人白细胞硫氧还蛋白还原酶的分子克隆和序列分析

目的：克隆人白细胞硫氧还蛋白还原酶（thioredoxin reductase,TR)编码区的cDNA并进行序列分析。方法：采用RT－PCR方法获得TR基因cDNA，选择阳性克隆并测序。结果：通过分子克隆和序列分析，发现TR基因长1554bp,ORF为1494bp，编码498个氨基酸，结论：确定了中国人白细胞TR的cDNA序列，并据此推导出相应的氨基酸序列。     

PC12细胞硫氧还蛋白cDNA的克隆及在大肠杆菌中的表达

硫氧还蛋白（Thioredoxin,TRX)是广泛存在于原核和真核细胞中的低分子量蛋白质，它含有保守的Cys-Gly-Pro-Cys活性位点，作为多效性细胞因子而具有重要的生物学功能，从PC12细胞中提取总RNA，经逆转录PCR（RT-PCR)扩增出硫氧还蛋白cDNA并克隆到PUC18质粒上，序列分析表明，克隆所得序列与GenBank中大鼠硫氧还蛋白cDNA序列完全一致。将该基因亚克隆到表达质粒pQE30上，质粒pQE30-TRX在大鼠杆菌M15中获得高效表达，带6His的融合蛋白约占总菌体蛋白的30％。分析鉴定表明，纯化的融合蛋白质分子量是14kD，且具有二硫键还原酶活性。     

硫氧还蛋白还原酶生物学活性及其与人类疾病的关系

硫氧还蛋白还原酶 (TrxR)是吡啶核苷酸和二硫化物氧化还原酶的黄素蛋白家族成员之一 ,包含保守的 Cys Val Asn Val Gly Cys 氧化还原位点 ,催化依赖硫氧还蛋白NADPH还原反应。TrxR含有存在于许多底物中的氧化还原活性位点 ,催化多种内源和外源性复合物。TrxR具有多种生物学活性 ,调节机体氧化还原反应和细胞生长与增殖 ,与人类某些疾病发病机制有关 ,可能是干预治疗的靶点     

硫氧还蛋白系统研究进展

硫氧还蛋白（Trx）系统由Trx、硫氧还蛋白还原酶（TR）和还原型烟酰胺腺嘌呤二核苷酸磷酸组成。Trx是一种重要的抗氧化分子,可抵抗多种应激引起的细胞死亡,在氧化还原反应中发挥着突出作用。TR是含硒（硒代半胱氨酸）的蛋白质,主要有3种形式：TR1（主要分布在胞质）、TR2（主要分布在线粒体）和TR3（主要分布在睾丸）。...     

硫氧还蛋白还原酶在大鼠阿尔茨海默病模型海马结构中的表达

目的 :探讨硫氧还蛋白还原酶 (thioredoxinreductase,TR)在阿尔茨海默病 (Alzheimer′sDisease,AD)大鼠模型发生中的作用。方法 :注射海人藻酸 (Kainicacid ,KA)毁损Meynert基底核建立AD大鼠模型 ,通过Y形迷宫判断动物模型成功与否 ,采用原位杂交方法检测AD大鼠模型中TRmRNA的表达。结果 :AD模型组大鼠短期学习记忆能力与对照组相比明显下降 (P <0 0 0 1 ) ,说明AD动物模型建立成功 ;TRmRNA原位杂交检测显示模型组大鼠海马各区TRmRNA杂交信号显著增强 ,与对照组相比有显著差异 (P <0 0 0 1 )。结论 :TR在AD发生发展中可能起神经元保护作用     

c-Jun对硫氧还蛋白还原酶1启动子转录的调控作用

目的: 探讨转录因子激活剂蛋白-1(activator protein-1,AP-1)家族成员c-Jun对硫氧还蛋白还原酶1(thioredoxin reductase 1, TrxR1 )启动子转录的调控作用。方法: 利用生物信息学技术分析调控 TrxR1 启动子区域的转录因子,构建 TrxR1 启动子区域一系列截短的萤光素酶报告基因载体,将含c-Jun的真核表达载体pcDNA3.1(+)-c-Jun和含有 TrxR1 启动子的萤光素酶报告基因载体共转染人胚肾HEK293细胞和鼠心肌H9c2细胞,检测转染细胞中萤光素酶活性。应用定点突变技术针对 TrxR1 启动子区域AP-1的可能结合位点进行突变,与c-Jun的真核表达载体共转染上述2种细胞,检测各组萤光素酶活性。利用染色质免疫共沉淀(ChIP),分析c-Jun与 TrxR1 启动子区域的AP-1结合位点结合情况。结果: 酶切及测序结果表明,获得的3个不同长度的 TrxR1 启动子克隆与GenBank DNA序列数据库对比分析序列一致,且插入方向正确;此3种质粒都有明显的启动子活性,在人胚肾HEK293和鼠心肌H9c2细胞中转染pcDNA 3. 1(+)-c-Jun可以上调 TrxR1 启动子活性。突变AP-1结合位点,导致 TrxR1 启动子活性明显降低;ChIP结果显示c-Jun结合在 TrxR1 启动子区域的AP-1结合位点上。结论: 转录因子AP-1家族成员c-Jun可能通过与 TrxR1 基因启动子区域AP-1结合位点相结合,上调 TrxR1 基因的转录。     

