SPR技术分析生物分子相互作用的研究方法

生物分子的活性功能是通过分子之间的相互作用来实现的,了解这种相互作用的关系时生命科学的研究及揭示生命发生发展的基本机制具有着重要的意义.基于表面等离子共振(SPR)的分析分子相互作用(BIA)的技术是新型的生物传感技术,其无需标、能实时跟踪检测生物分子间结合、解离的整个过程,通过分析传感图谱获取分子相互作用的模式和动力学常数等方面的信息.SPR是研究生物分子相互作用的强有力工具,SPR技术已被广泛应用于生命科学领域的研究,并且显示出广阔的应用前景.概述了SPR技术原理、分析方法及其评述了其存在的问题.     

SPR技术在免疫学研究中的应用

表面等离子共振(Surface Plasmon Resonance,SPR)技术是研究生物分子相互作用的强有力工具之一,该技术使生物分子之间相互作用的实时检测成为可能,并且灵敏度高、无需标记.通过分析传感图谱及分子相互作用的响应值获取分子相互作用的模式和动力学常数等方面的信息,并且获得的信息是能够定性和定量.SPR技术现在已广泛应用于生物、化学、免疫学研究及新药开发等领域.本文主要就SPR技术在免疫学研究中抗体活性检测、抗原表位预测等方面的应用进行了综述.     

血清蛋白与4,5-二溴荧光素相互作用及其分析应用的研究

在 0 .10mol/mL的醋酸溶液中 ,4,5 二溴荧光素能与血清蛋白形成稳定的复合物 ,最大吸收波长 482nm ,与试剂比较 ,红移了 12nm。据此建立了测定血清蛋白的方法 ,用于BSA和HSA的测定 ,分别在 2～ 14mg·L-1有线性关系 ,表观摩尔吸光系数分别为 3.12× 10 5L·mol-1·cm-1和 3.2 7× 10 5L·mol-1·cm-1。应用该法测定了人血清样品总蛋白含量 ,结果令人满意。     

表面等离子体共振技术在药物研究中的应用

表面等离子体共振(surface plasmon resonance,SPR)技术作为一种新型的免标记、实时在线研究生物分子间相互作用的高灵敏传感技术,已经在生命科学领域中得到了大量应用。该文简要介绍了SPR生物传感器的基本原理,重点评述了其在新药筛选和药物作用机制方面的研究进展,并对其前景进行了展望。     

SPR技术与功能基因组和蛋白质组研究

表面等离子体共振(surface plasmon resonance,SPR)依据光学—介质相互作用原理建立,属于实时和非标记的测试方法。SPR方法在研究分子间相互作用方面具有其独特的优势,其非标记和实时检测以及可以进行动力学分析的特点,给研究生物大分子的相互作用提供了诱人的解决方案。近来,随着SPR成像技术和SPR芯片制备技术的进展,将为功能基因组学和蛋白质组学研究提供重要的新的技术平台。     

蓖麻毒素检测技术研究进展

蓖麻毒素是从蓖麻种子的胚乳中提取的一种核糖体失活蛋白。基于其潜在的威胁,建立快速、灵敏的蓖麻毒素检测技术受到人们的高度关注。根据蓖麻毒素的理化性质、免疫原性,已经建立了免疫荧光技术、夹心免疫PCR技术、免疫胶体金标记技术、蛋白芯片技术和生物传感器技术等用于检测蓖麻毒素。     

表面等离子体共振技术在分子生物学中的应用

表面等离子体共振(SPR)技术可以实时、原位地测定生物分子间的相互作用而无需任何标记,可以连续监测吸附和解离过程,并可以进行多组分复合物的相互作用的研究。SPR技术在DNA的复制和转录、DNA的修复、核酸与药物的作用以及肽库和抗体库的筛选等分子生物学领域的应用研究取得了令人瞩目的进展,显示了常规技术无法比拟的优越性。     

基于SPR生物传感器的免疫学检测

利用一种全新的生物大分子相互作用检测仪表面等离子激元共振（SPR）生物传感器,对乙肝表面抗原,抗体,破伤风类毒素,抗体等生物制品进行生物特异性相互作用分析（BIA）,并对其在免疫学检测上的特征进行了探讨。     

