生物分子相互作用分析技术应用实例（二）——多聚物和转录蛋白的研究

BIA技术(biomolecular interaction analysis)即生物分子相互作用分析技术的应用范围相当广泛.这里介绍利用BIAcore研究信号传导中各蛋白质之间的相互作用及多聚物的形成及机理以及转录调节蛋白与启动子（DNA）的研究.     

基于表面等离子共振的新型生物传感技术及其在生命科学中的应用

生物分子的活性功能是通过分子之间的相互作用来体现的,了解这种相互作用的过程对于生命科学领域的研究及揭示生命发生发展的基本机制具有重要的意义。基于表面等离子共振(surface plasmon resonance,SPR)的新型生物传感技术——BIAcore(biomolecular interaction analysis)是研究生物分子相互作用的理想工具。它可以实时跟踪检测生物分子间结合、解离的整个过程,已被广泛应用于蛋白质组学、信号转导、新药开发、遗传学分析和食品检测等领域,并且显示出广阔的应用前景。     

靶向表皮生长因子受体的全新小分子配体筛选

肿瘤靶向分子的筛选一直是肿瘤治疗和早期诊断的研究热点 . 表皮生长因子受体 (EGFR) 在很多肿瘤细胞表面过量表达,是一个理想的药物输送靶点 . 选择了 EGFR 表面不同于表皮生长因子 (EGF) 结合位点的一个凹陷部位作为计算机模拟筛选的结合位点,然后使用 DOCK 软件包对 DTP-Plated 有机小分子数据库进行了两遍筛选,最后选择了 7 个有机小分子作为可能的靶向分子 . BIAcore 体外结合实验对所选择的小分子样品进行了进一步的验证,结果表明,小分子 NSC51186 能特异地与 EGFR 结合 . 小分子 NSC51186 和 EGFR 之间的动力学常数也得到进一步的测定 . 新的靶向分子和药物、纳米粒子或者基因载体相连,将有可能用于靶向于 EGFR 的肿瘤治疗和诊断 .     

Gβ1γ2蛋白的纯化及其与腺苷酸环化酶的互作关系

对G蛋白β₁γ₂亚基及偶联组分在信号传导中的作用进行了初步研究. 以离体昆虫细胞Sf9(Spodoptera frugiperda, 秋粘虫卵巢细胞)或H5(Trichoplusia ni, 粉纹夜蛾细胞)为生物反应器, 高效表达了G蛋白β₁γ₂亚基. 用Ni-NTA亲和层析柱, 经快速蛋白纯化技术(FPLC)获得高纯度的Gβ₁γ₂. 活性测定表明, 纯化的GGβ₁γ₂能显著刺激腺苷酸环化酶(AC2)的活力. 应用基于表面胞质团共振现象的BIAcore技术, 采用生物传感芯片NTA(biosensor chip NTA)直接证明腺苷酸环化酶2 (AC₂)的胞质末端C₂区域为G蛋白β₁γ₂亚基的结合区, 从而明确了二者互作的活性位点和结合位点同位于该区域.     

红外成像技术在生命科学中的应用

红外成像技术是利用物体自身各部分对红外热辐射的差异把红外辐射图像转换为可视图像的技术.对红外成像技术历史进行了简单介绍,对远红外成像技术在生命科学包括医学、植物、动物及农业中的应用进行了综述,并对红外成像技术在生命科学中的应用作了展望.     

光谱技术在血液诊断学上的应用

光谱技术能够了解物质结构、物质成分和它们的含量,且分析灵敏度和精度极高.光谱技术不仅可以测定生物组织的光学特性参数,而且光谱本身含有组织分子的许多信息.本文主要介绍:利用光谱技术应用于血液诊断学上的原理;医学上对癌症、白血病等疾病的传统诊断方法及其不足之处;应用光谱技术在诊断学上研究的现状,包括利用血清荧光光谱进行癌症的诊断、利用光声光谱进行白血病的诊断以及利用吸收光谱测量血液中微量元素的含量来诊断疾病;讨论利用光谱技术进行诊断存在的问题并展望其在疾病诊断上的应用前景.     

