基于序列剖面和可及表面积的蛋白质相互作用位点的预测

蛋白质相互作用位点的预测对于突变设计和蛋白质相互作用网络的重构都是至关重要的.由于实验确定的蛋白质复合物和蛋白质配体复合物的结构依然相当少,预测蛋白质相互作用位点的计算方法就显得十分重要.该文提出了一种以支持向量机为分类器,以邻近残基的序列剖面和可及表面积为输入数据来预测蛋白质相互作用位点的方法.计算结果显示,界面残基和非界面残基被识别的准确率为75.12%,假阳性率为28.04%.与输入数据仅有序列剖面的方法相比,界面残基和非界面残基被识别的准确率提高了4.34%,假阳性率降低了4.63%.     

抗体技术研究进展(1):人源抗体技术

100年来,抗体的发现为人类疾病诊断、治疗和有害物质的分析检测发挥了巨大的作用.特别是1975年发明了单克隆抗体技术以及1986年发明基因工程抗体技术,为研制特异性高、大量均一并大量生产抗体成为了现实,也使嵌合抗体、全人源抗体造福人类并产生巨大的经济效益.为了克服鼠源性单抗可诱发人抗鼠抗体(HAMA),通过嵌合抗体、改构抗体、小分子抗体等技术和改良抗体与抗原结合的特异性,已成为抗体技术研究的主要发展方向,本文主要就抗体人源化及抗体分子小型化,抗体功能复合化两个部分的进展进行综述.     

基于公共信息资源的p185抗体人源化设计

通过计算机辅助分子设计的手段，综合序列分析和结构分析的结果对抗体的结构完成了计算机模拟，并对其进行了人源化的模建和改造，从而建立了一套快速可行的抗体人源化设计方案。对一株抗p185^neu/Her-2单克隆抗体进行了克隆测序，并采用此方案进行了人源化设计。此设计方案完全使用可公共访问的生物信息学工具和数据库，具有良好的实用性和推广价值。     

基因工程抗体

目的 :了解新一代抗体的研究状况。方法 :通过计算机数据库检索相关文献 ,作出综合评述。结果 :利用基因工程技术制备的抗体为新一代的抗体 ,包括人源化的鼠源性抗体、重组抗体片段、转基因动物抗体、植物抗体和噬菌体抗体。结论 :基因工程抗体在疾病的诊断和预防 ,特别是治疗方面有广阔的应用前景。     

HIV-1逆转录酶非核苷类抑制剂MKC分子结合位点的理论研究

1RT1蛋白是一个HIV-1逆转录酶核心蛋白,是抑制剂药物分子的作用靶点.非核苷类抑制剂MKC分子是1RT1蛋白中的配体分子.采用量子力学密度泛函理论,B3LYP计算方法,结合6-31G(d,P)基组,逐个计算MKC分子与其周围半径0.7 nm结构范围内的34个氨基酸残基相互作用和结合能,发现在1RT1蛋白中103Lys残基是MKC分子的结合作用位点,结合能值为-46.73 kJ·mol-1.该结合位点的发现将为进一步设计和寻找抗HIV-1逆转录酶抑制剂药物分子提供一些理论基础.     

HIV-1 Tat与P-TEFb复合物的分子动力学模拟及结合自由能计算

P-TEFb结构中与Tat蛋白的结合位点是新型抗HIV-1药物设计和虚拟筛选的重要靶标.对含有两个锌指结构的Tat.P-TEFb复合物体系进行了14ns的分子动力学模拟,应用MM-PBSA/GBSA方法计算体系的结合自由能,以及通过基于氨基酸残基的能量分解来探究体系中蛋白质之间的相互作用.结果表明范德华作用能是复合物形成的主要驱动力,Cys261与ZN88之间的配位作用是结合自由能的最大贡献者.根据Tat.P-TEFb体系的动力学结构信息和残基能量分解结果预测了P-TEFb结构中可能的活性位点.位点Ⅰ和位点Ⅱ应可作为基于受体三维结构的抗HIV-1药物分子设计的起始位点.模拟计算的结果可以为进一步指导药物设计奠定基础.     