生物分子相互作用分析技术的一次突破

细胞生命活动的过程,无不涉及两个或多个生物分子之间相互作用,如信号传导、免疫反应、酶与底物作用等。因此,为了更好地研究生命过程,就迫切需要开发出一种能实时检测出生物分子间相互作用的系统,Phar－maca公司于90年代初就开发出了这样一种技术,即BM技术（BiornoecularInterationAnalssis）,该技术是基于表面等离子共振（Snd。PI。R。ce,简称SPR）的光物理现象［fi。在该现象中,所检测出的共振角与结合在传感片表面的生物分子的质量呈正比例关系。因此以时间对共振角（以共振单位表示）作图可记录相互作用的动态全过程,…     

SPR生物传感器的特点及其在生物特异性相互作用分析中的应用

利用表面等离子模共振技术（SPR）进行生物特异性相互作用分析（BIA）已成为现代基因工程技术中的一种先进的手段。与传统的研究方法如酶联免疫吸附测定（ELISA）相比,它具有方便快捷、灵敏度高,应用范围广,实时监控等多项特点,利用这种新型研究手段对于生命科学的基础研究。医学诊断以及治疗等方面有着十分重的意义。本粗略概括了近几年来利用SPR生物传感器进行基础研究的基本情况以及对其的改进,并简要分析了此项技术的优点以及发展前景。     

Preparation and characterization of monoclonal antibodies against adenylate kinase

Six hybridoma cell lines that can continuously secrete monoclonal antibodies against adenylate kinase (AK) have been produced. The characteristics including the subclass and molecular weight of monoclonal antibodies manufactured by these strains are also determined. Further studies show that the two monoclonal antibodies McAb3D3 and McAMD8 bind easily with AK absorbed on microtitration plates, with affinity constants of 8.4 × 108 M-1 and 9.6 × 108 M-1, while their interactions to AK in solution are much weaker, with affinity constants of 7.0 × 104 M-1 and 3.9×106M-1, respectively. Thus, McAb3D3 and McAMD8 react preferentially to the immobilized AKs. Since pro-teins are often partially denatured when absorbed on microtitration plates, it is suggested that both McAb3D3 and McAMD8 are directed against non-native AK.     

用ELISA法测定抗人心肌肌球蛋白轻链(CMLC)单克隆抗体的K_D值

人心肌肌球蛋白由轻链（ｃａｒｄｉａｃｍｙｏｓｉｎｌｉｇｈｔｃｈａｉｎｓ,ＣＭＬＣ）和重链（ｃａｒｄｉａｃｍｙｏｓｉｎｈｅａｖｙｃｈａｉｎｓ,ＣＭＨＣ）组成．当心肌细胞遭到破坏时,ＣＭＬＣ释放入血,可致血中ＣＭＬＣ浓度增高．血中ＣＭＬＣ的测定是一种高度...     

His-tag的人胞浆磷脂酶A_2的C2结构域的表达、纯化及性质

带有His tag的人胞浆磷脂酶A2 的C2结构域高效表达 ,用内源荧光的变化测定了其稳定性和其与钙离子结合的结合常数 .结果表明 ,带有His tag的C2结构域仍可有效用于研究其折叠及其与钙离子的协同性结合 ,温度从 2 2℃升高到 35℃时 ,C2结构域和钙离子结合的协同性程度显著增强 .     

梭曼和沙林对电鳐电器官乙酰胆碱酯酶抑制作用动力学

梭曼和沙林是毒性很强的有机磷神经毒剂,它们的共同特点是对胆碱酯酶（ＡＣｈＥ,ＥＣ３．１．１．７）都有很强的抑制作用,但两者又不完全相同．过去人们对梭曼和沙林的毒性、毒理研究得较多,而对其对ＡＣｈＥ抑制作用动力学研究得较少,且相互间差异较大．为了对这一...     

肌醇磷脂激酶(PI4-K)单克隆抗体的制备及其对细胞生长的抑制

制备了抗肌醇磷脂激酶 ( PI4- K)单克隆抗体 ( A6D)并测定了抗原 -抗体反应基本特性及功能 .结果表明 ,单克隆抗体与固相及溶液中肌醇磷脂激酶的亲和常数分别为 7.5× 1 0 6和 6× 1 0 8( mol/L) -1.单抗 1 .9× 1 0 -7mol/L可以抑制从细胞提取液的 PI4- K酶活力 50 % .用 FITC标记单抗在蛋白微球引导下进入细胞内 ,主要富集在细胞质膜区 ,并对 He La细胞和小鼠小脑细胞生长有明显抑制作用 .     

