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1.
小肽多拷贝基因表达载体的构建及其高效表达(英文)   总被引:6,自引:0,他引:6  
介绍一种快速、高效构建小肽多拷贝基因表达载体的策略 ,并构建了相应的表达载体pETE coT .用人工合成的编码 2 8个氨基酸残基的胸腺素α1基因为模型 ,采用限制酶EcoT14I识别序列CCAAGG为小肽基因两末端序列 ,利用其酶切后可产生非镜相对称粘性末端 ,一次连接反应就构建出一系列不同基因拷贝数的表达载体 ;在小肽基因两端分别引进编码FactorXa和羟胺蛋白切割位点的序列 ,表达出的融合蛋白可被FactorXa和羟胺剪切出不残留任何外源氨基酸的小肽 .不同拷贝数的小肽融合蛋白在大肠杆菌BL2 1(DE3)中均获得高效表达 .  相似文献   

2.
随着植物基因工程的发展,将多个基因转化植株已成为研究的热点之一,利用连接肽进行多个基因融合的策略巳引起广泛关注.利用连接多肽2A和LP4/2A分别将抗虫基因Bt(Bacillus thuringiensis,Bt) cry1Ah和耐革甘膦基因mG2epsps连接起来,构建了4个融合基因表达载体pHAG、pHLAG、pGAH、pGLAH和两个单基因载体pSAh、pSmG2,其中pHAG、pHLAG是Bt cry1Ah基因在前,mG2-epsps基因在后,pGAH、pGLAH是mG2-epsps基因在前,Bt cry1Ah基因在后,分别用2A和LP4/2A作为连接肽,由CaMV35S启动子驱动.利用农杆菌介导法将6个植物表达载体转入烟草,得到再生植株529株.经PCR检测,有261株再生苗为阳性植株,转化率达到49.3%.获得的转基因烟草可用于分析连接肽2A和LP4/2A的剪切效率以及不同基因与连接肽连接位置差异对基因表达的影响.  相似文献   

3.
张力  刘超  周昕  谢英  刘树锋 《四川动物》2015,(3):338-344
目的以Tol2为骨架载体,以绿色荧光蛋白(GFP)、Cherry为报告基因,探讨采用2A肽双基因载体构建策略构建单启动子双基因共表达质粒的方法;将B细胞刺激因子(BAFF)分别置于2A序列前后位置,分析位置效应对跨膜融合蛋白的表达与剪切的影响,探讨多基因共表达转基因斑马鱼构建技术。方法以In Fusion法将GFP-2A-Cherry序列构建到Tol2质粒上,所得p Tol-GFP-2A-Cherry质粒转染He La细胞、显微注射1-细胞期斑马鱼受精卵;倒置荧光显微镜观察He La细胞、斑马鱼幼鱼体内GFP与Cherry蛋白的表达,Western blot法验证GFP和Cherry蛋白的表达量与剪切情况;分别构建p Tol2-GFP-2A-BAFF与p Tol2-BAFF-2A-Cherry质粒,Western blot法检查BAFF的表达与剪切情况。结果 p Tol2-GFP-2A-Cherry质粒转染的He La细胞,GFP与Cherry均可单独表达且表达呈现时空一致性;GFP-2A-Cherry融合蛋白可被剪切为GFP与Cherry,且成等比例表达趋势。p Tol2-GFP-2A-Cherry质粒显微注射1-细胞期斑马鱼受精卵可获得可单独表达GFP与Cherry蛋白的转基因斑马鱼;p Tol2-GFP-2A-BAFF与p Tol2-BAFF-2A-Cherry于斑马鱼体内均有融合蛋白的表达,且BAFF序列位于2A序列后更易于融合蛋白的剪切。结论通过2A肽策略构建可实现在斑马鱼体内单一载体、单一启动子调控双基因表达目的。发现编码跨膜分泌蛋白的功能基因位于2A序列的不同位置会直接影响蛋白的剪切,功能基因位于2A序列后易于跨膜蛋白的剪切。  相似文献   

