并与磺胺噻唑( STZ)进行分子对接。间接竞争ELISA结果显示scFv4C7保留了亲本单克隆抗体的识别特性,分子对接结果显示STZ深陷入抗体的重链和轻链形成的“口袋”中,STZ分子更靠近重链,且主要与抗原互补决定区CDR H3相互作用。本研究为制备识别谱更广、亲和力更高的磺胺类药物抗体提供了必要的结构信息。     

迭代饱和突变提高Bacillus cereus胺脱氢酶对苯乙酮还原的催化效率

基于同源建模建立了Bacillus cereus胺脱氢酶(BcAmDH)的三维结构, 采用半理性设计方法, 对底物结合口袋附近的8个氨基酸残基(L42, G43, M67, A115, E116, T136, V293和V296)分别进行单点饱和突变, 通过显色法筛选出3个正向突变位点(116, 136和293). 进一步采用迭代饱和突变策略对这3个正向位点进行组合突变, 获得最优突变株V293A/E116V/T136S, 其对苯乙酮还原反应的催化效率达到2.54 L·min^-1·mmol^-1, 比BcAmDH提高了719%; 与BcAmDH相比, 最优突变株在催化苯乙酮的不对称还原反应时, 底物浓度由100 mmol/L提高至300 mmol/L, 转化率由42.1%提高至80.2%. 分子对接结果表明, 突变株底物结合口袋的位阻减小和底物进出通道的扩大是提高催化效率的主要原因.     

分子对接和动力学模拟提高嗜热蛋白酶PhpI的活力

通过分子对接和动力学模拟对嗜热蛋白酶的分子进行改造, 确定蛋白酶PH1704(PhpI)定点突变残基, 并通过分子生物学实验进行验证. 突变体K43C的蛋白酶活力提高了5.8倍. 分子动力学模拟结果表明, 经过8 ns的动力学模拟后, K43C突变体二级结构由野生型的S2片层(F11-E12-D13)变成环状结构. E12和K43均是活性位点的重要残基, 这种变化将导致活性位点的柔性增强, 有利于催化反应的发生.     

神经氨酸酶与3-(3-戊氧基)安息香酸作用机制的理论研究

运用柔性分子对接和分子动力学方法, 深入研究了4-(氮乙酰氨基)-5-胍基-3-(3-戊氧基)安息香酸(BA)与各类型神经氨酸酶(N1, N2, N9亚型和B型)间的作用机制. 结果显示, BA与各类型神经氨酸酶结合模式存在差异, 但作用机制比较相似: 与它们的活性腔均匹配良好, 并形成稳定的复合体系, 最大结合能分别等于－1233.62, －1385.72, －663.11, －1058.87 kJ•mol^－1. 这表明BA对各类型神经氨酸酶均有良好的抑制效果. 进一步分析发现, BA与各类型神经氨酸酶活性腔内保守关键氨基酸残基发生较强的静电和氢键作用, 而与易突变氨基酸残基作用较弱, 表明了活性腔内易突变氨基酸残基发生突变也不会对抑制效果造成明显影响. 因此, BA是一种极具应用前景的新型抗流感病毒药物. 结合以前的研究结果, 我们提出了以BA为底物的抗流感病毒药物的修饰方向.     

嘧啶并嘧啶酮类化合物作为L3MBTL1选择性络合剂的设计与合成

用嘧啶并嘧啶酮替换Lethal 3 malignant brain tumor 1(L3MBTL1)小分子络合剂UNC669分子中的芳香部分,合成了一系列嘧啶并嘧啶酮类化合物.采用同质邻近发光放大法(AlphaScreen^®)测试了其活性,得到IC₅₀值为1.21 μmol/L的化合物8a;通过对其5位基团进行改造,最终获得了3个选择性L3MBTL1络合剂8g, 8o与8p,它们仅对L3MBTL1有活性,对其同源蛋白L3MBTL3在内的其它甲基化识别蛋白则无活性.     