固有无序蛋白与结合配体作用位点的分析与预测

固有无序蛋白(简称IDPs)在生理条件下不具有稳定的二级或三级结构，但是在生物体内通过与结合配体相互作用来发挥重要的生物学功能，故研究固有无序蛋白与配体的相互作用，对理解这些蛋白的功能具有重要的生物学意义。本文基于IDPsBind数据库，获得固有无序蛋白与5类配体分子(DNA,RNA,金属离子，肽，小分子)结合的结合位点，然后对这些结合位点处残基出现在5类结合位点的倾向性进行分析，结果发现：5类配体分子的结合位点处氨基酸的分布是不一样的。然后，利用滑动窗口中心残基的结合配体类型，建立5类结合配体的结合位点数据集，并提取四种特征参数：位置特异性矩阵(PSSM),20种氨基酸组分(AAC),以及残基的疏水性(HP)和溶剂可及表面积(SASA)特征，结合机器学习算法对5类结合位点进行分类识别，在5折交叉检验结果中预测准确率(Acc)最高达到87%,当特征融合后，预测准确率(Acc)达到88.3%。该研究结果对固有无序蛋白与结合配体相互作用的分析提供了很好的参考。     

微分吸附加和法计算多孔材料的吸附特性

采用微分吸附加和法对4种具有代表性和独特孔结构的多孔材料MIL-101、活性炭、SBA-15和Na X分子筛的N2吸附等温线的一阶导数进行拟合。吸附位点方程采用带有吸附质分子相互作用力的Dubinin-Asthakov(DA)、Langmuir和Brunauer-Emmett-Teller(BET)方程。结果表明:微分吸附加和法求取的比表面积和BET、I点法一致。与BET理论相比,该方法考虑了多孔材料表面位点不均匀、吸附质分子间存在相互作用力等因素。拟合结果揭示多孔材料表面存在不同的吸附区域,每个吸附区域存在吸附质与吸附剂作用力不同的吸附位点,而且吸附质分子间的相互作用力不能忽略。此外拟合结果也揭示了导数拟合可以作为对普通吸附等温线的新的拟合途径。     

一种精细药物分子对接模型和优化方法

首先阐述了分子对接设计的基本原理,然后在蛋白质受体中引入关键残基的概念,建立了一个新的柔性分子对接模型．以配体的中心坐标以及它和受体关键残基的旋转键角为设计变量,以设计变量的尺寸为约束,通过最小化分子间相互作用能得到分子的最优取向和构象．一个自适应的遗传算法被用于求解上述优化模型,该算法采用多种群遗传策略、信息熵控制的空间减缩搜索技术以及拟精确罚函数方法,较好地平衡了效率与精度之间的关系,从而能够快速而稳定地逼近最优解．在此基础上,发展了新的精细分子对接程序,该程序可以进行受体与配体的柔性对接．药物分子对接实例证明,本文发展的精细药物分子对接算法和程序能够有效地用于药物分子设计．     

分子动力学研究抗体SPE7与抗原Alizarin red的相互作用机制

研究抗原与抗体的相互作用机制在治疗与抗体相关疾病药物的研发中起到重要作用.采用分子动力学模拟和MM-PBSA(molecular mechanics-Poisson Boltzmann surface area)以及溶解相互作用能SIE(solvated interaction energy)方法研究了抗原Alizarin red(AZN)与抗体SPE7的结合模式.研究结果证明范德华作用主导了抗原AZN与抗体SPE7的相互作用.基于残基能量分解的计算表明抗原AZN能与残基H-W35、H-Y105、L-Y34和L-W93产生较强的相互作用,此结果表明AZN与SPE7分离残基间的π-π相互作用驱动了AZN与SPE7的结合.期望这个研究能为治疗与抗体相关疾病药物的研发提供重要的理论指导.     

抗人胃腺癌细胞株AGS单克隆抗体的制备和初步鉴定

用人胃癌细胞株AGS作为抗原免疫Balb/c小鼠,通过杂交瘤技术制备抗人胃癌细胞mAb,应用间接ELISA法进行筛选,显微操作法进行亚克隆并用琼脂糖双向免疫扩散法鉴定亚类,免疫化学法检测特异性,获得l株分泌抗AGS细胞株的mAb的杂交瘤(命名为1B4).该杂瘤分泌的mAb与人宫颈癌细胞株Hela、肝癌细胞株HepG2和正常胎儿肺细胞均为阴性反应,而与AGS细胞呈阳性反应.结果表明,该mAb对AGS细胞株具有一定的特异性,可为胃癌相关抗原的筛选研究提供一定的帮助.     