4.
人组织激肽释放酶基因在哺乳动物细胞中的表达   总被引:3,自引:0,他引:3  
克隆人胰腺组织激肽释放酶基因 (hKK) ,构建融合荧光蛋白基因的真核表达载体 ,在CHO细胞中表达 ,为开发激肽释放酶基因工程产品以及开展基因治疗高血压研究奠定了基础。提取人胰腺组织总RNA后 ,RT PCR扩增KK ,构建中间载体KSKK。从KSKK中切出激肽释放酶基因 ,插入真核表达载体pEGFP C2 ,构建出激肽释放酶带有荧光蛋白报告基因的表达载体pEGC KK ,测序分析后转染CHO细胞 ,荧光显微镜观察激肽释放酶基因表达。并进行SDS PAGE及Westernblot分析。成功克隆激肽释放酶基因 ,并在CHO细胞获得表达 ,克隆的人组织激肽释放酶基因可用于激肽释放酶基因工程产品开发以及基因治疗研究。  相似文献   

5.
融合蛋白连接肽的研究进展   总被引:3,自引:0,他引:3  
融合蛋白是将两个或多个基因的编码区首尾连接,由同一调控序列控制构成的基因表达产物。对于有连接肽的融合蛋白,连接肽的设计在其中起着决定性的作用。该文从连接肽的设计、构建、分类、功能以及用连接肽构建融合蛋白时出现的问题和问题的解决等几个方面作了综述。  相似文献   

6.
IL-24和Smac为靶向癌症的多基因治疗研究中重要的候选基因.在构建IL-24和Smac双基因表达载体过程中,选择一个稳定高效的连接子非常关键.利用三个不同的连接子多肽序列IETD、EEED、F2A构建三个真核表达质粒pcDNA3.1(+)-IL-24-IETD-Smac、pcDNA3.1(+)-IL-24-EEED-Smac、pcDNA3.1(+)-IL-24-F2A-Smac,并联合5-氟尿嘧啶(5-FU)处理研究各连接子介导的融合蛋白剪切效果.上述实验结果表明,在三种IL-24-linker-Smac双基因表达载体中,F2A连接子剪切效果最佳且与caspase活性成正相关.IETD、EEED连接子介导的融合蛋白都有一定的剪切,但IETD/EEED多肽影响了上游IL-24蛋白的半衰期,加速了剪切后IL-24蛋白的泛素化降解.本研究为构建靶向癌症应用的双基因或多基因载体提供了设计参考.  相似文献   

7.
构建单纯疱疹病毒2型包膜糖蛋白D成熟肽基因毕赤酵母表达载体,并对序列进行分析,为进行高抗原性的真核表达重组gD蛋白奠定基础。采用PCR扩增HSV2-gD成熟肽基因,将该段基因克隆于pGEM-T克隆载体,转化鉴定后,与巴斯德毕赤酵母表达载体(pPIC9K)酶切连接,转化大肠杆菌DH5α,筛选测序确定构建了pPIC9K?gD的真核表达载体,对克隆的序列进行分析,预测表达产物的理化特性及抗原性。结果显示,获得的重组的酵母表达载体pPIC9K-gD,测序结果证实为HSV2-gD成熟肽基因,序列分析其高度保守,预测蛋白分子量40.63kD,等电点pI为7.15,包含完整成熟肽分值达1.7的多个抗原决定簇。成功构建了HSV2-gD成熟肽基因的毕赤酵母表达载体。  相似文献   

8.
多肽串联基因构建策略   总被引:3,自引:0,他引:3  
构建串联表达载体可有效提高小分子重组蛋白(肽)表达量和结构稳定性,并能屏蔽毒蛋白对宿主的伤害作用,因而被广泛采用。比较分析和综述了非对称粘性末端互补法、接头连接法、同尾酶法、表达盒串联法等4种多聚体、多拷贝基因串联表达载体的构建方法特点和适用范围。从效率、精度和产物切割等方面阐述了串联表达载体构建策略的选择依据。  相似文献   

9.
构建δ-睡眠肽(DSIP)蛋白与GFP的融合基因表达载体,高效表达和纯化GFP-DSIP融合蛋白。通过SOE-PCR拼接DSIP全长编码基因,并使得DSIP上游具有肠激酶识别位点,经双酶切定向克隆至表达载体pET-28a,构建重组载体pET-28a-DSIP,通过PCR扩增GFP全长编码基因,经双酶切定向克隆至pET-28a-DSIP,构建原核重组表达载体pET-28a-GFP-DSIP,通过双酶切和测序鉴定后,导入E.coli BL21宿主菌中,IPTG诱导表达融合蛋白,采用镍亲和层析和分子筛凝胶层析获得高纯度蛋白,SDS-PAGE分析鉴定。经测序鉴定成功构建了原核重组表达载体pET-28a-GFP-DSIP,在IPTG诱导下获得可溶性的绿色荧光蛋白与睡眠肽的融合蛋白,经Ni-NTA亲和层析纯化成功获得高纯度的融合蛋白。成功构建了DSIP与GFP融合基因的重组表达载体,确定了GFP-DSIP融合蛋白诱导表达的最佳条件,获得了较高纯度的融合蛋白,为进一步研究DSIP蛋白的生物学功能奠定了基础。  相似文献   