抗单纯疱疹病毒单克隆抗体的研究——Ⅱ.单克隆抗体介导细胞毒效应的研究

建立了单克隆抗体(McAb)介导细胞毒作用(ADCC)~(51)Cr释放试验的测定力法。确定了最适工作条件。ADCC测定结果表明,5株抗HSV McAb介导ADCC的活性不同:McAb 1A12、2A8和1G8无ADCC活性;而1D10和2C5两株McAb作1:10稀释时,~(51)Cr释放率分别为27.09％和25.07％,稀释至1:100或1:1000时仍有ADCC活性。结果提示,不同的McAb抗原决定族诱导产生的抗体,在介导ADCC免疫保护作用上有差异,并为McAh治疗临床单纯疱疹病毒感染的可能性提供了实验资料。     

Whole ricin and recombinant ricin A chain idiotype-specific immunotoxins for therapy of the guinea pig L2C B cell leukemia

The therapeutic efficacy of whole ricin, or recombinant ricin A chain, coupled to a monoclonal antibody that reacts with the idiotype of the surface IgM expressed on guinea pig L2C lymphoblasts, was assessed. In vitro studies were done to characterize the immunotoxins (IT) and to demonstrate their specificity before use in vivo. The concentration of whole ricin IT (M6-Ricin) that inhibited protein synthesis by 50% (IC50) in L2C cells was 1.4 X 10(-9) M, in a 5-hr assay, in the presence of lactose to block non-antibody-directed toxicity. M6-Ricin did not inhibit protein synthesis in two control guinea pig cell lines that did not express the idiotype, nor did a whole ricin IT prepared with an isotype-matched monoclonal antibody of irrelevant specificity inhibit protein synthesis in L2C cells. Two recombinant ricin A chain IT, which differed from one another by a factor of 2 to 3 in the number of A chains conjugated per antibody molecule, were less effective in vitro than M6-Ricin (IC50 of greater than 5 X 10(-8) M). For in vivo experiments, the IT were given by the i.p. route 24 hr after the i.p. inoculation of 1 X 10(5) L2C cells. The highest doses of M6-Ricin and M6-Ricin A chain IT tested, 30 micrograms/kg and 3000 micrograms/kg, respectively, were within fourfold to fivefold of their maximum tolerated doses; no deaths or ill effects due to ricin toxicity were noted. These doses increased the median survival time of L2C-bearing guinea pigs to 31 to 34 days, compared with 15 days for untreated animals. This magnitude of increase in survival indicates that 99.999% (5 logs) of injected tumor cells were eliminated, thus accounting for the 12% long-term survival rate obtained. Median survival times for guinea pigs treated with 30 micrograms/kg of the A chain IT were 18 and 21 days for the two conjugates tested, and the median survival for guinea pigs treated with 3000 micrograms/kg of unconjugated antibody was 18 days. Our data demonstrate that recombinant A chain IT are active in vivo and that the B chain of ricin can potentiate IT activity in vivo. Although the potency differs by 100-fold, the therapeutic index of the intact ricin IT is similar to that of the ricin A chain IT.     

