利用脊髓灰质炎病毒5‘NCR和RNA聚合酶基因可提高外源基因表达

将含脊髓灰质炎病毒（ＰＶ）ＲＮＡ聚合酶的不同长度基因片段克隆到载体ｐＳＧ５质粒上,分别构建了４个表达ＲＮＡ聚合酶的质粒。体外转录实验证明,ｐＳＧ５－ＰＯＬ１．９９和ｐＳＧ５－ＰＯＬ２．０３质粒转染细胞的提取物促进了特异的ＲＮＡ转录,表明两质闰可表达ＲＮＡ聚合酶。将ＰＶ的５’ＮＣＲ序列插在载体ｐＧＲＥＥＮ ＬＡＮＴＥＲＮ－１的ＣＭＶ启动子下游,构建了ｐＧＲＥＥＮ ＬＡＮＴＥＲＮ－１－５’ＮＣＲ质粒;     

转译优化对EPO基因在COS7细胞中表达的影响

利用ＣＯＳ７细胞暂时表达系统,研究转译起始序列对ＥＰＯ－ｃＤＮＡ表达的影响。通过ＤＮＡ重组技术,构建了原ＥＰＯ－ｃＤＮＡ表达载体ｐＣＳＶ－ＥＰＯ（１）,其转译起始序列为５＇ＡＡＴＴＣＡＴＧＧ３＇。同时通过定点突变技术,将起始序列改变成５＇ＣＣＡＣＣＡＴＧＧ３＇,而构建了另一表达载体ＰＣＳＶ－ＥＰＯ（２）。后经序列分析证明无误后和前均通过ＤＥＡＥ－ｄｅｘｔｒａｎ法转染ＣＯＳ７细胞上清,测定结果为     

小鼠白细胞介素12双顺反子真核表达载体的构建及其在COS-7细胞中的表达

克隆小鼠白细胞介素１２（ＩＬ－１２）ｐ４０及ｐ３５ｃＤＮＡ,并构建同时含ｍＩＬ－１２ｐ４０和ｐ３５ｃＤＮＡ的双顺反子真核表达载体及其在哺乳动物细胞中的表达．白细胞介素１２是由巨噬细胞,树突状细胞等抗原提呈细胞产生的一种异二聚体细胞因子,对机体的细胞免疫功能起着重要的调节作用．利用脂多糖（１００ｐｇ／ｍｌ）和小鼠重组干扰素（ＩＦＮ－γ５００Ｕ／ｍｌ）体外联合刺激小鼠腹腔巨噬细胞,从中提取总ＲＮＡ,经ＲＴ－ＰＣＲ扩增出含信号肽的小鼠白细胞介素１２（ｍＩＬ－１２）ｐ４０及ｐ３５全长ｃＤ－ＮＡ．ＰＣＲ产物经酶切后,分别克隆至ｐＢｌｕｅｓｃｒｉｐｔⅡＳＫ载体中,序列测定结果与文献报道序列一致．然后利用脊髓灰质炎（Ｐｏｌｉｏ）病毒内核糖体进入位点（ＩＲＥＳ）连接ｍＩＬ－１２ｐ４０及ｐ３５ｃＤＮＡ,亚克隆至ｐｃＤＮＡ３载体中,构建成含ｍＩＬ－１２ｐ４０及ｐ３５ｃＤＮＡ双顺反子真核表达载体,即ｐｃＤＮＡ３／ｍＩＬ－１２,ｐ４０及ｐ３５ｃＤＮＡ同时受ｐｃＤＮＡ３中ｈＣＭＶ启动子驱动,将ｐ４０及ｐ３５转录至同一ｍＲ－ＮＡ上．通过ＬｉｐｏｆｅｃｔＡＭＩＮＥ将ｐｃＤＮＡ３／ｍＩＬ－１２转染ＣＯＳ－７细胞,７２ｈ收集培养上清,测定ｍ     

高效毛细管电泳在DNA序列分析中的应用

高效毛细管电泳在ＤＮＡ序列分析中的应用种康（兰州大学化学系,兰州７３００００）ＡＰＰＬＩＣＡＴＩＯＮＯＦＨＩＧＨ－ＰＥＲＦＯＲＭＡＮＣＥＣＡＰＩＬＬＡＲＹＥＬＥＣＴＣＴＯＲＨＯＲＥＳＩＳＯＮＤＮＡＳＥＱＵＥＮＣＩＮＧ￥ＣｈｏｎｇＫａｎｇ（Ｄｅｐａｒｔ...     