具有谷胱甘肽过氧化物酶活性的含硒人源单链抗体的制备

以谷胱甘肽(GSH)为靶抗原, 从噬菌体展示人源单链抗体库中筛选人源单链抗体(scFv). 经3轮筛选后, 用ELISA方法检测出5个(2, 11, 16, 24, 32 )可以和GSH结合的克隆. PCR产物的电泳和测序结果表明, 只有3个克隆(11, 16, 24)具有完整的scFv编码基因. 选取和GSH结合力高的克隆11的scFv 编码基因组装到表达载体pPELB上, 在大肠杆菌Rosetta中进行可溶性表达, 用Ni2+螯合亲和层析纯化scFv-11, 免疫点印迹结果证实该抗体能与GSH特异结合. 通过化学突变将scFv-11的丝氨酸转变成硒代半胱氨酸(Sec)后, 获得了具有谷胱甘肽过氧化物酶(GPX)活力的含硒(Se)人源单链抗体(Se-scFv-11), 其活力为351 U/μmol.     

基于分子对接和自由能计算的高活性苯并噻唑类ROCK抑制剂的设计、合成和生物学评价

以3个已报道的苯并噻唑类Rho关联含卷曲螺旋蛋白激酶(ROCK)抑制剂(化合物1~3)为研究对象,经分子动力学模拟获得其在ROCK2蛋白结合口袋中的稳定结合构象,通过分子对接结果从氨基酸角度初步揭示了此类抑制剂的结构-活性关系(SAR);然后,对这3个抑制剂进行MM/GBSA结合自由能(ΔG_(bind))研究,结合自由能计算可知ΔG_(bind)与化合物活性之间具有良好的相关性,且范德华作用能(ΔG_(VDW))对ΔG_(bind)的贡献最大.通过自由能分解获得了对于高活性抑制剂具有重要影响的关键残基.最后,根据分子对接和自由能研究结果设计并合成了3类新型苯并噻唑类似物(D1~D10).生物学评价结果表明,这10个化合物分别具有11~288 nmol/L(ROCK1)和2~105 nmol/L(ROCK2)的抑制活性.其中,化合物D3~D5在人肝微粒体代谢研究中展现出比已报道化合物更高的代谢稳定性.本研究不仅为高活性ROCK抑制剂的设计提供了理论指导,也为ROCK的应用研究提供了一系列结构新颖的高活性抑制剂.     

用荧光光谱和紫外-可见差谱研究抗体-卟啉的相互作用

meso-四(α,α,α,α-O-苯乙酰苯)卟啉与其单克隆抗体1F2结合后,产生显著的增色效应,反映了卟啉与抗体之间刚性紧密的结合,在抗体中的抗原结合部位存在芳香族氨基酸.用同步荧光光谱结合荧光猝灭法得到抗体抗原结合部位的芳香族氨基酸主要为色氨酸和酪氨酸,但酪氨酸的残基数要少于色氨酸.卟啉与抗体的结合比为1:1,解离常数(2.084±0.246)×10^-10mol/L,可见卟啉与McAb₁F₂有很高的亲和力.     

毒素DON单链抗体的同源建模及与DON结合的分子模拟研究

通过同源模建及分子力学构建并优化了呕吐毒素DON单链抗体的三维结构, 结合Procheck和verify_3D方法评估得到合理的抗体模型. 利用分子对接方法研究了单链抗体与其抗原DON的识别及相互作用. 结果表明, 毒素结合到抗体轻链上, 通过轻重链的交界区残基与重链结合, 与残基Pro107之间存在氢键作用. 采用分子动力学模拟和MM/GBSA方法计算了毒素DON与抗体之间的结合自由能, 计算结果与实验值相吻合, 体系疏水相互作用是维持复合物稳定结构的主要驱动力. 动力学模拟氢键分析和能量分解结果共同表明, 残基Pro107参与稳定氢键的形成并贡献很强的范德华作用, 是毒素结合抗体最关键的残基. 本研究为该毒素抗体的结构设计提供了重要的线索和理论依据, 对毒素类分子新型抗体的研究和开发具有理论指导价值.     

