噬菌体展示技术及其在抗原表位方面的应用

噬菌体展示技术是一种研究基因功能、蛋白质表达及其相互作用的有效方法,具有大量快速检测、发现并鉴定功能基因及蛋白质之间相互作用的优点。目前,已被广泛应用于生命科学的许多领域,特别是在蛋白质相互作用方面的成功应用,为筛选新的结合蛋白提供了一种简单、有效的手段。     

噬菌体展示技术及其应用

噬菌体展示技术是一项新兴的分子生物学技术;该技术将基因型和表现型有效地联系起来,是后基因组时代的有力工具。目前用于构建展示文库的噬菌体主要有丝状噬菌体、λ噬菌体、T4噬菌体和T7噬菌体;它们所展示的外源蛋白可以保持相对独立的空间结构和生物活性。随着此项技术的不断完善和发展,噬菌体展示技术已在新型疫苗的研制、酶抑制剂的筛选、医学诊断和治疗、多肽药物的开发、蛋白质相互作用的研究等领域得到了广泛的应用,并显示了良好的应用前景。     

噬菌体展示文库技术及在疫苗研制中的应用现状

噬菌体展示文库技术已成为高效表达筛选功能性蛋白的新一代技术,广泛应用于药物筛选及多肽疫苗研制等研究领域。现对噬菌体展示技术及其在疫苗研制中的研究现状作简要的综述。     

噬菌体展示系统的构建及其在疾病防控领域应用研究进展

噬菌体展示技术作为目前应用广泛的展示抗体技术,逐渐成为生产基因工程抗体的重要工具。噬菌体展示技术是以噬菌体或噬菌粒为载体,通过将外源多肽基因整合到噬菌体基因中,以融合表达的形式将外源蛋白展示在噬菌体表面的分子生物学技术。近年来,噬菌体展示技术在抗体筛选领域的应用越来越广泛,与传统制备抗体的方法相比,噬菌体展示技术具有通量高、成本低、操作简单等特点,且通过该技术筛选得到的抗体不仅可在标签蛋白的辅助下进行选择和纯化,还可通过基因测序的方法得到单链抗体的完整基因序列。笔者首先对噬菌体抗体库进行分类,根据抗体的来源将噬菌体抗体库分为天然抗体库和免疫抗体库,通过免疫动物制备的抗体文库的特异性、抗体阳性率明显高于天然抗体文库;其次简述了噬菌体抗体库的构建流程,其中噬菌体表达载体的选择是展示技术的关键,整合了噬菌粒基因的辅助噬菌体侵染其特异性菌株,从而通过抗生素平板对该系统进行选择性筛选;最后讨论了噬菌体展示技术在疾病防控领域应用的研究进展,抗体的首次人源化使同源性抗体在临床上应用成为可能,后续用于预防和治疗病毒性疾病的动物同源性抗体的研发进一步证明了噬菌体展示技术用于生产诊断或潜在治疗试剂的能力。该综述主要聚焦于噬菌体展示系统的构建及其在疾病防控领域的应用,以期对后续通过噬菌体展示技术筛选单链抗体的应用提供指导。     

噬菌体展示技术在动物病毒研究中的应用

噬菌体展示技术是20世纪80年代逐步建立并发展起来的一项分子生物学新技术。它以噬菌体或噬粒为载体,使外源肽或蛋白基因与噬菌体表面特定蛋白基因在其表面进行融合表达,进而通过亲和富集法筛选表达有特异肽或蛋白质的噬菌体。利用噬菌体表面展示技术可以构建抗体库、随机肤库、蛋白质突变体库和cDNA文库,从而广泛用于抗原表位、抗体、配体、细胞内信号转导以及药物筛选等研究,具有广阔的应用前景。本文跟踪了目前噬菌体展示技术在动物病毒检测领域的最新研究进展和发展前景。     

噬菌体展示技术的研究与应用

噬菌体展示技术是通过将外源基因导入到噬菌体结构基因中,使其在外膜蛋白进行融合表达,表达的蛋白具有良好的生物学活性,可以用于多肽筛选、蛋白质相互作用、或新型药物开发等方面的研究。随着噬菌体载体系统的逐渐完善,这一技术已经被广泛应用于生物学领域,文章主要对噬菌体展示技术的原理、噬菌体载体系统及其应用作一简要综述。     