10.
目前双基因和多基因转基因植物已经商品化,并展现了广泛的应用前景。但在转基因植物研究中,使多个基因同时在植物体中表达调控依然很难实现,是植物基因工程和生物技术发展中的难点。融合基因表达载体作为一种新型的方法,弥补了获得双价或多价转基因植物传统方法的缺点,具有更高的应用价值。本文对目前构建融合基因的方法作了评述,并对比较新颖的连接肽2A和LP4做了详细介绍。  相似文献   

11.
Kim JH  Lee SR  Li LH  Park HJ  Park JH  Lee KY  Kim MK  Shin BA  Choi SY 《PloS one》2011,6(4):e18556
When expression of more than one gene is required in cells, bicistronic or multicistronic expression vectors have been used. Among various strategies employed to construct bicistronic or multicistronic vectors, an internal ribosomal entry site (IRES) has been widely used. Due to the large size and difference in expression levels between genes before and after IRES, however, a new strategy was required to replace IRES. A self-cleaving 2A peptide could be a good candidate to replace IRES because of its small size and high cleavage efficiency between genes upstream and downstream of the 2A peptide. Despite the advantages of the 2A peptides, its use is not widespread because (i) there are no publicly available cloning vectors harboring a 2A peptide gene and (ii) comprehensive comparison of cleavage efficiency among various 2A peptides reported to date has not been performed in different contexts. Here, we generated four expression plasmids each harboring different 2A peptides derived from the foot-and-mouth disease virus, equine rhinitis A virus, Thosea asigna virus and porcine teschovirus-1, respectively, and evaluated their cleavage efficiency in three commonly used human cell lines, zebrafish embryos and adult mice. Western blotting and confocal microscopic analyses revealed that among the four 2As, the one derived from porcine teschovirus-1 (P2A) has the highest cleavage efficiency in all the contexts examined. We anticipate that the 2A-harboring cloning vectors we generated and the highest efficiency of the P2A peptide we demonstrated would help biomedical researchers easily adopt the 2A technology when bicistronic or multicistronic expression is required.  相似文献   

12.
We present an update on the pTRIDENT multicistronic mammalian expression vectors and their implications in various metabolic engineering and therapeutic applications. The pTRIDENT vector family has been expanded by construction of a new set of pTRIDENT-based vectors containing constitutive promoters of human origin (ubiquitin C and EF-1alpha promoters) and selectable markers (zeocin resistance) and expressing different reporter genes (secreted alkaline phosphatase (SEAP) and the secreted single-chain urokinase-type plasminogen activator (low-M(r) u-PA)). In addition, we have constructed pTRIDENT derivatives with novel streptogramin-repressible and streptogramin-inducible promoters for simultaneous and adjustable expression of three different transgenes. Streptogramin-inducible and tetracycline-repressible pTRIDENT derivatives were used to simultaneously control expression of three fluorescent proteins in mammalian cells: the enhanced cyan fluorescent protein (CFP), the recently isolated red fluorescent protein (RFP, also designated dsRed), and the enhanced yellow fluorescent protein (YFP). Owing to their modular structure, the pTRIDENT vector family represents a construction kit for the design of novel multicistronic expression constructs.  相似文献   

13.
2A Peptide sequences are now being widely used to construct multicistronic expression vectors. It is suggested that when only the first 2A-linked protein bears a signal sequence, the signal-less protein(s) downstream of 2A can also be translocated into the mammalian endoplasmic reticulum system through a “slipstreaming” mechanism. By using flow cytometry and cell surface CD90 as a localization indicator, we show here that slipstreaming translocation does not occur in mammalian cells; that is, the second protein downstream of 2A still requires signal sequence for secretary or membrane-anchored expression.  相似文献   

14.
Attempts to generate reliable and versatile vectors for gene therapy and biomedical research that express multiple genes have met with limited success. Here we used Picornavirus 'self-cleaving' 2A peptides, or 2A-like sequences from other viruses, to generate multicistronic retroviral vectors with efficient translation of four cistrons. Using the T-cell receptor:CD3 complex as a test system, we show that a single 2A peptide-linked retroviral vector can be used to generate all four CD3 proteins (CD3epsilon, gamma, delta, zeta), and restore T-cell development and function in CD3-deficient mice. We also show complete 2A peptide-mediated 'cleavage' and stoichiometric production of two fluorescent proteins using a fluorescence resonance energy transfer-based system in multiple cell types including blood, thymus, spleen, bone marrow and early stem cell progenitors.  相似文献   

