基因工程抗体表达系统研究进展

本文对大肠杆菌，酵母、昆虫细胞及哺乳动物细胞表达系统进行了比较，并介绍其研究进展。     

基因工程抗体表达系统研究进展

为使临床上能广泛应用基因工程抗体，当务之急是研究方便、快捷、经济和批量制备活性抗体的技术，关键是选择高表达系统。本文对大肠杆菌、酵母、昆虫细胞及哺乳动物细胞表达系统进行了比较，并介绍其研究进展。     

基因工程药物

随着人类基因组逐渐被破译 ,人们的生活将发生巨大变化 ,这对医药行业也是巨大的冲击。基因工程药物已经走进人们的生活 ,利用基因治愈更多的疾病不再是一个奢望。随着人们对自身的了解迈上新台阶 ,很多病因将被揭开 ,药物就会设计得更好些 ,治疗方案就能“对因下药” ,生活起居、饮食习惯有可能根据基因进行调整 ,人类的整体健康状况将会提高 ,2 1世纪的医学基础将由此奠定。1　什么是基因工程药物所谓基因工程药物就是先确定对某种疾病有预防和治疗作用的蛋白质 ,然后将控制该蛋白质合成过程的基因取出来 ,经过一系列基因操作 (图 1) ,最…     

应用巴氏毕赤酵母表达基因工程药物的影响因素

巴氏毕赤酵母（Pichia pastoris，Pp）表达体系是近年来刚发展起来的真核表达体系，其操作技术和酿酒酵母非常相似，相比而言Pp表达体系的应用还不够广泛，但这一体系已经显示出很好的优势和应用前景，已经成为目前分子生物学领域中用于表达重组蛋白的标准工具之一，广泛用于基因重组药物的表达。本文就采用这一表达体系表达外源蛋白所应考虑的因素进行综述。     

基因工程药物

基因工程已诞生20余年,原理已清楚,技术已定型,在医药、化工、食品、电子、环保及农业各领域已开展了广泛的应用研究.但其成就最为显著者乃是医药行业.自从基因工程人脑激素获得成功以来,已有数十种基因工程药物及近200种单克隆抗体(McAb)面市,社会经济效益极为显著.1 基因工程原理和技术     

色氨酸酶基因工程菌的活力及其与表达关系研究

选择并改进以Ｌ－色氨酸为底物测定吲哚的生成量作为色氨酸酶活力测定方法,应用改进的方法测定了７株工程菌色氨酸酶的活力,也对不同诱导时间色氨酸酶的表达与活力关系进行了比较,结果表明工程菌色氨酸酶的活力比宿主菌均有不同程度的提高,其中１１号工程菌比宿主菌高１６倍,诱导表达１ｈ时酶活最高,但酶活高低并不与其表达量呈平行关系。     

基因工程药物的治疗进展

基因工程药物在传统化学药物难以攻克的疾病治疗方面显示了较大作用，本文综述了基因工程药物在肿瘤，类风湿性关节炎、器官移植免疫排斥、心血管疾病、病毒感染性疾病（包括艾滋病）及糖尿病等疾病治疗方面的进展，为我国新药研究开发提供思考。     

Expression without boundaries: Cell-free protein synthesis in pharmaceutical research

Proteins are an increasingly important class of new drugs. Pharmaceutical proteins are usually expressed in cell based systems in the development phase and in production, and although cell free methods have recently emerged they have not been used widely for therapeutic protein development or production. Cell free expression methodology is well suited for pharmaceutical protein expression and engineering and will probably become more commonly used in the future. Cell free expression allows protein engineering in high throughput format, flexible strategies for glycosylation and chemical conjugation, and allows easy use of unnatural amino acids as building blocks of proteins. Thus, cell free expression can be used to modify protein solubility, stability, and pharmacokinetics of therapeutic proteins. Likewise, it is potentially useful in protein development for biomaterial matrices, nanoparticles, and vaccines. This review illustrates the potential of cell free expression in pharmaceutical protein research and development while highlighting both advantages and limitations of the method.     