噬菌体展示技术及其应用研究进展

噬菌体展示技术(Phage Display techniques,PDT)起源于1985年,是一种用于筛选和改造功能性多肽、蛋白质强有力的生物技术,广泛应用于研究蛋白质的结构与功能,蛋白质间的识别与作用,分离体内特异蛋白质与基因以及实验进化等多个分子生物学领域.目前噬菌体展示技术的研究进展非常迅速,在抗原决定簇的定位、蛋白质相互作用位点的确定、特异调节分子的分离和人工抗体和疫苗的制备、诊断技术、酶抑制剂的研究开发、多肽药物的研制等生物技术研究的不同领域得到了应用,并对这些领域产生了深远的影响.     

噬菌体展示技术及其应用研究进展

噬菌体展示技术（Phage Dlsplay techniques，PDT）起源于1985年，是一种用于筛选和改造功能性多肽、蛋白质强有力的生物技术，广泛应用于研究蛋白质的结构与功能，蛋白质间的识别与作用，分离体内特异蛋白质与基因以及实验进化等多个分子生物学领域。目前噬菌体展示技术的研究进展非常迅速，在抗原决定簇的定位、蛋白质相互作用位点的确定、特异调节分子的分离和人工抗体和疫苗的制备、诊断技术、酶抑制剂的研究开发、多肽药物的研制等生物技术研究的不同领域得到了应用，并对这些领域产生了深远的影响。     

噬菌体展示技术及其在单克隆抗体制备中的应用

噬菌体展示技术是一种强大的高通量多肽筛选技术,常用于确定靶标蛋白的亲和力与结合位点,并广泛应用于单克隆抗体的制备。随着下一代测序和微流控技术的出现,噬菌体展示技术已成为制备单克隆抗体更为强大和流行的工具。通过对噬菌体展示技术的历史发展与分类、噬菌体展示技术制备单克隆抗体的过程以及其在制备单克隆抗体上的应用进行论述,以期为相关研究人员提供参考。     

噬菌体展示技术及其在动物病毒性疾病中的应用

噬菌体展示技术是在20世纪80年代中期兴起的一项技术,该技术可以实现外源蛋白质或多肽基因的表达产物与噬菌体衣壳蛋白融合,进而展示到噬菌体表面。近年来,噬菌体展示技术在动物病毒性疾病中的应用范围越来越广,在筛选动物病毒抗原表位及抗病毒多肽、疫苗研制等领域显示出了明显的优势。文章就噬菌体展示技术的基本原理及噬菌体展示技术在动物病毒性疾病方面的应用作一综述,以期对后续噬菌体展示技术在病毒性疾病的诊断、防制等方面的应用作指导。     

A health index to evaluate clinically a ß-hemolytic streptococcal infectious disease model in the horse

Quantification of the clinical manifestations of a disease has been a serious problem particularly as related to clinical trials or drug efficacy studies. Historically, this quantification has been limited to categorizing each patient into one of three or four groups, e.g. worse, no improvement, improved. This problem becomes serious when an investigation utilizes an experimentally induced animal disease model. A health index, which quantifies the clinical state of horses which have an experimentally induced beta-hemolytic streptococcal infection, is described. Aspects of experimental design and statistical analysis are also discussed in relationship to the use of the index for drug efficacy studies.     

溶出度测定在药物制剂研发与质量评价中的应用进展

介绍了药物溶出度及溶出度试验的概念,并着重阐述了溶出度在新药研发的处方筛选、工艺设计、剂型选择和生物利用度预测以及在药物质量控制和再评价方面的应用.     

Target-based and whole-worm screening approaches to anthelmintic discovery

Experimental approaches for identifying new anthelmintics include target-based and whole-worm screening methods. The former involves basic research into characterising and validating new targets, mostly proteins, followed by identification of inhibitors or agonists through the use of target-based screening assays and/or in silico drug design. The latter experimental approach uses whole-worm assays to identify anthelmintic agents with unknown modes of action, or where the primary interest lies in whether analogues are able to kill (or disable) worms rather than in measuring their direct impact on their likely target. This paper focuses initially on the intestine and external layers of nematodes as potential drug targets. Specific anthelmintic agents targeting either tissue are discussed to illustrate the impact of disruption to these structures. In both cases, the activity of these agents against insects was known, and activity against nematodes was identified using whole worm screening assays. Recent literature identifying ecdysone signalling pathway receptors in nematodes is then used to provide an example of basic research into a specific target that may lead to the development of high-throughput target-based drug screening assays. Finally, the role of whole-worm screening approaches versus target-based screening is discussed briefly.     