猪脑抗吗啡镇痛肽的分离纯化及其抗血清对吗啡耐受影响的初步…

猪脑组织提取液经ＳｅｐｈａｄｅｘＧ－５０分子筛层析,Ｓ－ＳｅｐｈａｒｏｓｅＦａｓｔＦｌｏｗ阳离子交换柱层析及两次ＨＰＬＣ分离得到一分子量为１２０００,等电点ＰＩ７．１的多肽,并测定了其氨基酸组成和Ｎ末端部分序列：Ｎ－Ｐｈｅ－Ｌｙｓ－Ｇｌｙ－Ｐｈｅ－Ｐｒｏ－Ａｓｐ－Ａｓｐ／（Ｌｙｓ）－Ｌｙｓ／（Ａｓｐ）－Ａｓｐ－Ｔｙｒ,给昆明小鼠脑室注射或尾静脉注射肽均能抑制吗啡引起的镇痛作用,其作用随着注射剂量的     

大鼠诱导型一氧化氮合酶基因转录调控区的克隆与鉴定

采用单特异引物ＰＣＲ克隆法,得到大鼠诱导型一氧化氮合酶（ｉＮＯＳ）基因转录调控区ＤＮＡ片段。核酸序列分析证实,大鼠ｉＮＯＳ基因的５＇－侧翼区含有ＩＦＮ－γ和ＴＮＦ－α应答元件及ＮＦ－ｋＢ结合位点的保守序列。这些保守序列的位置及排列显区别于人和小鼠的ｉＮＯＳ基因。电泳迁移率改变分析（ＥＭＳＡ）表达,ＶＳＭＣ受ＩＬ－１和ＩＦＮ－γ刺激后,细胞核内产生某种可与ｉＮＯＳ基因５＇－侧翼区特异结合的核蛋白因     

大鼠内侧隔核、斜角带中NOS与ChAT、NGF-R、AChE的共存神经元

用酶组织化学和免疫组织化学双标技术,观察了正常ＳＤ大鼠基底前脑内侧隔核（ＭＳ）、斜角带垂直支（ＶＤＢ）和水平支（ＨＤＢ）中ＮＯＳ阳性神经元的形态和分布及ＮＯＳ与胆碱能神经元标志物ＣｈＡＴ、ＮＧＦ受体（ＮＧＦ－Ｒ）和ＡＣｈＥ之间的共存关系。结果发现,ＭＳ、ＶＤＢ和ＨＤＢ的头端ＮＯＳ阳性神经元较多、胞体较大、突起多,尾端ＮＯＳ阳性神经元数目较少、胞体较小、突起少而短。ＮＯＳ＋ＣｈＡＴ双标神经元占ＮＯＳ阳性神经元总数的９０％,占ＣｈＡＴ阳性神经元总数的３９％;ＮＯＳ＋ＮＧＦ－Ｒ双标神经元占ＮＯＳ阳性神经元总数的８３％,占ＮＧＦ－Ｒ阳性神经元总数的４０％;ＮＯＳ＋ＡＣｈＥ双标神经元占ＮＯＳ阳性神经元总数的９６％,占ＡＣｈＥ阳性神经元总数的３９％。这些结果为研究Ａｌｚｈｅｉｍｅｒ＇ｓｄｉｓｅａｓｅ病理过程中基底前脑隔区胆碱能神经元退变与ＮＯ的关系提供了形态学依据。     

大鼠诱导型一氧化氮合酶基因转录调控区的克隆与鉴定

采用单特异引物ＰＣＲ克隆法,得到大鼠诱导型一氧化氮合酶（ｉＮＯＳ）基因转录调控区ＤＮＡ片段．核酸序列分析证实,大鼠ｉＮＯＳ基因的５′－侧翼区含有ＩＦＮ－γ和ＴＮＦ－α应答元件及ＮＦ－κＢ结合位点的保守序列．这些保守序列的位置及排列显著区别于人和小鼠的ｉＮＯＳ基因．电泳迁移率改变分析（ＥＭＳＡ）表明,ＶＳＭＣ受ＩＬ－１和ＩＦＮ－γ刺激后,细胞核内产生某种可与ｉＮＯＳ基因５′－侧翼区特异结合的核蛋白因子．     

