哺乳动物细胞表达外源基因常用病毒载体的研究进展

文章概述了逆转录病毒载体、腺病毒载体、腺相关病毒载体、慢病毒载体等四种常用哺乳动物细胞表达外源基因的病毒载体系统的来源、组成、特点及应用方面研究的进展.     

动物基因工程慢病毒载体--一种将传染性病毒颗粒转变成基因治疗的载体

慢病毒载体是一类逆转录载体,它既能感染分裂细胞,也能够感染非分裂细胞.也就是说,它能够广泛地应用于基因治疗,构建转基因动物和结合RNAi技术进行基因功能研究.     

动物基因工程慢病毒载体——一种将传染性病毒颗粒转变成基因治疗的载体

慢病毒载体是一类逆转录载体,它既能感染分裂细胞,也能够感染非分裂细胞.也就是说,它能够广泛地应用于基因治疗,构建转基因动物和结合RNAi技术进行基因功能研究.     

使用病毒载体的罕见病基因疗法临床进展

 很大一部分的罕见病由遗传因素决定，难以用普通的小分子或大分子药物治愈，而基因治疗有望从根本上修正人体功能的缺失或异常，给罕见病患者带来改善生活质量的希望。目前许多基因疗法的临床试验正在开展，病毒载体是常用的基因递送方法，本文讨论了用于临床基因递送的多种病毒载体，包括腺相关病毒、逆转录病毒和慢病毒，重点列举了这些病毒在罕见病临床试验中的研究、应用和进展，评价了这些病毒的优缺点，并简述了基因疗法的研究方向及应用前景。     

慢病毒及其载体系统研究进展

基于慢病毒的病毒载体的研究已经发展到一定水平,因此用慢病毒作为基因转移媒介的临床研究已经开始,在实验室和临床应用中,慢病毒具有特别的优点,尤其是能整合未分裂细胞的独特能力.现在基于HIV的慢病毒载体的临床研究已经开始应用于人体.本文介绍基于HIV-1的慢病毒和慢病毒载体的结构,慢病毒载体的优点以及慢病毒载体应用的展望.     

基因治疗中目的基因的导入方法

基因治疗正从实验室研究走向临床应用，它给医学界引入了一种全新的概念，基因治疗主要是利用病毒介导的基因转移。文中介绍了目前已应用和将要应用于人类基因治疗的逆转录病毒载体、腺病毒载体、腺相关病毒载体、疱疹病毒载体等的构建方法，优缺点和适于治疗的疾病。扼要地介绍了目的基因导入的理化方法。     

病毒载体滴度对基因转移效率的影响研究

用改良的经典方法测定包装ＬａｃＺ基因的腺病毒和逆转录病毒载体的病毒滴度，以及用新建的简便方法测定逆转录病毒载体的病毒滴度，应用此两种病毒载体介导ＬａｃＸＺ基因分别导入１５种人或小鼠靶细胞，并以不同滴度的病毒上清液转染靶细胞Ｋ５６２，探讨病毒滴度对基因转移效率的影响，实验结果表明，病毒滴度与转染效率呈正相关，逆转录病毒滴度由１０＾６ＣＦＵ／ｍＬ。     

重组慢病毒对不同哺乳动物细胞基因转移及表达效率的研究

慢病毒是一种具有独特优点和巨大应用潜力的哺乳动物细胞基因转移载体,我们对慢病毒载体对不同哺乳动物细胞的基因转移及表达效率进行了平行比较研究.应用第三代重组慢病毒系统构建了携带CMV启动子-EGFP报告基因表达元件的重组慢病毒Lenti-EGFP,分别对多种不同哺乳动物细胞进行转导实验,在转导48 h后应用流式细胞仪检测报告基因在不同细胞株中的转移及表达效率.我们共使用了29种哺乳动物细胞株,包括14种人类组织细胞,5种猴组织细胞,9种鼠组织细胞,1种兔组织细胞.结果显示,重组慢病毒具有良好的基因转移能力,可有效进入多数哺乳动物细胞,对不同种属来源的细胞没有表现出特别的偏嗜性,但对贴壁培养细胞的基因转移效率明显高于对悬浮培养细胞.本研究为重组慢病毒系统的合理使用提供了基础.     

慢病毒载体及其介导的RNA干扰在基因治疗中的应用研究

主要就慢病毒载体及其介导的RNA干扰技术在基因治疗中的应用研究进行了综述,并对其在该领域具有的广阔前景进行了展望.慢病毒载体作为一种新型的载体,可有效地将携带的目的基因导入宿主细胞,并将其整合到宿主细胞基因组中,从而使目的基因得以持久稳定地表达.该载体因具有高感染性、高表达效率及不易诱发宿主免疫反应等优点,已成为基因治疗研究中的一个重要工具.RNA干扰技术可以特异性抑制或关闭特定基因的表达,因此该技术可广泛应用在基因功能探究和恶性肿瘤治疗等领域.由慢病毒载体介导的RNA干扰技术能持久、稳定、特异性地抑制各类细胞中特定基因的表达,在病毒感染、肿瘤等疾病的基因治疗中被广泛应用,成为生物医学领域的新热点.     

