双功能基因工程抗体在不同系统中表达的研究进展

基因工程双功能抗体是近年来抗体研究的一个热点,利用分子生物学技术,在体外构建基因工程抗体基因片段,与各种类型的表达载体相连,使重组抗体在原核细胞,哺乳类动物细胞,昆虫细胞表达系统中获得表达,其表达量,功能蛋白活性各具特点,显示出在临床肿瘤、病毒病的治疗和诊断方面的应用前景,本文就双功能抗体的构建方式及多种表达系统的研究进展进行综述。     

体外翻译系统在分子生物学研究中的应用

体外翻译系统又称无细胞蛋白质合成系统,是分子生物学中一种常规的表达系统,该系统可用于蛋白质快速分析鉴定,基因转录和翻译的调控机理的研究以及分子间的相互作用的研究,如蛋白质和蛋白质的相互作用,蛋白质与DNA的相互作用,蛋白质与RNA的相互作用等。     

无细胞蛋白表达系统新进展及在生物制药工程中的应用

无细胞蛋白表达系统作为体外合成蛋白表达技术的一种,其基础是细胞抽提物。具备反应操控简便和遗传背景简单等诸多优点。经过大量研究人员的努力,无细胞蛋白表达系统取得新进展,具有较大潜力,可应用于生物制药工程。现就无细胞蛋白表达系统发展进程进行分析,总结该系统在生物制药工程中的应用。     

小鼠胚胎干细胞体外发育分化模型

胚胎干细胞 (Embryonicstemcell ,EScell)是多潜能性细胞 ,它在体外既可维持不分化而无限增殖 ,又能参与胚胎发育分化为各种类型细胞和组织而形成器官 ;小鼠ES细胞可供的数量大、在体外培养条件可进行精确调控、实验比较经济加上现代基因及其他生物操作等技术 ,因此小鼠ES细胞体外发育分化系统被广泛地作为模型系统加以利用。小鼠ES细胞体外发育分化研究为推动其他哺乳类动物以及人的ES细胞研究 ,从而将更好地进行细胞、组织工程实验为人类细胞组织和基因治疗服务创造了有利条件。本文将ES细胞体外发育分化情况加以概述 ,以便更好地开展ES细胞体外研究     

体外展示技术及其在抗体工程中的应用

体外展示技术包括核糖体展示技术、mRNA展示技术、DNA展示技术,是在无细胞蛋白质表达体系内将基因型和表型通过一定的方法连接在一起,体外高通量的筛选多肽和蛋白质的技术。抗体的产生是一个不断选择的过程,利用体外展示技术在体外选择针对某一抗原的抗体分子,并结合基因工程技术对抗体进行改造,以产生高亲和力、高特异性的抗体。体外展示技术的研究和应用已越来越广泛,有望成为下一代的抗体制备技术。     

分析超速离心技术及其在分子生物学中的应用

分析超速离心技术是生物化学和分子生物学中的一种经典方法。该技术是基于热力学和流体动力学第一定律,利用分析超速离心机研究蛋白、核酸和聚合物及其他各种复合物在溶液状态下的行为。从该技术利用的仪器、原理和数据分析方法及其在分子生物学中的应用等方面进行了综述,并对这一技术在现阶段的应用进行了总结和展望,以期为国内相关领域研究人员提供参考。     

文库筛选与分子进化的核糖体展示新方法

利用适当的文库筛选技术快速、简便地从DNA文库、随机肽库、抗体库或其它蛋白文库中筛选生物活性物质是目前分子生物学研究的一个热点.核糖体展示是一种完全离体进行的功能蛋白筛选和进化鉴定的新技术,避免了传统的活体筛选技术的缺陷,使得文库容量增大、分子多样性加强.本文系统地评述了核糖体展示技术在制备ScFv单链抗体方面的应用,包括ScFv单链抗体模板的构建、体外转录与体外翻译、亲和筛选及筛选效率的测定以及分子多样性和体外进化研究,讨论了核糖体展示技术目前的发展动态、存在问题及发展趋势.     

无细胞蛋白质合成系统的研究进展

无细胞蛋白质合成系统是一种以外源mRNA或DNA为模板 ,通过在细胞抽提物的酶系中补充底物和能源物质来合成蛋白质的体外系统 .与传统的体内重组表达系统相比 ,体外无细胞合成系统具有多种优点 ,如可表达对细胞有毒害作用或含有非天然氨基酸 (如D 氨基酸 )的特殊蛋白质 ,能够直接以PCR产物作为模板同时平行合成多种蛋白质 ,开展高通量药物筛选和蛋白质组学的研究等 .本文综述了无细胞蛋白质合成系统的发展历史、系统中合成蛋白质所需的能量供应、遗传模板的稳定性和微型无细胞生物反应器等多方面的研究 ,并探讨了无细胞蛋白质合成系统中存在的难点、研究方向和广泛的应用前景     