A camelid antibody fragment inhibits the formation of amyloid fibrils by human lysozyme

Amyloid diseases are characterized by an aberrant assembly of a specific protein or protein fragment into fibrils and plaques that are deposited in various organs and tissues, often with serious pathological consequences. Non-neuropathic systemic amyloidosis is associated with single point mutations in the gene coding for human lysozyme. Here we report that a single-domain fragment of a camelid antibody raised against wild-type human lysozyme inhibits the in vitro aggregation of its amyloidogenic variant, D67H. Our structural studies reveal that the epitope includes neither the site of mutation nor most residues in the region of the protein structure that is destabilized by the mutation. Instead, the binding of the antibody fragment achieves its effect by restoring the structural cooperativity characteristic of the wild-type protein. This appears to occur at least in part through the transmission of long-range conformational effects to the interface between the two structural domains of the protein. Thus, reducing the ability of an amyloidogenic protein to form partly unfolded species can be an effective method of preventing its aggregation, suggesting approaches to the rational design of therapeutic agents directed against protein deposition diseases.     

The three-dimensional structure of an intact monoclonal antibody for canine lymphoma.

Crystal structures of Fab antibody fragments determined by X-ray diffraction characteristically feature four-domain, beta-barrel arrangements. A human antibody Fc fragment has also been found to have four beta-barrel domains. The structures of a few intact antibodies have been solved: in two myeloma proteins, the flexible hinge regions that connect the Fc to the Fab segments were deleted so the molecules were non-functional, structurally restrained, T-shaped antibodies; a third antibody, Kol, had no hinge residues missing but the Fc region was sufficiently disordered that it was not possible to relate its disposition accurately with respect to the Fab components. Here we report the structure at 3.5 A resolution of an IgG2a antitumour monoclonal antibody which contains an intact hinge region and was solved in a triclinic crystal by molecular replacement using known Fc and Fab fragments. The antibody is asymmetric, reflecting its dynamic character. There are two local, apparently independent, dyads in the molecule. One relates the heavy chains in the Fc, the other relates the constant domains of the Fabs. The variable domains are not related by this 2-fold axis because of the different Fab elbow angles of 159 degrees and 143 degrees. The Fc has assumed an asymmetric, oblique orientation with respect to loosely tethered yet almost collinear Fabs. Our study enables the two antigen-binding segments as well as the Fc portion of a functional molecule to be visualized and illustrates the flexibility of these immune response proteins.     

A gamma-chain complex forms a functional receptor on cloned human lymphocytes with natural killer-like activity

We have recently derived from human fetal blood (25 wks) a series of cloned cell lines that were selected for their ability to kill the conventional natural killer (NK) target cell K562. It was found that a fraction of these clones express CD3 proteins but not the monomorphic Ti alpha beta determinant recognized by WT31 antibody. One interleukin-2-dependent CD3+ WT31- clone, termed F6C7, was used for immunization of mice to generate monoclonal antibodies directed at a potentially novel recognition receptor. It was shown that F6C7 cells, which transcribe Ti beta but not Ti alpha genes, surface-express a clonotypic structure, termed NKFi. Immunoprecipitations performed with anti-NKFi monoclonal antibody (mAb) indicated that the corresponding molecule is resolved in SDS-polyacrylamide gel electrophoresis (PAGE) as a single band of relative molecular mass approximately 85,000 (Mr approximately 85K). After reduction, a major band was detected at 44K and a faint band was present at 41K. The present study was designed to characterize this structure. It was found that NKFi represents either two 44K disulphide-linked gamma (TCR) chains, or possibly one gamma chain associated to an additional undetected molecule, and that the 41K material corresponds to a partially glycosylated fraction of the gamma protein. Anti-NKFi mAb both induces a specific autocrine proliferative response and blocks cytotoxic function, demonstrating that gamma chains serve as functional receptor structures on subpopulations of normal human lymphocytes.     