人硫氧还蛋白cDNA的克隆、表达及其多克隆抗体制备

目的制备硫氧还蛋白1(thioredoxin-1,Trx-1)多克隆抗体。方法从乳腺癌细胞系MCF-7中用RT-PCR的方法得到了Trx-1全长基因,将它克隆到原核表达载体上进行大量的表达和纯化,纯化的蛋白对新西兰大白兔进行背部多点注射,40d后取其血清用梯度饱和硫酸铵沉淀的方法进行多克隆抗体的纯化。用ELISA和Western印迹实验测定抗体效果。结果成功获得了Trx-1全长cDNA,通过原核表达得到了大量Trx-1蛋白,并制备了高效价的多克隆抗体。结论此多克隆抗体对Trx-1蛋白具有良好的识别能力,可以应用于Trx-1的功能研究。     

硫氧还蛋白与肺部疾病的研究进展

硫氧还蛋白系统是一个广泛存在于自然界的巯基氧化还原系统,它能够维持细胞的氧化还原动态平衡及阻止炎症反应的发生。它由硫氧还蛋白（thioredoxin,Trx）、硫氧还蛋白还原酶（thioredoxin reductase,Tr x R）和还原型辅酶II（reduced coenzy me II,NADPH）三部分组成。人类Tr x作为一种分泌性蛋白,在细胞内外发挥着多种氧化还原调控作用。Trx通过直接抑制氧化应激和间接与关键信号转导分子结合而发挥了抗氧化、抗凋亡和抗炎症作用。最近的研究显示Trx作为机体内主要的氧化应激相关因子,可能在肺部疾病中发挥了一定作用,且有可能成为临床干预治疗的靶点。本文对Trx在呼吸系统的表达与慢性阻塞性肺疾病、支气管哮喘、急性肺损伤（acute lung disease,ALI）和间质性肺疾病（interstitial lung disease,ILD）的关系进行简述。     

硫氧还蛋白-1与衰老的研究进展

过去的几十年里,虽然对衰老的氧化应激理论进行了广泛的研究,但是目前仍然存在着许多争议,特别在哺乳动物中。最近关于硫氧还蛋白-1(Trx1)在氧化应激和衰老中的研究提示：1)氧化应激及后续信号通路的改变可能在不同年龄阶段有着不同的病理生理作用;2)与氧化损伤的累积相比,氧化应激和氧化还原状态导致氧化还原敏感信号通路的改变可能在衰老的病理生理中扮演着更重要的角色。因此,本文对硫氧化蛋白-1与衰老的关系进行简述。     

TR基因重组腺病毒载体的构建和鉴定

目的 :构建含人硫氧还蛋白还原酶 (TR)基因的重组腺病毒载体 ,探讨TR的抗氧化功能与神经退行性疾病的相关性。方法 :从重组质粒pGEM TR上用内切酶切下编码 5 0 0个氨基酸的全长TRcDNA片段 ,并连接穿梭质粒pShuttle,再双酶切pShuttle TR。将带有CMV启动子的目的片段 ,插入E1、E3缺失的Adeno X病毒DNA中 ,以Adeno TRDNA通过脂质体转染HEK2 93细胞 ,获得重组腺病毒Adeno TR进行PCR鉴定及病毒滴度测定。用重组腺病毒感染CV1细胞 ,通过免疫荧光染色与Westernblot分别检测重组腺病毒感染的细胞上和裂解液中TR蛋白的表达。结果 :重组腺病毒Adeno TR的病毒滴度为 4 .4× 10 11pfu/L。PCR、荧光显微镜证实 ,以及Westernbolt分析 ,在相对分子质量 (Mr)约 5 5 0 0 0处均出现特异性条带。结论 :成功地构建了重组腺病毒载体 ,并能介导外源基因TR表达 ,为进一步研究TR的功能及其与疾病的相关性奠定了基础。     

