Stathmin特异性siRNA表达质粒抑制stathmin的表达

目的:构建微管解聚蛋白stathmin基因的特异性siRNA质粒表达载体,以便研究stathmin在鼻咽癌中的生物学作用。方法:合成stathmin特异性DNA片段,退火形成的双链DNA片段克隆于质粒表达载体pGenesil-1.3;载体经扩增后,进行酶切和测序鉴定;采用QIAGEN公司脂质体(effectenetransfectionreagent)将鉴定后的重组质粒转入鼻咽癌CNE2细胞;RT-PCR与Westernblot检测stathmin基因表达。结果:酶切和测序分析表明,插入siRNA质粒表达载体中的DNA片段,其碱基序列和插入方向正确。表达分析证实特异性siRNA质粒表达载体能有效抑制鼻咽癌CNE2细胞中stathmin的表达。结论:构建的siRNA质粒表达载体对stathmin的表达有很好的抑制作用。     

SPACR原核表达载体的构建和表达

目的构建Sialoprotein associated with cones and rods（SPACR）原核表达载体并高效表达。方法根据基因文库中SPACR基因的cDNA序列设计引物,采用PCR方法扩增SPACR cDNA片段,并克隆进原核表达载体pGEX-6P-1中,转化于大肠杆菌BL21,测序鉴定后,用IPTG诱导表达出目的蛋白,经谷胱甘肽琼脂糖柱柱层析纯化,收获表达产物进行SDS-PAGE电泳分析和Western blot鉴定。结果 PCR后扩增得到的目的片段,经酶切鉴定及DNA序列测定,证实重组载体pGEX-6P-1-SPACR构建正确,表达蛋白分子量约为分别为21.2、22.6、36.2及20.8kDa。结论在大肠杆菌中获得了SPACR片段的高效表达,为研究其蛋白功能奠定了基础。     

人Regucalcin cDNA真核表达载体的构建及表达

目的构建人regucalcin（RGN）全长cDNA的真核表达载体，观察其在人肾皮质近曲小管上皮细胞（HK-2细胞）中表达。方法用RT-PCR技术扩增获得人HK-2细胞RGN全长cNDA，将扩增的产物连接入pMD18-T克隆载体上。将重绢质粒转化人大肠杆菌DHSa内并提取质粒，双酶切鉴定、DNA测序鉴定准确，再用双酶切方法将RGN基因定向连接到真核表达载体DECFP-NI中，构成pEGFP-RGN，将pEGFP-RGN转化人大肠杆菌DHSa内并提取质粒，双酶切鉴定、DNA测序鉴定准确。再用双酶切方法将RGN基因与EGFP定向连接到真核表达载体pcDNA3．1（＋）-zeocin中，构建真核表达pcDNA3．1-RGN-EGFP-zeo的重组体。将pcDNA3．1-RGN．EGFP-zeo转化人大肠杆菌DHSa内并提取质粒，双酶切鉴定、DNA测序鉴定准确，用脂质体转染入HK-2细胞。荧光显微镜、激光共聚焦显微镜观察pcDNA3．1-RGN-EGFP-zeo表达情况。结果RGNcDNA全长是897bp。以此构建的pcDNA3．1-RGN-EGFP-zeo真核表达载体，经DNA测序与GenBank中的人RGNcDNA序列一致。荧光显微镜、激光共聚焦显微镜观察到pcDNA3．1-RGN-EGFP-zeo转染的细胞中有绿色荧光蛋白的表达。结论本实验成功构建了pcDNA3．1-RGN．EGFP．zeO真核表达质粒，并在HK-2细胞中表达。     

人regucalcin cDNA高表达重组质粒的构建及其在原核细胞中的表达

目的 原核表达获得大量人regucalcin（RGN）蛋白。方法 用逆转录聚合酶链反应（RT-PCR）技术从人。肾皮质近曲小管上皮细胞（HK-2）中扩增RGN全长cDNA，将扩增的产物连接于测序载体pMD18-T，测序鉴定准确后，进行酶切，将正确的RGN全长cDNA与原核表达载体pET42b（＋）构建成重组质粒pET42b（＋）-RGN。将pET42b（＋）-RGN先转化入大肠杆菌DH5α，提取质粒经双酶切鉴定正确后，再转化入表达宿主大肠杆菌DE3菌株。对转化菌株进行诱导、破菌，进行十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）与免疫印迹分析，鉴定RGN融合蛋白质表达的正确性。RGN重组融合蛋白经Ni^2＋亲和层析纯化，达电泳纯。结果 构建了重组质粒pET42b（＋）-RGN并获得高效表达，RGN重组融合蛋白经异丙基硫化-β-D-半乳糖苷（IPTG）诱导后以包涵体形式存在，表达的蛋白质相对分子质量为34000。结论 人RGN蛋白在pET42b（＋）原核表达载体可获得高效表达，为进一步了解RGN蛋白质的结构、功能及其抗体的制备等奠定了基础。     