大鼠iNOS基因上游调控区在转录激活中的作用

为了探讨大鼠ｉＮＯＳ基因上游调控区不同部位在对细胞因子诱导应答中所起的作用,将调控区不同部位插入ｐＳＶ０－ＣＡＴ报告基因载体,转染体外培养的血管平滑肌细胞（ＶＳＭＣ）,经ＩＬ－１β诱导后,采用氯霉素乙酰转移酶（ＣＡＴ）活性测定和Ｎｏｒｔｈｅｒｎ印迹杂交,检查了调控区各部位在ＩＬ－１β诱导ｃａｔ表达中所起的作用．结果表明,被转染的细胞在未经ＩＬ－１β刺激时,各种调控区序列启动ｃａｔ表达的活性均很低．在ＩＬ－１β作用下,调控区远端序列（－１０３７～－４３８）、近端序列（－４３７～＋４６）和全长序列（－１０３７～＋４６）均能独立激活ｃａｔ表达,其中以全长序列的作用最强,表明ｉＮＯＳ基因表达调控区远端和近端序列均具有启动子和增强子样功能．同时证实,远端序列和近端序列单独启动ｃａｔ表达的活性分别为全长序列的９１．３％和６７．１％,揭示大鼠ｉＮＯＳ基因调控区远端序列在介导ＩＬ－１β的应答反应中发挥更重要作用．     

池杉─水稻系统的生态效应（Ⅱ）系统的生态环境效应

池杉┐水稻系统的生态效应（Ⅱ）系统的生态环境效应黄兆祥郑珍贵朱笃（南昌大学生物科学工程系,南昌３３００４７）ＥＣＯＬＯＧＩＣＡＬＥＦＦＥＣＴＯＦＴＡＸＯＤＩＵＭＡＳＣＥＮＤＥＮＳ－ＯＲＹＺＡＳＡＴＩＶＡＥＣＯＳＹＳＴＥＭ（Ⅱ）ＥＣＯＬＯＧＩＣＡＬ－...     

Direct immunologic activities of CpG DNA and implications for gene therapy

Vertebrate immune systems have evolved the ability to detect and be activated by most microbial and viral DNAs by virtue of their content of unmethylated 'CpG motifs', which are selectively suppressed in vertebrate DNA. Because their CpGs are also unmethylated, the DNA in gene therapy vectors routinely induces direct immune stimulation through activating this host defense mechanism. Administration of such 'CpG DNA' by injection or inhalation triggers rapid activation of B cells, monocytes, macrophages, dendritic cells, and natural killer cells, along with the release of pro-inflammatory cytokines. These immune stimulatory effects can be prevented by chloroquine and other drugs that interfere with endosomal maturation or by the presence of certain neutralizing DNA sequences, which block the immune stimulatory CpG motifs. Aside from serving as the genetic code, DNA can have direct immune activities. Vertebrate immune systems have evolved a defense mechanism that is able to broadly detect most microbial and viral DNAs because of differences in the frequency and methylation of CpG dinucleotides in particular base contexts. B cells, monocytes, macrophages, and dendritic cells spontaneously take up DNA of any type. If the DNA contains these immune stimulatory 'CpG-S motifs', the cells become activated within minutes and begin producing pro-inflammatory cytokines such as IL-6 and IL-12 and upregulate expression of co-stimulatory molecules. This results in the activation of both innate and acquired immune responses. The pro-inflammatory effects of CpG-S motifs are opposed by CpG dinucleotides in certain distinct base contexts, termed neutralizing or CpG-N motifs. Increasing the ratio of CpG-S to CpG-N motifs enhances the immune stimulatory effects of DNA, even if the total level of CpGs in the DNA is not altered. While this is useful in generating enhanced genetic vaccines, the opposite strategy is likely to become useful for the generation of gene therapy vectors with reduced inflammatory effects.     

Requirements for effective inhibition of immunostimulatory CpG motifs by neutralizing motifs

The DNA of bacteria and many viruses contain unmethylated CpG dinucleotides in particular sequence contexts that activate vertebrate immune cells. A subset of these CpG motifs was previously found to oppose the effects of immunostimulatory (CpG-S) motifs and has been termed neutralizing (CpG-N) motifs. Here we show that oligodeoxynucleotides (ODNs) composed of clusters of CpG-N motifs could partially inhibit the induction of interleukin-12 (IK-12) from mouse spleen cells by ODN containing CpG-S motifs. However, non-CpG-containing ODN were also inhibitory, suggesting that neutralization of CpG-S ODNs by CpG-N ODNs in trans was nonspecific. Neutralization of CpG-S motifs by CpG-N motifs in cis was specific, but the degree of inhibition was strongly dependent on the particular CpG-S motif being neutralized, with motifs having an A residue 5' to the CG being much more resistant to inhibition than motifs having a T residue 5' to the CG. The degree of inhibition was dependent on the spacing between the CpG-S and CpG-N motifs, with the ability to neutralize inversely correlating with distance. In addition, whereas ODNs containing extended clusters of CpG-N motifs were nonstimulatory, isolated CpG-N motifs remained stimulatory in most sequence contexts. Finally, CpG-N ODNs were shown to be nonstimulatory when instilled into the lungs of BALB/c mice, but the ability of CpG-N motifs to neutralize CpG-S motifs in cis was not observed. These results show that there are precise and fairly complex interactions between immunostimulatory and inhibitory sequence motifs that govern whether a given DNA is able to activate the vertebrate immune system.     