人脂联素原核表达载体PQE30/ADPN的构建及在大肠杆菌中的表达

目的:构建人脂联素重组表达载体PQE30/ADPN,并在大肠杆菌宿主系统中表达出脂联素蛋白,对表达产物进行鉴定。方法:将构建好的人脂联素克隆载体PUC57/ADPN与原核表达载体PQE30通过双酶切方法位点特异地连接在一起,再转化入大肠杆菌JM109感受态细胞中。通过筛选得到含有人脂联素基因的重组载体PQE30/ADPN,并用IPTG在大肠杆菌M15中诱导表达。结果:PCR获得长度为753bp目的片段,经PQE30原核表达载体连接、筛选及序列分析后,证实所插入的目的片段与GenBank检索的人脂联素cDNA序列(Accession NM-004797)100%匹配;含重组Adiponectin质粒的大肠杆菌经0.4mmol/L的IPTG诱导6h后有表达。结论:应用并成功构建了人脂联素原核表达载体PQE30/ADPN,且在大肠杆菌中获得有效表达。     

Transgenic fish systems and their application in ecotoxicology

Abstract

The use of transgenics in fish is a relatively recent development for advancing understanding of genetic mechanisms and developmental processes, improving aquaculture, and for pharmaceutical discovery. Transgenic fish have also been applied in ecotoxicology where they have the potential to provide more advanced and integrated systems for assessing health impacts of chemicals. The zebrafish (Daniorerio) is the most popular fish for transgenic models, for reasons including their high fecundity, transparency of their embryos, rapid organogenesis and availability of extensive genetic resources. The most commonly used technique for producing transgenic zebrafish is via microinjection of transgenes into fertilized eggs. Transposon and meganuclease have become the most reliable methods for insertion of the genetic construct in the production of stable transgenic fish lines. The GAL4–UAS system, where GAL4 is placed under the control of a desired promoter and UAS is fused with a fluorescent marker, has greatly enhanced model development for studies in ecotoxicology. Transgenic fish have been developed to study for the effects of heavy metal toxicity (via heat-shock protein genes), oxidative stress (via an electrophile-responsive element), for various organic chemicals acting through the aryl hydrocarbon receptor, thyroid and glucocorticoid response pathways, and estrogenicity. These models vary in their sensitivity with only very few able to detect responses for environmentally relevant exposures. Nevertheless, the potential of these systems for analyses of chemical effects in real time and across multiple targets in intact organisms is considerable. Here we illustrate the techniques used for generating transgenic zebrafish and assess progress in the development and application of transgenic fish (principally zebrafish) for studies in environmental toxicology. We further provide a viewpoint on future development opportunities.     

人脂联素基因在L-02细胞中表达及其功能检测

目的:在人正常肝细胞株L-02中表达外源人脂联素蛋白,并观察其生物学活性。方法:采用脂质体法将成功构建的人脂联素真核表达质粒pcDNA3.1/CT-GFP-TOPO-Adiponectin转染L-02细胞;免疫组织化学定位观察脂联素的表达;Western blot检测培养上清及细胞沉淀中脂联素水平;酶联免疫法测定培养基血糖。结果:构建的真核表达质粒能有效转染L-02细胞,并在培养上清及细胞沉淀中检测到脂联素蛋白,该重组蛋白能够降低高糖处理的L-02细胞的血糖水平。结论:重组人脂联素蛋白能在L-02中有效表达,该蛋白定位在细胞质,并且能分泌到胞外,表达产物经体外实验证实具有生物学活性。     