大肠杆菌细胞抽提物制备方案的简化与优化

无细胞蛋白表达系统是一种将目的蛋白在体外进行表达的新技术和新方法,已广泛应用到蛋白质组学、蛋白质结构和功能等领域的研究中。在无细胞蛋白表达系统中,细胞抽提物的制备是关键因素之一。通过对大肠杆菌细胞抽提物制备过程中离心速度、预孵化和透析等参数的考察,利用绿色荧光蛋白作为报告蛋白,可以得到一个细胞抽提物制备的简化方案。采用相对低的转速(12 000×g,10 min),简易空孵化即可制备出活性高的细胞抽提物,用于无细胞体系蛋白表达,其表达的绿色荧光蛋白产量为209μg/mL。与传统的大肠杆菌细胞抽提物S30相比较,新方案将使时间与成本节省62%,产量是传统方法的2.6倍,使无细胞蛋白表达技术的操作快速、高通量的优势更加明显。     

用重组酶介导扩增技术快速扩增核酸

体外核酸快速扩增技术是一种可使微量核酸在体外高效快速扩增的技术, 自问世以来, 被广泛应用于分子生物学、医学和法理鉴定等领域, 并被不断改进, 以使其功能和适应性更为广泛. 重组酶介导扩增法是在现有体外核酸扩增原理的基础上发展起来的恒温体外快速扩增核酸技术. RAA法利用重组酶、单链结合蛋白和DNA聚合酶代替了传统PCR的热循环解链过程, 实现了在37℃恒温下的核酸快速扩增, 无需特殊的辅助仪器, 对操作人员的要求也不高, 具备简单、节能、便携、快速等特点, 有望在不远的将来取代传统的热循环PCR反应.     

Design and implementation of an introductory course for computer applications in molecular genetics

Formal training in computational biology was initiated at Wayne State University in 1990 to meet the needs of the faculty. This was still at a time when the molecular databases and analysis tools could be housed in what is now equivalent to a modern but dated desktop computer. In 1995 the course was expanded to include graduate students to provide these senior students with a foundation in computational biology. This course has armed our students with a requisite set of basic skills that are necessary for a successful career in molecular genetics. It is now an integral component of the graduate program of the Center for Molecular Medicine and Genetics and our experiences in course delivery have been detailed (BioInformatics Methods and Protocols, S. Misener and S. A. Krawetz, eds., Humana Press, Totowa, NJ, 2000.). The course was expanded to a campus-wide unlimited enrollment program for the summer of 2000 to address the needs of our student body. In this review we present our experience with delivering a multidisciplinary campuswide computational biology course to a new and widely diverse student body.     

Effective Binding Force Calculation in Dimeric Proteins

We apply the Blue Moon constrained Molecular Dynamics technique to study a particular case of molecular recognition, one of the main issues of modern molecular biology. We investigate the effects of mutation of interface residues on the binding strength of the dimeric protein superoxide dismutase from Photobacterium leiognathi. With our technique we produce a specific path describing the separation of the dimers and we calculate the effective mean force involved in the process. We apply the method to two mutants and compare the results with those obtained in an earlier calculation on the native enzyme. The method is sensitive to the mutations and allows us to establish a semi-quantitative hierarchy for the association strengths of the three enzymes.     

基于能力培养的分子生物学实验教学改革与探索

分子生物学技术已成为生命科学领域普遍运用的方法和手段,分子生物学实验课是一门让学生理解、掌握分子生物学技术的课程。系统总结了近年来我们以学生的能力培养为宗旨,在分子生物学实验教学中进行的建立“3＋1”的新课程体系、更新教学内容、尝试新的教学方法和手段、改革考核方式等探索,并就下一步改革进行了展望。     

Studies on DNA-related enzymes to elucidate molecular mechanisms underlying genetic information processing and their application in genetic engineering

ABSTRACT

Recombinant DNA technology, in which artificially “cut and pasted” DNA in vitro is introduced into living cells, contributed extensively to the rapid development of molecular biology over the past 5 decades since the latter half of the 20^th century. Although the original technology required special experiences and skills, the development of polymerase chain reaction (PCR) has greatly eased in vitro genetic manipulation for various experimental methods. The current development of a simple genome-editing technique using CRISPR-Cas9 gave great impetus to molecular biology. Genome editing is a major technique for elucidating the functions of many unknown genes. Genetic manipulation technologies rely on enzymes that act on DNA. It involves artificially synthesizing, cleaving, and ligating DNA strands by making good use of DNA-related enzymes present in organisms to maintain their life activities. In this review, I focus on key enzymes involved in the development of genetic manipulation technologies.     