抑制性寡脱氧核苷酸的生物学效应及研究进展

DNA对于机体免疫系统具有很多复杂的作用。存在于细菌DNA中的刺激性CpG基序能够促进机体免疫细胞分泌多种细胞因子,使机体产生偏向Th1方向的免疫应答,而抑制性寡脱氧核苷酸（oligodeoxynucleotide,ODN）可以选择性阻断刺激性CpG诱导的免疫激活作用。抑制性ODN按其结构不同大致分为三类,它可能通过影响细胞对CpG-S的结合及摄取、降低CpG-S特异性受体TLR9的表达发挥抑制作用。本综述主要介绍抑制性ODN的结构特征、作用机制和它在一些疾病中发挥的作用。     

CpG penta- and hexadeoxyribonucleotides as potent immunomodulatory agents

We demonstrate a new design for immunomodulatory CpG DNA containing two sequences each with as few as five or six-nucleotides joined together via 3(')-3(') linkers. These do not require the -PuPu(Py)CGPyPy- hexameric motif generally found essential for CpG DNA immune stimulation. These novel, short-immunomers show potent immunostimulatory activity manifested by IL-12 and IL-6 secretion in murine spleen cell and PBMC cultures and splenomegaly in vivo. Short-immunomers show strong activation of NF-kappaB and stress-activated signaling pathways and induce cytokines in J774 cell cultures. The same sequences also induce cytokines in healthy human PBMC cultures whereas conventional CpG DNA requires different optimal sequences for murine and human immune cells. Additionally, short-immunomers inhibit IL-5 secretion and induce IFN-gamma secretion in conalbumin-sensitized mouse spleen cell cultures, suggesting reversal of established Th2 responses to Th1 type responses. Short-immunomer also inhibits growth of MCF-7 human tumor xenograft in nude mice. This is the first report of activity with such short DNA sequences and also of sequences lacking hexameric motifs proposed in earlier studies.     

Effect of suppressive DNA on CpG-induced immune activation

Bacterial DNA and synthetic oligodeoxynucleotides (ODN) containing unmethylated CpG motifs stimulate a strong innate immune response. This stimulation can be abrogated by either removing the CpG DNA or adding inhibitory/suppressive motifs. Suppression is dominant over stimulation and is specific for CpG-induced immune responses (having no effect on LPS- or Con A-induced activation). Individual cells noncompetitively internalize both stimulatory and suppressive ODN. Studies using ODN composed of both stimulatory and suppressive motifs indicate that sequence recognition proceeds in a 5'-->3' direction, and that a 5' motif can block recognition of immediately 3' sequences. These findings contribute to our understanding of the immunomodulatory activity of DNA-based products and the rules that govern immune recognition of stimulatory and suppressive motifs.     

以CpG为佐剂的犬细小病毒基因疫苗的初步研究

目的研制犬细小病毒(CPV)基因疫苗。方法以CPV VP2基因为基因免疫的目的基因,以pcDNA3和pcDNAK质粒为基因免疫的载体,以非甲基化的胞嘧啶鸟嘌呤二核苷酸(CpG)为核心的免疫刺激序列为免疫佐剂,构建重组质粒并免疫BALB/c小鼠和毕格犬。结果经pcDNA3-VP2C1(含1个拷贝CpG基序)基因免疫的BALB/c小鼠能产生抗CPV血凝抑制抗体;对于经CPV灭活苗初次免疫的毕格犬,用pcDNAK-VP2C2(含2个拷贝CpG基序)质粒免疫产生的再次免疫应答优于pcDNA3-VP2C1。结论VP2基因、pcDNAK和犬源CpG可用于CPV基因疫苗的进一步研究。     

Microbial DNA induces a host defense reaction of human respiratory epithelial cells