国内外基因工程药物研究开发的现状和展望

本文论述了国内外基因工程药物研究和开发的最新进展，并对我国基因工程药物的发展提出了建议。     

重组人干扰素大肠杆菌高效表达系统的建立

目的:构建重组人干扰素-α2b(rhIFN-α2b)原核表达系统,以获得rhIFN-α2b在大肠杆菌中的高效表达。方法:依据大肠杆菌遗传密码子频率表,在不改变氨基酸组成的情况下,人工半合成适于在大肠杆菌中表达的rhIFN-α2b编码序列;经PCR扩增后,克隆人表达型质粒载体pDH;筛选阳性菌落,温度诱导表达,表达产物行聚丙烯酰胺凝胶电泳(SDS-PAGE)、蛋白质印迹(WesternBlot)鉴定,并以人羊膜细胞-水泡性口炎病毒(WISH-VSV)系统鉴定rhIFN-α2b抗病毒活性。结果:PCR产物经琼脂糖凝胶电泳鉴定可见与预期大小符合的512bp的DNA条带;重组质粒pDH/IFN-α2b经序列分析,证实含有与预期相符且读码框架正确的hIFN-2b编码序列;SDS-PAGE可见与IFN-α2b分子质量大小一致的蛋白质条带,WesternBlot鉴定此条带为rhIFN-2b,其比活性达(1.8～2)×108U/mg。结论:rhIFN-α2b在大肠杆菌中的高效表达系统构建成功,且能高水平地表达出具有生物活性的rhIFN-α2b。     

Expression and characerization of human kainate receptor subunits in Escherichia coli and mammalian cells

Cellular expression systems for human kainate receptor subunits (EAA1 and EAA2) have been developed as tools to support drug screening and rational drug design. EAA1 and EAA2 sequence-specific polyclonal antibodies were generated to characterize polypeptide expression on introduction of appropriate plasmid expression constructs to Escherichia coli (E. Coli), chinese hamster ovary (CHO) and human embryonic kidney (HEK-293) cells. A polypeptide with an apparent molecular mass of ～ 120 kilodaltons (kDa) was identified by Western blot analysis in CHO and HEK-293 cells expressing EAA2. Three major immunoreactive bands of 116, 110, and 90 kDa were identified in HEK-293 cells expressing EAA1. The polyclonal antibodies will allow the direct determination of EAA1 and EAA2 expression in human brain. Pharmacological characterization of a stable CHO cell line expressing EAA2 revealed a dissociation constant for kainate (K_D) of 1.92 ± 0.28 nM (n = 3). This is the first report describing a stable cell line expressing EAA2 and the third report describing a stable cell line expressing a human glutamate receptor subunit. These studies are an important prelude to discovery of EAA1 and/or EAA2 specific drugs. © 1995 Wiley-Liss, Inc.     

重组人白细胞介素21表达条件的探讨

目的探讨重组人白细胞介素21工程菌诱导表达的最佳条件。方法用摇瓶发酵的方法探索不同诱导条件(诱导温度、诱导时间、接种量I、PTG浓度等)对目的蛋白质表达的影响。结果该工程菌表达目的蛋白质的最佳温度是42℃;高密度接种更有利于目的蛋白质的表达。结论建立了BL21/pET-30a(+)-IL21工程菌最佳诱导表达条件,为重组人白细胞介素21的制备奠定了基础。     

Osx cDNA反义表达载体的构建

目的：构建反义Osterix(Osx)cDNA表达载体。方法：快速提取人全血基因组DNA。以DNA为模板，经过两步PCR扩增获得Osx编码序列，与pGEM-T Easy连接获得重组克隆载体。将Osx编码序列反向克隆人真核表达载体pcDNA3，进行序列测定。结果：经序列分析证实，Osx扩增序列完全正确，且反向插入到pcDNA3载体。结论：成功构建了Osx反义表达载体，为研究Osx对成骨细胞分化的影响奠定了基础。     

小鼠微透析技术及其在药物传递系统中的应用

微透析技术（mierodialysis，MD）是由早期神经化学实验室中的灌流取样技术发展而来的一项新技术，后逐渐应用于药学领域，并得到快速发展。早期MD主要以大鼠为实验动物，进行药物传递系统（drug delivery system，DDS）的研究。但是大鼠的实验模型较匮乏，小鼠基因工程（mice genetic engineering，MGE）的出现和迅速发展则弥补了这一不足。