基于变结构控制的模糊逻辑控制

在现有滑模变结构控制的基础上,引入模糊逻辑决定控制器参数.讨论了如何选择变结构滑动轨线,获得较好的系统快速性,使变结构控制的设计具有更大的灵活性和实用性.通过以矢量控制为速度环的交流伺服系统的仿真和实验结果,说明此方法是很有前途的。     

Molecular cloning of cDNA encoding human interleukin-2 receptor

The human interleukin-2 (IL-2) receptor was purified by affinity chromatography using the anti-Tac monoclonal antibody, and its N-terminal amino acid sequence was determined. Complementary DNA clones were isolated and sequenced to reveal the primary structure of the IL-2 receptor precursor, which has 272 amino acid residues. The receptor is separated into two domains by a putative 19-residue transmembrane region. Two mRNAs (1.4 and 3.5 kilobases) hybridizing to the cDNA clone were found in human T cells bearing the IL-2 receptor. The cDNA directed synthesis of the IL-2 receptor in COS cells.     

基于多维视角的河南省城市-区域系统空间结构特征分析

城市-区域系统的空间结构与区域的协同、综合发展相互依赖、密不可分,整合与优化空间结构可有效地促进区域系统中城市的协调、持续发展.基于扩展断裂点、分形、城市空间分布、空间相互作用等空间结构分析方法,对河南省城市-区域系统空间结构特征进行分析与评价.研究表明河南省城市-区域系统空间结构具有以下特征:城市空间影响范围具有不均衡性;城市之间的空间关联性不够强;城镇点在空间上随机、均匀分布;城市相互作用强度梯度差异大.     

氟苯尼考残留的酶联免疫检测方法的研究

对氟苯尼考进行结构改造制备半抗原及抗原,免疫家兔得到多克隆抗体。经测定,该抗体对氟苯尼考和氟苯尼考胺均有反应,且灵敏度较高,效价达到1∶480 000;在此基础上通过对各实验条件参数的优化建立了间接竞争酶联免疫检测方法,该方法检测限为0.5μg/kg,以20,50μg/kg进行空白样品添加实验,回收率为82.5%～96.0%,批间变异系数在4.1%～15.2%,可初步用于动物组织样本中氟苯尼考残留的检测.     

Replacing the complementarity-determining regions in a human antibody with those from a mouse

The variable domains of an antibody consist of a beta-sheet framework with hypervariable regions (or complementarity-determining regions--CDRs) which fashion the antigen-binding site. Here we attempted to determine whether the antigen-binding site could be transplanted from one framework to another by grafting the CDRs. We substituted the CDRs from the heavy-chain variable region of mouse antibody B1-8, which binds the hapten NP-cap (4-hydroxy-3-nitrophenacetyl caproic acid; KNP-cap = 1.2 microM), for the corresponding CDRs of a human myeloma protein. We report that in combination with the B1-8 mouse light chain, the new antibody has acquired the hapten affinity of the B1-8 antibody (KNP-cap = 1.9 microM). Such 'CDR replacement' may offer a means of constructing human monoclonal antibodies from the corresponding mouse monoclonal antibodies.     

Immunocytochemical localization in rat brain of a prolactin release-inhibiting sequence of gonadotropin-releasing hormone prohormone

The structure of a precursor protein for gonadotropin-releasing hormone (GnRH) of relative molecular mass 10,000 has recently been deduced from cloned complementary DNA sequences derived from human placental messenger RNA. The 56-amino-acid peptide representing residues 14-69 of this prohormone exhibits potent inhibition of prolactin secretion. To investigate whether the same prohormone is synthesized in mammalian brain and describe the anatomical distribution of the prolactin-inhibiting region of this molecule, we have generated antiserum to a synthetic peptide containing residues 40-53 of the human placental precursor. We report here that a substance recognized by this antibody is present in GnRH-containing neurones of the rat brain and appears to coexist with GnRH in secretory granules of nerve terminals in the median eminence. These results indicate homology between hypothalamic and placental prohormones for GnRH and are consistent with the suggestion elsewhere in this issue that a prolactin-inhibiting factor (PIF) is generated from this prohormone and cosecreted with GnRH by nerve terminals in the median eminence.