基于同源建模和定点突变技术研究嗜热型L-阿拉伯糖异构酶与D-半乳糖的亲和作用

通过同源建模分析选取对Lactobacillus fermentum CGMCC2921来源的L-阿拉伯糖异构酶(简称L-AI酶)催化D-半乳糖生产D-塔格糖起重要作用的氨基酸位点进行突变,发现当Q16,M311,K423和Q438位点的氨基酸突变为丙氨酸时,突变酶Km值降低,其中突变酶M311A降至本体的51.6%,对D-半乳糖的转化率提高了18.7%.当K423位点的氨基酸残基分别突变为丙氨酸、天冬酰胺或精氨酸时,突变酶与底物的亲和力以及D-半乳糖的转化率随着423位点突变氨基酸侧链长度的增加而降低.运用计算机分子模拟技术分析表明,当M311位点氨基酸突变为丙氨酸以后,催化位点氨基酸残基与底物D-半乳糖之间的氢键作用增强,导致与底物亲和力增大,从而提高了酶活力.     

Analysis of Ah receptor binding affinities of polybrominated diphenyl ethers via in silico molecular docking and 3D-QSAR

Polybrominated diphenyl ethers (PBDEs) have become ubiquitous contaminations due to their use as flame retardants. The structural similarity of PBDE to some dioxin-like compounds suggested that they may share similar toxicological effects: they might activate the aryl hydrocarbon receptor (AhR) signal transduction pathway and thus might have adverse effects on wildlife and humans. In this study, in silico computational workflow combining molecular docking and three-dimensional quantitative structure–activity relationship (3D-QSAR) was performed to investigate the binding interactions between PBDEs and AhR and the structural features affecting the AhR binding affinity of PBDE. The molecular docking showed that hydrogen-bond and hydrophobic interactions were the major driving forces for the binding of ligands to AhR, and several key amino acid residues were also identified. The CoMSIA model was developed from the conformations obtained from molecular docking and exhibited satisfactory results as q ² of 0.605 and r ² of 0.996. Furthermore, the derived model had good robustness and statistical significance in both internal and external validations. The 3D contour maps generated from CoMSIA provided important structural features influence the binding affinity. The obtained results were beneficial to better understand the toxicological mechanism of PBDEs.     

Interactions between anti-ErbB2 antibody A21 and the ErbB2 extracellular domain provide a basis for improving A21 affinity

Anti-ErbB2 antibodies are well researched for the therapy of ErbB2-overexpressing tumors. The therapeutic potential and efficacy of these antibodies are closely related to their affinities to ErbB2. Previously we reported that an anti-ErbB2 antibody A21 targeting a conformational epitope comprising several loops in ErbB2 extracellular subdomain I and II could inhibit the proliferation of ErbB2-overexpressing cancer cells in vitro and in vivo. Here we found that another structureless and non-conserved loop in subdomain I of ErbB2 extracellular domain (ECD) was important for binding to A21, and then the antigen-contact sites on A21 were determined by site-directed mutation. The loop was constructed by molecular modeling, and a new model of A21-ErbB2 complex was generated by docking using the crystal structure of the scfv A21 and the model of ErbB2 ECD with the loop built. Based on the complex model, computational design for A21 affinity improvement was performed to enhance its affinity to ErbB2. Two mutants with about 1.7-fold improvement in affinity were obtained. Our study provided a rational molecular basis for affinity improvement and mechanism investigation of A21.     

In silico mutagenesis and docking study of Ralstonia solanacearum RSL lectin: performance of docking software to predict saccharide binding

In this study, in silico mutagenesis and docking in Ralstonia solanacearum lectin (RSL) were carried out, and the ability of several docking software programs to calculate binding affinity was evaluated. In silico mutation of six amino acid residues (Agr17, Glu28, Gly39, Ala40, Trp76, and Trp81) was done, and a total of 114 in silico mutants of RSL were docked with Me-α-L-fucoside. Our results show that polar residues Arg17 and Glu28, as well as nonpolar amino acids Trp76 and Trp81, are crucial for binding. Gly39 may also influence ligand binding because any mutations at this position lead to a change in the binding pocket shape. The Ala40 residue was found to be the most interesting residue for mutagenesis and can affect the selectivity and/or affinity. In general, the docking software used performs better for high affinity binders and fails to place the binding affinities in the correct order.     