The role of molecular biology in veterinary parasitology

The tools of molecular biology are increasingly relevant to veterinary parasitology. The sequencing of the complete genomes of Caenorhabditis elegans and other helminths and protozoa is allowing great advances in studying the biology, and improving diagnosis and control of parasites. Unique DNA sequences provide very high levels of specificity for the diagnosis and identification of parasite species and strains, and PCR allows extremely high levels of sensitivity. New techniques, such as the use of uniquely designed molecular beacons and DNA microarrays will eventually allow rapid screening for specific parasite genotypes and assist in diagnostic and epidemiological studies of veterinary parasites. The ability to use genome data to clone and sequence genes which when expressed will provide antigens for vaccine screening and receptors and enzymes for mechanism-based chemotherapy screening will increase our options for parasite control. In addition, DNA vaccines can have desirable characteristics, such as sustained stimulation of the host immune system compared with protein based vaccines. One of the greatest threats to parasite control has been the development of drug resistance in parasites. Our knowledge of the basis of drug resistance and our ability to monitor its development with highly sensitive and specific DNA-based assays for 'resistance'-alleles will help maintain the effectiveness of existing antiparasitic drugs and provide hope that we can maintain control of parasitic disease outbreaks.     

Evidence‐Based Medicine: The Design and Interpretation of Noninferiority Clinical Trials in Veterinary Medicine

Noninferiority trials are clinical studies designed to demonstrate that an investigational drug is at least as effective as an established treatment within a predetermined margin. They are conducted, in part, because of ethical concerns of administering a placebo to veterinary patients when an established effective treatment exists. The use of noninferiority trial designs has become more common in veterinary medicine with the increasing number of established veterinary therapeutics and the desire to eliminate potential pain or distress in a placebo‐controlled study. Selecting the appropriate active control and an a priori noninferiority margin between the investigational and active control drug are unique and critical design factors for noninferiority studies. Without reliable historical knowledge of the disease response in the absence of treatment and of the response to the selected active control drug, proper design and interpretation of a noninferiority trial is not possible. Despite the appeal of conducting noninferiority trials to eliminate ethical concerns of placebo‐controlled studies, there are real limitations and possible ethical conundrums associated with noninferiority trials. The consequences of incorrect study conclusions because of poor noninferiority trial design need careful attention. Alternative trial designs to typical noninferiority studies exist, but these too have limitations and must also be carefully considered.     

Demonstrating bioequivalence using clinical endpoint studies

For drug products not amenable to blood level studies, clinical endpoint studies have been used as an indirect measure of formulation difference in bioavailability between test and reference products. However, clinical endpoint studies are not as sensitive in detecting formulation differences as blood level studies and offer numerous challenges to both regulatory authorities and sponsors. The objective of this article is not to suggest new regulatory policies, but to explore new methodologies and alternative solutions to clinical endpoint bioequivalence (BE) studies, which are used when a blood level study is not considered to be appropriate. To achieve this objective, this article identifies situations where a clinical endpoint study might be appropriate, lists the advantages and disadvantages of this type of study design, and discusses possible alternative solutions. It is concluded that future evidence-based research is needed to explore new methodologies such as clinical trial simulations of various study designs, new statistical methods, and new in vitro methods to demonstrate BE.     

Oocyte-based screening system for anti-microtubule agents

Taxol and vinblastine have been widely used in cancer chemotherapy as anti-microtubule agents. However, there are on-going efforts to find new anti-microtubule agents with fewer of the side effects associated with these drugs, such as toxicity or the development of resistance. The standard method used to identify anti-microtubule agents has been the in vitro microtubule polymerization assay. One limitation of this system is that the only compounds selected are those that act on tubulin. Novel compounds whose targets are upstream or are related unknown molecules are not detected. Therefore, many researchers have recently tried to develop novel, phenotype-based drug screening systems. In this study, we developed an oocyte-based screening system for anti-microtubule agents. Dramatic phenotypic changes in microtubules can easily be observed in ovulated oocytes treated with microtubule-stabilizing or -destabilizing agents, such as taxol or vinblastine. After culturing with test samples for 5 h, oocytes were analyzed with fluorescence microscopy after immunostaining. In the oocyte-based screening system, the effective dose (ED50) of taxol for microtubule polymerization is approximately 5 nM, and the ED50 of vinblastine for microtubule depolymerization is approximately 2.5 nM. In addition, taxol-like and vinblastine-like compounds can be evaluated simultaneously in a single assay using this system.     