实时荧光定量PCR在猪肺炎支原体检测中的应用

实时荧光定量PCR技术是一种利用荧光检测方法来定量核酸的技术,具有高度的灵敏性、特异性和精确性.综述了荧光定量PCR技术的基本原理及其在猪肺炎支原体检测中的应用.     

噬菌体抗体库技术筛选抗VEGF165单克隆抗体

目的:获得抗血管内皮生长因子165(VEGF165)单克隆抗体,并对其功能进行初步验证。方法:利用噬菌体抗体库展示技术筛选与VEGF165结合的噬菌体克隆并测序,以测序正确的阳性克隆质粒为模板,PCR扩增抗体的轻重链可变区基因,并克隆至哺乳动物细胞表达载体中,构建全抗体表达载体;将全抗体表达载体转染293E细胞,收取培养细胞上清,利用ProteinA亲和纯化抗体;通过结合ELISA、表面等离子共振检测抗体的亲和力,以人脐静脉内皮细胞(HUVEC)为模型验证抗体功能。结果:经过噬菌体抗体库展示技术筛出1个与VEGF165特异性结合的抗体序列VG2;293E细胞表达了VG2全抗体蛋白,SDS-PAGE显示VG2抗体纯度较高;BIAcore检测结果表明该抗体具有较高亲和力(KD=0.56nmol/L),竞争抑制ELISA结果表明VG2抗体能抑制VEGF与VEGF受体(VEGFR)的结合(IC50为1.470μg/mL),进一步实验结果表明VG2能够抑制VEGF引起的HUVEC增殖。结论:制备了靶向VEGF165的全人源单克隆抗体VG2,该抗体具有较高的亲和力,能阻断VEGF165/VEGFR2的结合,并抑制HUVEC的增殖,可以作为潜在药物应用于肿瘤治疗。     

PEG修饰对PLL-EGF基因载体靶向结合作用的影响

聚阳离子基因载体系统由于安全性好和便于设计等优点,近年来在基因治疗中的应用发展迅速.在进行基因药物的体内靶向输送时,目前国际上主要通过在基因输送系统中修饰聚乙二醇(PEG)和靶向分子来提高体内输送的稳定性和靶向性.PEG的修饰可能会遮蔽靶向分子的功能呈现,因此建立定量分析方法评价PEG修饰对靶向结合作用的影响非常重要.将连接有表皮生长因子(EGF)的聚赖氨酸(PLL)基因载体作为研究模型,建立BIAcore检测方法,比较PLL-EGF,PEG7000修饰的PLL-EGF,PEG20000修饰的PLL-EGF对表皮生长因子受体(EGFR)的结合和解离速率,评价PEG修饰对PLL-EGF靶向功能呈现的影响.结果表明,PEG7000的修饰降低了EGF和EGFR之间的结合速率,提高了解离速率,整体减弱了靶向分子的靶向结合能力.PEG20000的修饰进一步减弱靶向分子功能的呈现.因此在进行靶向型聚阳离子基因输送系统设计时,考察PEG修饰对靶向结合能力的影响程度非常重要.该研究结果也对其他基因载体系统的设计提供必要的参考.     

小麦、黑麦FISH技术特异序列探针的研究进展

荧光原位杂交(fluorescence in situ hybridization,FISH)技术在染色体定位、基因克隆、遗传标记以及染色体畸变研究中得到了广泛的应用.随着分子生物学的飞速发展,用于FISH技术的染色体特异重复序列探针的开发和利用有了巨大进展.就近年来小麦、黑麦等FISH技术中染色体特异重复序列探针的研究进展及应用作一综述.     