Novel Selenium-containing Human Single-chain Variable Fragment with Glutathione Peroxidase Activity from Computer-aided Molecular Design

In order to enhance the glutathione peroxidase(GPX) catalytic activity of the selenium-containing single-chain variable fragments(Se-scFv), a novel human scFv was designed on the basis of the structure of human antibody and optimized via bioinformatics methods such as homologous sequence analysis, three-dimensional(3D) model building, binding-site analysis and docking. The DNA sequence of the new human scFv was synthesized and cloned into the expression vector pET22b(+), then the scFv protein was expressed in soluble form in Escherichia coli BL21(DE3) and purified by Ni2+-immobilized metal affinity chromatography(IMAC). The serine residue of scFv in the active site was converted into selenocysteine(Sec) with the chemical modification method, thus, the human Se-scFv with GPX activity was obtained. The GPX activity of the Se-scFv protein was characterized. Compared with other Se-scFv, the new human Se-scFv showed similar efficiency for catalyzing the reduction of hydrogen peroxide by glutathione. It exhibited pH and temperature dependent catalytic activity and a typical ping-pong kinetic mecha- nism.     

The Interaction Between Crystal Violet and Bovine Serum Albumin: Spectroscopic and Molecular Docking Investigations

The present work reported the investigations on the interaction between a triphenylmethane industrial dye—crystal violet (CV)—and bovine serum albumin (BSA) by spectroscopic methods and molecular docking calculation. The static quenching mechanism of the intrinsic fluorescence of BSA by CV was deduced by the fluorescence measurements and the ground-state complex formation was confirmed from the UV-vis spectra. The site maker competition binding experiments together with the molecular docking showed that the CV molecule specifically bound on the subdomain IIA of BSA. The obtained values of thermodynamic properties of binding suggested that the hydrophobic interaction was dominated as suggested by molecular docking results that the CV molecule was surrounded by hydrophobic amino acid residues. The conformation change of BSA in the binding process was detected by circular dichroism spectra and Fourier-transform infrared (FTIR) spectra and also reflected by the size change of BSA from the measurements by dynamic light scattering (DLS).     

The synergistic effect between hydrophobic and electrostatic interactions in the uptake of amino acids by strongly acidic cation-exchange resins

The goal of this work was to investigate the synergistic effect between the electrostatic and hydrophobic interactions upon the uptake of organic ions with hydrophobic moieties by ion-exchange resins with hydrophobic matrixes. The uptake of neutral amino acids by a macroporous polystyrene-based strongly acidic cation-exchange resin (D001) and two strongly acidic cation-exchange resins (poly(2-acrylamido-2-methyl propanesulfonic acid) and poly(vinylsulfonic acid)) with much less hydrophobic matrixes essentially follow an ion exchange stoichiometry. However, the thermodynamic parameters of the uptakes indicate that besides electrostatic interaction, hydrophobic interaction also contributes to the affinity of the amino acids with hydrophobic side chains for D001. No detectable uptake capacities for the amino acids by D001AM, which was obtained by amidation of the sulfonic acid groups of D001, can be determined. Thus, it is deduced that the hydrophobic interaction alone contributes little to the uptake of these amino acids by D001, of which hydrophobicity is the same with or lower than that of D001AM. These results indicate that synergistic effect exists between the electrostatic and hydrophobic interactions when the two interactions exist in a chelate manner and the hydrophobic interaction contributes to the uptake even if the hydrophobic interaction is so weak that it contributes little to the uptake when it acts alone.     