原位PCR技术及其应用前景

原位PCR是分子生物学领域中一种崭新的技术,它结合了PCR技术和原位杂交技术的优点.该文介绍了原位PCR技术的起源、发展及方法学,并简要描述了该技术的应用现状及前景．     

Listeria monocytogenes,a model in infection biology

Listeria monocytogenes causes listeriosis, a systemic infection which manifests as bacteremia, often complicated by meningoencephalitis in immunocompromised individuals and the elderly, and fetal‐placental infection in pregnant women. It has emerged over the past decades as a major foodborne pathogen, responsible for numerous outbreaks in Western countries, and more recently in Africa. L. monocytogenes' pathogenic properties have been studied in detail, thanks to concomitant advances in biological sciences, in particular molecular biology, cell biology and immunology. L. monocytogenes has also been instrumental to basic advances in life sciences. L. monocytogenes therefore stands both a tool to understand biology and a model in infection biology. This review briefly summarises the clinical and some of the pathophysiological features of listeriosis. In the context of this special issue, it highlights some of the major discoveries made by Pascale Cossart in the fields of molecular and cellular microbiology since the mid‐eighties regarding the identification and characterisation of multiple bacterial and host factors critical to L. monocytogenes pathogenicity. It also briefly summarises some of the key findings from our laboratory on this topic over the past years.     

Industrial practice and the biology of leaching of metals from ores The 1997 Pan Labs Lecture

Biomining processes have been used successfully on a commercial scale for the recovery of metals, the most important of which are copper, uranium and gold. These processes are based on the activity of chemoautolithotrophic bacteria which are able to use either iron or sulfur as their energy source and which grow in highly acid conditions. In general, low-rate dump and heap leaching processes are used for copper recovery while the biooxidation of difficult-to-treat gold-bearing arsenopyrite ores is carried out commercially in highly aerated stirred tank reactors. Because of the high levels of bacterial activity required, limitations in the growth rate of the microorganisms which were not apparent in low-rate processes have become an important factor. A key to the commercialization of the gold-bearing arsenopyrite biooxidation process was the development of a rapidly-growing, arsenic-resistant bacterial consortium. The empirical technique of mutation and selection in a continuous-flow system was used to improve the ability of the bacteria to decompose the ore. This approach resulted in a dramatic initial enhancement in growth rate but a plateau in improvement of performance has been reached. Further advances will require a more direct approach based on an understanding of the underlying physiological mechanisms and an application of the tools of molecular biology. Considerable advances have been made in our understanding of the molecular biology of Thiobacillus ferrooxidans. However much less is known about the other biomining bacteria. Recent studies using 16S rRNA analysis techniques have indicated that T. ferrooxidans may play a smaller role in continuous flow stirred tank biomining processes than was previously thought. Received 20 November 1997/ Accepted in revised form 2 March 1998     

An Example of Circular Statistics in Chronobiological Studies: Analysis of Polymorphism in the Emergence Rhythms of Schistosoma Mansoni Cercariae

In the not too distant past, it was common belief that rhythms in the physical environment were the driving force, to which organisms responded passively, for the observed daily rhythms in measurable physiological and behavioral variables. The demonstration that this was not the case, but that both plants and animals possess accurate endogenous time-measuring machinery (i.e., circadian clocks) contributed to heightening interest in the study of circadian biological rhythms. In the last few decades, flourishing studies have demonstrated that most organisms have at least one internal circadian timekeeping device that oscillates with a period close to that of the astronomical day (i.e., 24h). To date, many of the physiological mechanisms underlying the control of circadian rhythmicity have been described, while the improvement of molecular biology techniques has permitted extraordinary advancements in our knowledge of the molecular components involved in the machinery underlying the functioning of circadian clocks in many different organisms, man included. In this review, we attempt to summarize our current understanding of the genetic and molecular biology of circadian clocks in cyanobacteria, fungi, insects, and mammals. (Chronobiology International, 17(4), 433–451, 2000)     

Harnessed viruses in the age of metagenomics and synthetic biology: an update on infectious clone assembly and biotechnologies of plant viruses

Recent metagenomic studies have provided an unprecedented wealth of data, which are revolutionizing our understanding of virus diversity. A redrawn landscape highlights viruses as active players in the phytobiome, and surveys have uncovered their positive roles in environmental stress tolerance of plants. Viral infectious clones are key tools for functional characterization of known and newly identified viruses. Knowledge of viruses and their components has been instrumental for the development of modern plant molecular biology and biotechnology. In this review, we provide extensive guidelines built on current synthetic biology advances that streamline infectious clone assembly, thus lessening a major technical constraint of plant virology. The focus is on generation of infectious clones in binary T‐DNA vectors, which are delivered efficiently to plants by Agrobacterium. We then summarize recent applications of plant viruses and explore emerging trends in microbiology, bacterial and human virology that, once translated to plant virology, could lead to the development of virus‐based gene therapies for ad hoc engineering of plant traits. The systematic characterization of plant virus roles in the phytobiome and next‐generation virus‐based tools will be indispensable landmarks in the synthetic biology roadmap to better crops.