人regucalcin cDNA高表达重组质粒的构建及其在原核细胞中的表达

目的原核表达获得大量人regucalc in(RGN)蛋白。方法用逆转录聚合酶链反应(RT-PCR)技术从人肾皮质近曲小管上皮细胞(HK-2)中扩增RGN全长cDNA,将扩增的产物连接于测序载体pMD18-T,测序鉴定准确后,进行酶切,将正确的RGN全长cDNA与原核表达载体pET42b( )构建成重组质粒pET42b( )-RGN。将pET42b( )-RGN先转化入大肠杆菌DH5α,提取质粒经双酶切鉴定正确后,再转化入表达宿主大肠杆菌DE3菌株。对转化菌株进行诱导、破菌,进行十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDS-PAGE)与免疫印迹分析,鉴定RGN融合蛋白质表达的正确性。RGN重组融合蛋白经N i2 亲和层析纯化,达电泳纯。结果构建了重组质粒pET42b( )-RGN并获得高效表达,RGN重组融合蛋白经异丙基硫化-β-D-半乳糖苷(IPTG)诱导后以包涵体形式存在,表达的蛋白质相对分子质量为34 000。结论人RGN蛋白在pET42b( )原核表达载体可获得高效表达,为进一步了解RGN蛋白质的结构、功能及其抗体的制备等奠定了基础。     

Imaging gene expression

Genome-wide sequencing and increasing use of microarrays has resulted in the identification of a large number of new genes which may have important functional roles in development and onset of disease. Classical approaches in gene expression studies fall short in providing information about cellular localization of these genes and their relative levels of expression in tissues. Rapid determination of gene expression can be achieved with in situ methods of localization of gene expression in combination with recent developments in imaging technology.     

An enhanced autogene-based dual-promoter cytoplasmic expression system yields increased gene expression

The relatively low levels of transfection that can be achieved by current gene-delivery systems have limited the therapeutic utility of gene transfer. This is especially true for nonviral gene-delivery systems, where the levels of gene expression achieved are usually below the levels achieved by viral gene transfer systems. One strategy for increasing gene expression is to design a cytoplasmic expression system that does not require nuclear delivery for gene expression to occur. This can be achieved through the use of an autocatalytic cytoplasmic expression system using phage RNA polymerases. Here we describe cytoplasmic expression systems that yield increased levels of gene expression following in vitro transfection. We demonstrate direct evidence for an exponential, autocatalytic increase in gene expression using autogenes, as well as levels of reporter gene expression that are 20-fold higher than standard CMV-based nuclear expression systems. The development of a high-efficiency plasmid-based expression system could significantly improve the gene expression properties of nonviral gene-delivery systems, thereby increasing their clinical utility.     

SARS病毒M蛋白真核表达载体的构建与表达

目的 构建SARS病毒M蛋白片段真核表达载体,并检测其在Vero细胞中的表达.方法 在PCR引物下游引入Flag序列,以PCR从质粒pGEX-6P-1-SARS-M中扩增M蛋白编码基因片段,将酶切后的PCR产物克隆至pcDNA3.1(+)中,构建并鉴定重组质粒pcDNA3.1(+)-SARS-M.重组质粒经Superfect转染Vero细胞,Western blot检测基因表达情况.结果 重组质粒经酶切鉴定和基因测序显示构建正确;Western blot检测表明重组M蛋白片段在Vero细胞中获得正确表达.结论 成功构建SARS病毒M蛋白片段真核表达载体,并在Vero细胞中获得正确表达,为进一步研究M蛋白的功能奠定了基础.     

真核表达载体pcDNA3-ICOSIg的构建及表达

背景：人可诱导共刺激分子0cos）是迄今发现的重要共刺激分子家族成员,可促进激活T细胞的增殖和分泌、调节Th1／Th2细胞的极化、增强依赖T细胞的B细胞功能,阻断ICOS共刺激信号会导致T细胞的克隆失活或克隆无反应,从而诱导肿瘤对机体的免疫逃逸。目的：构建表达ICOSIg的质粒,观察其在小鼠体内的表达。方法：克隆编码ICOS的胞外片段,将其与编码小鼠免疫球蛋白IgG恒定片段（Ig）的基因融合,构建ICOSIg融合基因及其分泌型真核表达载体pcDNA3-ICOSIg,酶切鉴定重组子,测序,利用阳性脂质体载体包被pcDNA3-ICOSlg转染小鼠右侧大腿肌肉组织,Westernblot法检测血清ICOSIg水平。结果与结论：经测序鉴定证实pcDNA3．ICOSIg质粒目的基因片断与Genbank上公布的ICOS序列完全一致,说明质粒构建成功。脂质体载体包被pcDNA3-ICOSIg转染小鼠7d,在小鼠血清中检测到ICOSIg的阳性表达,说明pcDNA3．ICOSlg能够在小鼠肌细胞内表达。证实利用基因合成和重组技术可成功构建真核表达载体pcDNA3-ICOSIg。f201     

Ecdysone-controlled expression of transgenes

Inducible expression systems show great potential for use in human gene therapy and systems based on insect ecdysone receptors are particularly promising candidates. This article describes such systems and reviews actual and potential uses of ecdysone-controlled transgenes in vitro and in vivo. The ligand specificity of ecdysone receptor-based systems is considered, along with the safety and efficacy of the ecdysteroid and non-steroidal compounds used to activate them.     

Regulated gene expression systems

Most gene therapy research to date has focused on solving the delivery problem--getting genes efficiently and stably into target cells and tissues. Those working on systems for regulating the expression of genes once delivered have often been accused of trying to run before they can walk. Yet regulation is likely to be essential if gene therapy is to realize its full potential as a mainstream clinical option for delivering proteins. Dramatic progress has been made in designing and testing systems in which expression is controlled by orally active drugs. The next few years should see the first clinical trials of drug-regulated gene therapies.     

Language and pain expression

This paper arose during work for a BSc(Hons) dissertation, and considers the theoretical approach of Wittgenstein to pain analysis This paper seeks to discuss one aspect of the research undertaken representing some of the findings which illustrate the association between pain and language