Epithelial cells represent the initial site of bacterial colonization in the respiratory tract. TLR9 has been identified in B cells and CD 123(+) dendritic cells and found to be involved in the recognition of microbial DNA. It was the aim of the study to investigate the role of TLR9 in the host defense reactions of the respiratory epithelium. Respiratory epithelial cell lines (IHAEo(-), Calu-3) or fully differentiated primary human cells as air-liquid interface cultures were stimulated with bacterial DNA or synthetic oligonucleotides containing CpG motifs (CpG oligodeoxynucleotides). Expression of TLR9, cytokines, and human beta-defensin 2 was determined by quantitative RT-PCR or by ELISA. We found that TLR9 is expressed by respiratory epithelial cell lines and fully differentiated primary epithelial cells at low levels. Stimulation of the above-mentioned cells with bacterial DNA or CpG oligodeoxynucleotide resulted in an inflammatory reaction characterized by a dose-dependent up-regulation of cytokines (IL-6, IL-8) and human beta-defensin 2. Up-regulation of NF-kappaB in epithelial cells in response to the CpG motif containing DNA was inhibited by overexpression of a dominant negative form of MyD88. These results provide clear evidence that the human respiratory epithelium is capable of detecting microbial DNA by TLR9. The respiratory epithelium has an important function in triggering innate immune responses and therefore represents an interesting target for anti-inflammatory therapy.     

Cytokine induction by a bacterial DNA-specific modified base

Unmethylated CpG dinucleotides in DNA contribute to a rapid inflammatory response in mammals. Here we show that N(6)-methyladenine (N(6)-MeA), a bacterium-specific modified base, also causes cytokine production. An oligodeoxyribonucleotide (ODN) containing N(6)-MeA induced cytokines when injected into mice. Co-injection of N(6)-MeA and CpG ODNs enhanced cytokines 2- to 3-fold, as compared with the injection of a CpG ODN alone. Plasmid DNA containing N(6)-MeA, complexed with cationic lipids, induced IL-12. These results indicate that the bacterium-specific base, in addition to the unmethylated CpG motif, triggers the mammalian immune response, and suggest that N(6)-MeA-containing DNA could be useful for cellular immunotherapy and DNA vaccine.     

Therapeutic effect of CpG motifs on the development of chronic graft-versus-host disease in mice

Transferring DBA/2 spleen cells into (C57BL/10xDBA/2) F1 (referred to as BDF1) mice induces a chronic graft-versus-host disease (GVHD), characterized by the production of Th(2) cytokines, hypergammaglobulinemia, and immune complex-mediated glomerulonephritis that resembles systemic lupus erythematosus. DNA motif consisting of an unmethylated CpG dinucleotide flanked by two 5' purines and two 3' pyrimidines (CpG ODN) induces Th(1) cytokine production in mice. This study examines the effect of administering CpG ODN to mice undergoing chronic GVHD, based on the premise that altering Th(1)/Th(2) activity might beneficially impact on disease progression.GVHD BDF1 mice injected with DBA/2 spleen cells were treated with weekly intraperitoneal injection of 50 microg CpG ODN. This treatment significantly suppressed the production of IgG anti-DNA autoantibody and reduced the development of glomerulonephritis. Serum IgG2a titers were higher in the CpG ODN than in non-CpG control group, whereas IgG1 titers were unchanged. As predicted, IFN-gamma levels were significantly higher in the CpG ODN-treated group, while IL-4 levels were lower, resulting in a shift in the Th(1)/Th(2) cytokine ratio. Results suggest that CpG ODN administration may be of therapeutic benefit in chronic GVHD.     

Plasmid DNA activates murine macrophages to induce inflammatory cytokines in a CpG motif-independent manner by complex formation with cationic liposomes

Plasmid DNA (pDNA) is very important in non-viral gene therapy and DNA vaccination. Unmethylated CpG motifs in bacterial DNA, but not in vertebrate DNA, are known to trigger an inflammatory response, which inhibits gene expression while improving immunological consequences. In this report, we investigated the cytokine secretion induced by pDNA/cationic liposome complexes using murine macrophages. Naked CpG DNA induced tumor necrosis factor-alpha (TNF-alpha) secretion from the macrophages, but DNA without CpG motif did not, demonstrating that the cytokine induction was mediated by CpG motifs. pDNA complexed with cationic liposomes, but not the cationic liposomes alone, produced a significant amount of TNF-alpha from the macrophages. Surprisingly, methylated pDNA and calf thymus DNA complexed with the cationic liposomes were also able to induce TNF-alpha production, indicating that these responses were not dependent on CpG motifs. Taken together, the present study demonstrated that for the first time DNA can stimulate murine macrophages in a CpG motif-independent manner when it is complexed with the cationic liposomes.