An affinity improved single-chain antibody from phage display of a library derived from monoclonal antibodies detects fumonisins by immunoassay

Fumonisin B analogs, particularly FB₁, FB₂, and FB₃, are major mycotoxins found in cereals. Single-chain fragment variable (scFv) antibodies represent a promising alternative immunoassay system. A phage-displayed antibody library derived from four monoclonal antibodies (mAbs) generated against FB₁ was used to screen high binding affinity scFv antibodies; the best candidate was designated H2. Surface plasmon resonance measurements confirmed that the H2 scFv displayed a 82-fold higher binding affinity than its parent mAb. Direct competitive enzyme-linked immunosorbent assay demonstrated that the H2 antibody could competitively bind to free FB₁, FB₂, and FB₃, with an IC₅₀ of 0.11, 0.04, and 0.10 μM, respectively; it had no cross-reactivity to deoxynivalenol, nivalenol and aflatoxin. Validation assays with naturally contaminated samples revealed a linear relationship between the H2 antibody-based assay results and chemical analysis results, that could be expressed as y = 1.7072x + 5.5606 (R² = 0.8883). Homology modeling of H2 revealed a favorable binding structure highly complementary to the three fumonisins. Molecular docking analyses suggested that the preferential binding of the H2 scFv to FB₂ was due to the presence of a hydrogen radical in its R1 position, leading to a proper electrostatic matching and hydrophobic interaction. The H2 scFv antibody can be used for the rapid, accurate, and specific detection of fumonisin contamination in agricultural samples.     

基于VHSE结构表征的TAP亲和活性预测及选择特异性分析

应用氨基酸描述子VHSE(Principal component score vector of hydrophobic, steric, and electronic properties)对613个抗原9肽进行结构表征, 在此基础上, 采用支持向量机结合逐步回归变量筛选方法, 成功建立了抗原肽抗原处理相关转运蛋白(Transporter associated with antigen processing, TAP)亲和活性预测模型, 最优线性支持向量机模型的R2, Q2和R2ext分别为0.7386, 0.7270和0.6057. 模型结果分析表明, 影响TAP亲和活性的首要因素是电性, 其次是立体和疏水性质; 底物9肽的P1(N端)及P2, P7和P9(C端)位氨基酸物化性质对TAP亲和活性有重要影响, 而P3, P4, P5和P6位对模型贡献相对较小, P8位则与活性无关. 依据最优模型对模拟点突变9肽的TAP亲和活性的预测结果, 并结合变量载荷分析, 对TAP底物选择特异性进行了分析和总结.     

Development of sensitivity-improved fluorescence-linked immunosorbent assay using a fluorescent single-domain antibody against the bioactive naphthoquinone, plumbagin

A fluorescent single-domain antibody (fluobody), a fusion protein of a green fluorescent protein extracted from Aequorea coerulescens (AcGFP), a mutant that has been codon-optimized for mammalian expression, and a single-chain variable fragment antibody (scFv), against plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone; PL) was successfully constructed and expressed in Escherichia coli. The expressed fluobody was purified, refolded, and characterized to develop a speedy, simple, and sensitive fluorescence-linked immunosorbent assay (FLISA) for the determination of PL. In this study, two kinds of fluobody containing PL-scFv at the N-terminus of AcGFP (N fluobody) or the C-terminus of AcGFP (C fluobody) were constructed with flexible amino acid linker (Gly₄Ser)₂ between PL-scFv and AcGFP for comparative purposes. Characterization of the fluobodies revealed that the C fluobody has better properties as a probe for FLISA than the N fluobody because the fluorescence intensity of C fluobody was 18-fold higher than that of N fluobody. Moreover, C fluobody exhibited a fourfold-higher binding affinity than the N fluobody. More interestingly, the limit of detection for PL measurement in FLISA (24 ng mL⁻¹) was improved to eightfold higher than that in conventional ELISA (0.2 μg mL⁻¹), indicating that a sensitive immunoassay could be developed by using fluobody instead of monoclonal antibody or scFv.