生物分子相互作用分析法测定甜菜组织中的钙调素

以甜菜为实验材料,经甜菜坏死黄脉病毒（beet necrotic yellow vein virus, BNYVV）侵染后,用生物分子相互作用分析（biomolecular interaction analysis,BIA）技术测定植物组织中钙调素（calmodutin,CaM）含量,分析在BNYVV侵染下植物组织中CaM的变化。该方法可以实时检测生物分子之间的相互作用,受杂质影响小,可简化样品的前处理,能够快速高通量分析大量的蛋白质样品,灵敏度高,准确性好。     

No apparent progress in bioelectrical impedance accuracy: validation against metabolic risk and DXA

Bioelectrical impedance (BIA) is quick, easy, and safe when quantifying fat and lean tissue. New BIA models (Tanita BC-418 MA, abbreviated BIA(8)) can perform segmental body composition analysis, e.g., estimate %trunkal fatness (%TF). It is not known, however, whether new BIA models can detect metabolic risk factors (MRFs) better than older models (Tanita TBF-300, abbreviated BIA(4)). We therefore tested the correlation between MRF and percentage whole-body fat (%BF) from BIA(4) and BIA(8) and compared these with the correlation between MRF and dual-energy X-ray absorptiometry (DXA, used as gold standard), BMI and waist circumference (WC). The sample consisted of 136 abdominally obese (WC >or= 88 cm), middle-aged (30-60 years) women. MRF included fasting blood glucose and insulin; high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, and triglycerides; high sensitive C-reactive protein, plasminogen activator inhibitor-1 (PAI-1), and fibrinogen; and alanine transaminase (ALT) liver enzyme. We found that similar to DXA, but in contrast to BMI, neither %BF BIA(4) nor %BF BIA(8) correlated with blood lipids or ALT. In the segmental analysis of %TF, BIA(8) only correlated with inflammatory markers, but not insulin, blood lipids, or ALT liver enzyme (in contrast to WC and %TF DXA). %TF DXA was associated with homeostatic model assessment insulin resistance (HOMA-IR) independently of WC (P = 0.03), whereas %TF BIA(8) was not (P = 0.53). Receiver-operating characteristic (ROC) curves confirmed that %TF BIA(8) did not differ from chance in the detection of insulin resistance (P = 0.26). BIA estimates of fatness were, at best, weakly correlated with obesity-related risk factors in abdominally obese women, even the new eight-electrode model. Our data support the continued use of WC and BMI.     

Advances in surface plasmon resonance biomolecular interaction analysis mass spectrometry (BIA/MS)

Ongoing, worldwide efforts in genomic and protein sequencing, and the ability to readily access corresponding sequence databases, have emphatically driven the development of high‐performance bioanalytical instrumentation capable of characterizing proteins and protein–ligand interactions with great accuracy, speed and sensitivity. Two such analytical techniques have arisen over the past decade to play key roles in the characterization of proteins: surface plasmon resonance biomolecular interaction analysis (SPR‐BIA) and matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF). SPR‐BIA is used in the real‐time investigation of biomolecular recognition events, and is thereby capable of providing details on the association and dissociation kinetics involved in the interaction, information ultimately leading to the determination of dissociation constants involved in the event. MALDI‐TOF is used in the structural characterization, identification and sensitive detection of biomolecules. Although the two techniques have found many independent uses in bioanalytical chemistry, the combination of the two, to form biomolecular interaction analysis mass spectrometry (BIA/MS), enables a technique of analytical capabilities greater than those of the component parts. Reviewed here are issues of concern critical to maintaining high‐levels of performance throughout the multiplexed analysis, as well as examples illustrating the potential analytical capabilities of BIA/MS. Copyright © 1999 John Wiley & Sons, Ltd.     