干涉UBXN1慢病毒颗粒制备及稳定细胞系的建立

构建并鉴定UBXN1-shRNA慢病毒表达载体,以便应用RNAi技术以及慢病毒感染系统建立稳定干涉细胞系并进一步研究UBXN1的功能。将携带不同特异性干涉序列的DNA片段插入PLKO.1载体中构建慢病毒表达载体,并制备慢病毒颗粒。将慢病毒颗粒感染U2OS细胞,建立稳定干涉细胞系,应用real-time PCR和western blot技术分别检测U2OS细胞中UBXN1 mRNA和蛋白质水平的表达差异。重组克隆经酶切证实shRNA正确插入慢病毒载体,DNA测序证实插入的序列正确,western blot检测证实设计的五条shRNA干扰序列有效的敲低U2OS细胞中内源性UBXN1的表达。成功制备UBXN1的慢病毒干涉颗粒,并建立UBXN1稳定下调的U2OS细胞系。     

通用型奶牛多位点基因打靶载体系统的构建

以奶牛为研究对象,以其重复的rRNA基因间的间隔序列为靶位点,基于BAC重组酶系统构建多位点基因打靶载体,为建立体内多位点基因打靶技术获得关键材料.首先构建BAC-TDN筛选载体,然后构建pYLVS-GD表达载体.将BAC-TDN筛选载体和pYLVS-GD表达载体共转化至大肠杆菌NS3529中,通过Cre重组酶的作用形成BAC-TDN-VS-GD质粒,采用归位内切酶I-SceI切除pYLVS载体骨架,构建奶牛多位点基因打靶载体BAC-TDN-GD,利用接头LS使之环化.BAC-TDN-GD打靶载体与pYLSV质粒组成了通用型奶牛多位点打靶载体系统.以重复序列为靶位点的多位点基因打靶技术,将部分解决目前存在的打靶效率低、安全性差等问题.     

植物基因工程研究现状与展望

植物基因工程与人类生活密切相关,它将给植物育种带来革命性的变革。植物基因工程研究的关键问题是向植物中转移外源基因,它主要涉及植物受体的选择和基因转移方法的探索。由于Ti质粒载体的建立和应用,双子叶植物中的应用性研究已经取得了突破性的进展。单子叶植物方面的工作尚处于基础理论研究阶段。     

带有绿色荧光蛋白基因(gfp)的植物重组表达质粒pBI-GFP的构建

利用 DNA重组技术 ,将经定点突变改造的绿色荧光蛋白基因 ( gfp)克隆到植物表达载体p BI-1 2 1中 ,成功地构建了植物重组表达质粒 p BI-GFP.     

A novel nonviral nanoparticle gene vector: Poly-L-lysine-silica nanoparticles

DNA delivery is a core technology for gene structure and function research as well as clinical settings. The ability to safely and efficiently targeted transfer foreign DNA into cells is a fundamental goal in biotechnology. With the development of nanobiotechnology, nanoparticle gene vectors brought about new hope to reach the goal. In our research, silica nanoparticles (SiNP) were synthesized first in a microemulsion system polyoxyethylene nonylphenyl ether (OP-10)/cyclohexane/ammonium hydroxide, at the same time the effects of SiNP size and its distribution were elucidated by orthogonal analysis; then poly-L-lysine (PLL) was linked on the surface of SiNP by nanoparticle surface energy and electrostatically binding; lastly a novel complex nanomateial—poly-L-lysine-silica nanoparticles (PLL-SiNP) was prepared. The analysis of plasmid DNA binding and DNase I enzymatic degradation discovered that PLL-SiNP could bind DNA, and protect it against enzymatic degradation. Cell transfection showed that PLL-SiNP could efficiently transfer PEGFPC-2 plasmid DNA into HNE1 cell line. These results indicated that PLL-SiNP was a novel nonviral nanoparticle gene vector, and would probably play an important role in gene structure and function research as well as gene therapy.     

拟南芥HSP70基因启动子表达载体的构建及在烟草BY2细胞中的应用

探讨了拟南芥的HSP70基因在液体悬浮培养的烟草BY2细胞中的表达及应用.用PCR扩增的方法从拟南芥col生态型基因组中扩增获得HSP70基因启动子序列,将其连入p1300表达载体且以GFP为报告基因,将该表达载体采用农杆菌转基因转入液体悬浮培养的烟草BY2细胞中,观察转基因细胞中报告基因GFP的表达情况.结果显示:HSP70:GFP转基因液体悬浮培养的烟草BY2细胞中有GFP的表达.该表达载体可在液体悬浮培养的BY2细胞中正常表达,且可在较短时间内获得大量实验材料,对拟南芥的HSP70基因启动子的进一步研究提供理论依据和丰富的实验材料,且可明显缩短实验周期.     