研究报告: 基于分子对接和步进序列群的蓖麻毒素核酸适配体序列优化

目的 有效结合分子对接预测和表面等离子体共振实验评价技术，获得亲和力更强、序列最短的最优适配体。方法 针对前期筛选出的靶向蓖麻毒素的3条80 nt单链DNA适配体（L14、P3、L7），在明确各自二维随机区茎环序列与靶蛋白结合能力的基础上，以H-DOCK分子对接为指导，分别确定蓖麻毒素适配体随机区的最短结合单元，从而构建两端延长步进序列群，以表面等离子体共振技术测定序列群序列的亲和力和动力学参数，明确适配体的结合关键结构，从而筛选得到最优适配体。结果 3条全长适配体的随机区适配体L14r、P3r、L7r均可形成一定的茎环结构，其中L14r较L14的亲和力增强9倍、L7r增强2倍、P3r基本不变。对随机区适配体和蓖麻毒素进行分子对接，结果显示，L14r、P3r、L7r的对接分数值皆优于阴性序列40T，结合关键氨基酸个数分别为11、8、9个，存在距离小于5 ?的预测结合位点分别为20、12、15个，具有良好的与蓖麻毒素的结合能力。进一步明确了蓖麻毒素活性口袋所容纳的适配体最短结合单元L14rm、P3rm、L7rm的序列构成，在此基础上构建出两端延长步进序列群。针对该步进群，基于结合关键氨基酸个数、结合位点个数、对接得分等参数的变化和表面等离子体共振测定结果筛选出最优适配体。所获得的最优适配体L14rm、L7rm-2亲和力继续增强了1~2倍。结论 随机区适配体能有效地与蓖麻毒素结合，较之全长适配体亲和力更强，分子对接结合步进序列群设计，仅使用17条序列，便有效获得了3条最优适配体并明确其结合作用。3条结合蓖麻毒素的最优适配体——L14rm、P3r、L7rm-2的K_D值分别为（64±30）、（167±19）、（120±1）nmol/L，亲和力提高到全长适配体的14、1、4倍。     

Utilization of an Enantioselective Surface Plasmon Resonance Electrode for the Selection of the Best C70 Fullerene as Chiral Selector for the Enantioanalysis of L-Cysteine

Abstract

Two novel enantioselective surface plasmon resonance (SPR) sensors based on self-assembled monolayer of (5-6)-fullerene-C₇₀ and diethyl (1,2-methanofullerene C₇₀)-71-71-dicarboxylate as chiral selectors are proposed. Binding assay, apparent affinity constant, and apparent dissociation binding constant have been used to analyze and study the enantioselectivity of C₇₀ fullerene-octadecanethiol film for L-cysteine which was chosen as model analyte. The apparent affinity constant for the complex formed by L-cysteine with (5-6)-fullerene-C₇₀ and diethyl (1,2-methanofullerene C₇₀)-71-71-dicarboxylate films were 1 × 10¹⁰ L/mol, and 6.7 × 10⁸ L/mol, respectively. Accordingly, the chiral selector of choice will be (5-6)-fullerene-C₇₀. No binding was recorded between any of the tested C₇₀ fullerenes and D-cysteine, this proving the enantioselectivity of the proposed SPR sensors.     

Analysis of the binding of phenylalanine to phenylalanyl-tRNA synthetase

Using the complete rate equation for the PP_i-ATP exchange reaction at equilibrium, the dissociation constants of phenylalanine (10^?5m), phenylalanine butyl ester (8 × 10^?5m), benzyl alcohol (6 × 10^?4m), phenylalaninol (2 × 10^?4m), hydrocinnamic acid (3 × 10^?3m) and glycine (>1 m) with the phenylalanyl-tRNA synthetase (Escherichia coli K12) were determined. Taking the model of Koshland (1962) for the estimation of the configurational free energy change due to proximity and orientation, and decomposing the process of binding into several thermodynamic steps, the contribution to binding of the benzyl group, glycine unit, protonated amino group, carboxylate group and joint interactions were estimated. The results are: (1) the standard free energy contributions for binding phenylalanine are benzyl group (?8.2 kcal/mol), glycine unit (?2.5 kcal/mol), protonated amino group (?0.8 kcal/mol) and carboxylate group (1 kcal/mol). (2) The standard free energy change due to the change in the interaction between the protonated amino group and carboxylate group when they are transferred from the aqueous environment to the enzyme environment is ?2.7 kcal/mol. (3) A dissociation constant for glycine of 7.5 m is calculated without the hypothesis that a conformational change occurs in the enzyme when the benzyl unit of phenylalanine binds, permitting an interaction of the enzyme with the protonated amino and/or carboxylate groups.The detection of E·AA² and E·ATP shows that a sequential addition of substrates is not necessary for binding. A comparison of the dissociation constants of E·AA (10^?5m), E·ATP (1.5 × 10^?3m), E·PP (5.5 × 10^?4m), E·I (8 × 10^?5m) and the mixed complexes E·I·ATP (6 × 10^?8m²), E·I·PP (5 × 10^?8m²) and E·AA·PP (7 × 10^?9m²), with phenylalanine butyl ester as the inhibitor, indicates no strong interaction between the binding of ATP or PP_i with the binding of phenylalanine.