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扫描模型和遗漏扫描模型是真核生物mRNA翻译起始的两种主要机制,但其仍存在某些例外情况,如对具有多顺反子结构的mRNA,选择性翻译起始的发生机制目前仍不清楚.本研究基于GFP蛋白开放表达框(ORF)构建了一系列重组表达载体,用以转录在移码翻译顺序及同一翻译顺序下,AUG起始密码子处于不同序列背景,以及间隔不同距离的多顺反子结构mRNA.通过转染人Bel 7402细胞系,研究了这些多顺反子结构mRNA的翻译起始模式.结果表明,在移码翻译顺序下,多顺反子mRNA可翻译出对应的不同蛋白质,而在同一翻译顺序下,GFP蛋白表达框中的多个AUG密码子,仅有首位起始密码子可发挥作用,提示核糖体在从首位起始密码子开始翻译的同时,可能会有部分核糖体继续向下扫描并识别下游的起始密码子,而这种选择性的翻译起始效率,主要取决于密码子所处的序列背景及间隔距离等因素.  相似文献   

18.
Recent reports on various cancer models demonstrate a great potential of cytosine deaminase/5-fluorocytosine suicide system in cancer therapy. However, this approach has limited success and its application to patients has not reached the desirable clinical significance. Accordingly, the improvement of this suicide system is an actively developing trend in gene therapy. The purpose of this study was to explore the cytotoxic effect observed after co-expression of hepatitis A virus 3C protease (3C) and yeast cytosine deaminase/uracil phosphoribosyltransferase fusion protein (FCU1) in a bicistronic vector. A set of mono- and bicistronic plasmid constructs was generated to provide individual or combined expression of 3C and FCU1. The constructs were introduced into HEK293 and HeLa cells, and target protein synthesis as well as the effect of 5-fluorocytosine on cell death and the time course of the cytotoxic effect was studied. The obtained vectors provide for the synthesis of target proteins in human cells. The expression of the genes in a bicistronic construct provide for the cytotoxic effect comparable to that observed after the expression of genes in monocistronic constructs. At the same time, co-expression of FCU1 and 3C recapitulated their cytotoxic effects. The combined effect of the killer and suicide genes was studied for the first time on human cells in vitro. The integration of different gene therapy systems inducing cell death (FCU1 and 3C genes) in a bicistronic construct allowed us to demonstrate that it does not interfere with the cytotoxic effect of each of them. A combination of cytotoxic genes in multicistronic vectors can be used to develop pluripotent gene therapy agents.  相似文献   

19.
To allow for structural analysis of the human acetylcholinesterase (hAChE) subunit, a series of eukaryotic vectors was designed for efficient expression. Several eukaryotic multicistronic expression vectors were tested in various mammalian cell lines. All expression vectors contained the selectable neo gene under control of a weak promoter, while the hAChE cDNA was under control of the cytomegalovirus (CMV) immediate-early or Rous sarcoma virus long terminal repeat (RSV LTR) or simian virus 40 (SV40) early promoters. Optimal production and secretion of recombinant hAChE (rehAChE) was achieved in the embryonal kidney 293 cell line transfected either with the RSV-hAChE or with CMV-hAChE expression vectors. Clones expressing and secreting as much as 5-25 pg of enzyme per cell per 24 h were obtained without resorting to coamplification techniques or continuous maintenance of cells under selective pressure. The purified (specific activity of 6000 units per mg protein) homodimer and tetramer enzyme molecules displayed typical AChE biochemical properties: a Km value of 120 microM for acetylthiocholine; a kcat value of 3.9 x 10(5)/min, and selective by AChE-specific inhibitors. Catalytic subunit dimers (130 kDa) exhibit differential N-glycosylation patterns, and upon reduction resolve into 67- and 70-kDa monomeric subunits. These two forms appear as a single discrete 62-kDa band following deglycosylation by N-glycanase. The N-terminal amino acid sequence analysis of the purified mature enzyme suggests the existence of two alternative cleavage sites for the removal of the signal peptide, in which the 'mature' position 1 is either Ala31 or Gly33. Both of these positions conform with the consensus signal peptide recognition sequences and demonstrate bidirected processing of signal peptides on a native molecule.  相似文献   

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