Epitope Mapping by Label-Free Biomolecular Interaction Analysis

The diversity of B-cell response to a large immunogen gives rise to a series of antibodies that can be used for epitope mapping of an antigen. This is based on the relative reaction pattern for all antibodies in relation to each other and other ligands to the studied protein. With the introduction of an instrument system, BIAcore, label-free real-time biomolecular interaction analysis (BIA) was made possible. It is based on biosensor technology, with a carboxymethyl-dextran-coated gold surface and an integrated fluidics for transport of liquid. The basic idea is to measure label-free binding of an analyte from a continuous flow to an immobilized ligand in real time. With an automatic approach, quantitative analysis and sequential injection characteristic biospecific binding parameters such as affinity and kinetic constants can be measured. The instrument system was adopted at an early stage for epitope mapping. With label-free detection, antibodies from tissue culture media can be analyzed without purification. Binding of both antigen and a series of antibodies can be individually determined in molar ratio by sequential injections. The quantitative aspects of BIA offer the possibility of further refined epitope mapping. The relative binding pattern for 30 monoclonal antibodies against HIV-1 p24 core protein has been analyzed. Multideterminant analysis and peptide identification of binding sites were performed. Verification of the binding pattern has also been performed in relation to mapping with ELISA as well as the binding to peptides derived from the antigen sequence. Functional domains of proteins in relation to an epitope map have been identified forTaqpolymerase.     

Practical considerations in BIA/MS: optimizing the biosensor-mass spectrometry interface

Biomolecular interaction analysis mass spectrometry (BIA/MS) is a multiplexed analytical technique that utilizes a unique combination of surface plasmon resonance (SPR) and matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for the detection and analysis of small amounts of proteins residing in complex biological systems. In order to achieve high sensitivity during BIA/MS, certain experimental parameters and sequences of events need to be optimized and maintained. Immobilized ligand density, flow rate and biosensor control (in SPR-BIA) and matrix choice and application (in MALDI-TOF MS) have significant influence on the final outcome of the BIA/MS analysis and, consequently, need to be optimized and carefully controlled. In addition, chip washing and cutting are essential in converting the SPR-active sensor chips into target surfaces amenable to MALDI-TOF MS. Reviewed here are the prerequisites for successfully interfacing SPR-BIA with MALDI-TOF MS.     

Chemiluminometric biochemical induction assay (CBIA) for the detection of DNA-damaging agents

A microbroth chemiluminometric version of the biochemical induction assay (BIA) was developed using a chemiluminescent substrate widely used to detect beta-galactosidase in high-throughput screening (HTS) laboratories. The assay was run in both 96-well and 384-well plate formats using the Zymark RapidPlate liquid handling system to transfer samples and reagents. Chemiluminescence was read using the Victor-2 multilabel counter. The new microbroth chemiluminometric method, the CBIA, allowed rapid screening of samples, crude extracts, and pure compounds for their DNA-damaging effects in bacteria. In screening a small subset of our natural products library samples by the agar plate BIA and the CBIA, the latter yielded a higher hit rate, suggesting it is more sensitive than the agar plate assay. The CBIA was unaffected by the colored samples often encountered during screening of crude natural products extracts.     

生物传感技术的新应用——生物分子相互作用分析技术（BIA）

BIA 技术(biomolecular interaction analysis)——生物分子相互作用分析技术是利用生物传感技术发展的全新概念.生物分子间的相互作用可在免标记的状态得到实时的追踪和分析.文章简单介绍BIA的原理和应用范围.在以后的几期杂志中,还将介绍一系列应用实例.结合已发表的近400篇应用文献,读者可以看到此新技术应用范围之广,得到的信息之多,堪称开创了生物技术的新纪元.     

Detection of mutations in PCR products from clinical samples by surface plasmon resonance

Two different strategies for scanning and screening of mutations in polymerase chain reaction (PCR) products by hybridization analysis are described, employing real-time biospecific interaction analysis (BIA) for detection. Real-time BIA was used to detect differences in hybridization responses between PCR products and different 17-mer oligonucleotide probes. For the analysis using a biosensor instrument, two different experimental formats were investigated based on immobilization of either biotinylated PCR products or oligonucleotide probes onto a sensor chip. Applied on the human tumour suppressor p53 gene, differences in hybridization levels for full-match and mismatch situations employing both formats allowed the detection of point mutations in exon 6 PCR products, derived from a breast tumour biopsy sample. In addition, a mutant sample sequence could be detected in a 50/50 background of wild type exon 6 sequence. The suitability of the different formats for obtaining a regenerable system and a high throughput of samples is discussed. © 1997 John Wiley & Sons, Ltd.