携带p53基因重组腺病毒载体的构建

将pCMVp53重组转移载体经BamHI和NheI酶切,得到p53基因cDNA,然后将cDNA片段克隆到转移载体pCMV5GFP,使其受CMV5启动子的调控,获得pCMV5p53重组转移载体.用该线状重组转移载体与腺病毒右臂DNA经磷酸钙共转染293细胞获得重组腺病毒,经酶联免疫吸附法(ELISA)测定p53蛋白含量,证明外源p53基因在含重组腺病毒的293细胞中得到表达.     

Specific anti-tumor effect induced by attenuated Salmonella typhimurium vaccine expressing extracellular region of vascular endothelial growth factor receptor 2

The purposes of this research were to study the stable expression of exogenous gene encoding therapeutic protein in attenuated Salmonella typhimurium, observe the metabolism of oral gene vaccine carried by attenuated Salmonella typhimurium in BALB/c mouse, and investigate the feasibility of prevention and treatment of tumors by the recombinant bacteria. Recombinant plasmid pcDNA3.1 VEGFR2(n1-7) was transformed into competent attenuated Salmonella typhimurium SL3261 to develop oral DNA vaccine SL3261-pcDNA3.1 VEGFR2(n1-7). To observe whether the exogenous gene can be expressed in the recombinant bacteria, PCR was performed to amplify the CMV promoter of the eukaryotic expression vector as the proof of stable expression of exogenous protein; transmission elec- tron microscopy (TEM) was applied to observe the morphology of the recombinant bacteria to confirm that the exogenous gene has no impact on the growth of the bacteria, and then BALB/c mice were immunized with the gene vaccine. After inoculation of the gene vaccine, the recombinant bacteria SL3261 could be detected in the tissues such as small intestine, colon, liver and spleen. And then, mice in each group were challenged with tumor cells. The results of animal experiment showed that tumor growth of the mice in experimental group was inhibited and survival time of immunized mice was prolonged compared with control groups. A higher lymphocyte infiltration in tumors from animals treated with DNA vaccine was observed. Immunohistochemical analysis of tumor samples revealed an en- hanced accumulation of CD8 cytotoxic T lymphocytes, as well as an increase in CD4 cells in the tumors of animals treated with the oral gene vaccine compared to tumors from control group mice. Ultrastructure of the tumor tissue showed that tumor cells in the samples of the immunized mice were well-differentiated. Our research confirmed that the exogenous gene can be stably expressed in the attenuated Salmonella typhimurium and has no impact on the growth of the recombinant bacteria; the exogenous gene can de delivered to the host by attenuated Salmonella typhimurium to produce anti-tumor effect with no obvious cytotoxity to the host. In this study, it is established that attenuated Salmonella typhimurium could be used as a vector for oral gene vaccine, and our study provided a theoretical basis for the body distribution and the metabolism of the recombinant bacteria. This strategy may provide a simple, safe and effective way for the prevention and treatment of tumors.     

Production of human alpha-interferon in silkworm using a baculovirus vector

Microorganisms are generally used for mass production of foreign gene products, but multicellular organisms such as plants have been proposed as an economical alternative. The silkworm may be useful in this context as it can be cultured easily and at low cost. We have therefore developed a virus vector to introduce foreign genes, for example, the gene for human alpha-interferon (IFN-alpha), into silkworms. We used the baculovirus Bombyx mori nuclear polyhedrosis virus (BmNPV) which has a large (greater than 100 kilobases, kb) double-stranded circular DNA genome within its rod-shaped capsid. Baculoviruses have been used previously as vectors for expression of beta-interferon and beta-galactosidase in established cell lines. Although BmNPV has not been used previously as an expression vector, it has an advantage over the baculovirus Autographa californica NPV in that it has a narrower host range and will not grow in wild insect pests in the field. In the present study, the polyhedrin gene encoding the major inclusion body protein of BmNPV was identified by hybridization with complementary DNA and cloned in a plasmid. For insertion of foreign genes, we constructed a recombinant plasmid carrying a polylinker linked to the promoter of the polyhedrin gene, and inserted the IFN-alpha gene into this plasmid. The resulting plasmid and the BmNPV genomic DNA were co-transfected into BM-N cells, and stable recombinant viruses isolated by plaque assay on BM-N cells. The recombinant virus replicated in silkworm larvae, which synthesized as much as 5 X 10(7) units (approximately 50 micrograms) of interferon in their haemolymph.     

基于Normalized Cut的基因表达数据聚类

利用基因表达数据进行聚类分析可提高肿瘤诊断的正确率,对生物医学研究具有重要意义.该文将Normalized Cut应用于基因表达数据的聚类中,将样本映射为高维空间的点,利用亲近矩阵和度矩阵构造正规Laplacian矩阵,经SVD分解得到反映原始样本类别信息的指示向量,利用指示向量各分量的符号差异实现基因表达数据的聚类.通过对白血病和结肠癌数据集的实验,证明了该文方法的有效性.