寄生虫乳酸脱氢酶研究进展

大多数体内寄生虫的生存必须依赖糖酵解途径将葡萄糖代谢为乳酸以提供能量。乳酸脱氢酶（lactate dehydrogenase, LDH）是糖酵解途径的末端酶,催化丙酮酸还原为乳酸及乳酸氧化为丙酮酸的可逆反应,与寄生虫生存密切相关。研究表明,各种寄生虫LDH在理化性质和分子结构方面均有独特的特性,是良好的诊断分子和潜在的药物作用靶标。对寄生虫LDH功能的研究,对于促进寄生虫病诊断、疫苗研究以及新抗虫药物的研发具有重要意义。本文对国内外寄生虫LDH的研究现状进行了综述。     

人兽共患寄生虫病候选疫苗分子筛选方法研究进展

人兽共患寄生虫种类多、宿主广泛且危害严重。血吸虫病、棘球蚴病、囊尾蚴病、旋毛虫病、弓形虫病等是常见的重要人兽共患寄生虫病。人类和家畜饱受寄生虫病的危害,这对公共卫生和畜牧业造成了很大的影响。控制传染源、切断传播途径和保护易感群是控制人兽共患寄生虫病流行的综合防控措施。在综合防控策略中,疫苗的使用是切断循环链、控制乃至消灭人兽共患寄生虫病的理想和有效途径之一。选用高效的抗原筛选方法挖掘潜在的疫苗候选分子是开发疫苗的前提和关键。抗原筛选技术的更新换代使得研究者发掘出了更多新抗原和保护性多肽。现有的抗原筛选方法主要包括传统的粗抗原筛选法、cDNA文库筛选法、蛋白质组学筛选法、生物信息学及多组学技术联合筛选法。很多抗原筛选的方法是伴随寄生虫疫苗研究的发展应运而生的,粗抗原筛选法是基于抗原抗体相互反应的免疫学原理而设计的,此方法筛选的天然抗原可引起机体较强的免疫反应;cDNA文库筛选抗原的优势在于筛选更有针对性,所以候选产物的成分更单一、明确;蛋白质组学筛选法是基于质谱而兴起的一种筛选技术,它既可对未知蛋白组分进行鉴定,还可对鉴定结果进行差异比较,在未知分子的发现和功能特殊的靶分子筛选中发挥着重要作用;随着后基因时代的到来,生物信息学及多组学联合筛选技术使得抗原筛选逐步进入了多维、立体的筛选模式,也使得候选抗原及其表位的功能研究更加深入,这为基因工程疫苗和多肽疫苗候选分子的筛选提供了技术手段。     

家蚕模式化研究进展

建立具有中国特色的新型模式生物系统,是我国模式生物研究与发展的重要方向之一。家蚕(Bombyx mori)不仅具备模式生物的基本要素,而且还有其突出的特点和优势,2002年国际无脊椎动物协会正式将家蚕确定为鳞翅目模式昆虫。家蚕代表了生物界的一个大类群,易于在实验室内饲养和繁殖,世代短,子代群体大,遗传背景清楚,便于进行实验操作,并且遗传资源丰富,具有悠久的基础研究历史和现代科学研究基础,所以家蚕作为实验动物,在经典遗传学、动物遗传学、发育生物学、细胞生物学、昆虫免疫学、鳞翅目害虫防治、环境监测乃至医药学等研究领域受到广泛的关注。家蚕是少数完成基因组计划从框架图到精细图、重测序的代表物种,家蚕生物信息资源及分析平台以及功能基因鉴定和功能分析的平台日趋完善,使家蚕已经成为基因组学研究的典型范例之一。后基因组时代即使一个科学目标也有多种模式生物进行综合化分析,新的模式生物系统将逐渐形成。中国家蚕基因组计划推动了家蚕模式生物系统化的研究。建立具有中国特色的家蚕模式生物系统,并推进在鳞翅目害虫防治、人类疾病模型、医药筛选及环境检测等领域的实际应用,将有助于我国生命科学和生物产业相关领域形成优势竞争力。