Expression of the thioredoxin system in interstitial lung disease

The thioredoxin system containing thioredoxin (Trx) and thioredoxin reductase (TrxR) has profound effects on cell proliferation and protection against exogenous oxidants. The significance of the Trx system in human lung and lung diseases is, however, largely unresolved. Altogether, 66 specimens of human lung were investigated by immunohistochemistry for their expression of Trx and TrxR. The diseases included interstitial pneumonias such as usual interstitial pneumonia (UIP), desquamative interstitial pneumonia (DIP), and UIP associated with collagen vascular diseases (CVD-ILD), and granulomatous diseases such as sarcoidosis and allergic alveolitis. The ultrastructural localization of Trx and TrxR was analysed by immunoelectron microscopy. In healthy lung, Trx and TrxR were expressed in bronchial epithelium and alveolar macrophages. Trx and TrxR were highly concentrated in areas of metaplastic epithelium in UIP and in alveolar macrophages in DIP, though fibrotic areas in UIP were mainly negative. The expression of both enzymes was clearly weaker in CVD-ILD than in UIP. Granulomas of sarcoidosis showed moderate to intense Trx immunoreactivity. Ultrastructurally, Trx and TrxR were expressed diffusely in the cytosolic compartment and plasma membrane of metaplastic type II pneumocytes, macrophages, and bronchial epithelial cells. This study highlights the importance of Trx and TrxR in primary defence in bronchial epithelium, alveolar epithelium, and macrophages in human lung, but also indicates that elevated expression of these proteins may serve as markers of ongoing cell regeneration and inflammation.     

Expression of genes for thioredoxin 1 and thioredoxin 2 in multidrug resistance ovarian carcinoma cells SKVLB

The expression of genes for thioredoxin isoforms Trx1 and Trx2 was studied in sensitive SKOV-3 and resistant SKVLB human ovarian carcinoma cells. The development of doxorubicin resistance was accompanied by a significant increase in the expression of TRX1 gene and less pronounced increase in TRX2 gene expression. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 144, No. 9, pp. 274–276, September, 2007     

抗人CD154单抗轻重链可变区基因克隆与序列分析

目的:克隆抗人CD154抗体轻重链可变区基因,并分析其核苷酸序列,为基因工程抗体的构建奠定基础。方法:从分泌能抑制免疫反应的抗人CD154单克隆抗体杂交瘤细胞株 7E8中提取总RNA,合成cDNA第一链后,经PCR扩增获得抗人CD154单抗轻链可变区 (VL)和重链可变区 (VH)基因,分别克隆入pUC18载体,并进行序列分析。结果:①抗体的轻链可变区基因全长为 341bp,编码113个氨基酸,归属于Ig的Vκ2基因,氨基酸序列分析结果显示轻链可变区含有明确的 4个骨架区和 3个抗原决定簇互补区,在第 2 3位和第 93位氨基酸为半胱氨酸,是与抗体二硫键形成有关的两个特征性氨基酸;②抗体的重链可变区基因全长为 354bp,编码118个氨基酸,归属于小鼠IgVH基因,D、J区基因分别属于DSP2.9和JH2,氨基酸序列分析结果显示,重链可变区含有明确的 4个骨架区和 3个抗原决定簇互补区,在第 2 3和第 97位氨基酸为半胱氨酸,是与抗体二硫键形成有关的两个特征性氨基酸。结论:经核苷酸序列分析证明所克隆的基因分别为抗体的轻、重链可变区基因.     

Efficiency of selenocysteine incorporation in human thioredoxin reductase

Thioredoxin reductase (TR) is a flavoenzyme, containing one selenocysteine (Sec) residue at the penultimate carboxyl-terminus, that catalyzes the NADPH-dependent reduction of oxidized thioredoxin. Sec is encoded by the UGA stop codon in the open reading frame of the mRNA, and the conserved stem-loop structure in 3'-untranslated regions functions as the determinant of Sec incorporation instead of termination of translation. The efficiency of Sec incorporation in Sec-containing enzymes in physiological or selenium (Se)-deficient condition remains unclear. To clarify this, we have developed monoclonal antibodies to human TR, and established a sandwich enzyme-linked immunosorbent assay to determine TR protein content. We observed that the specific activity of cytosolic TR in NCI-H441 cells increased with increasing concentrations of Se in a serum-free medium. The specific activity of TR purified from each cytosol was essentially equal to the calculated specific activity of each cytosolic TR. The Se content of TR increased with increasing concentration of Se in the medium, from 0.32 mol/mol of TR subunit (no SE) to 0.98 mol/mol of TR subunit (500 nM Se), and was directly correlated with the specific activity of TR. When calculated from the cytosolic TR specific activity of human peripheral mononuclear cell, the theoretical efficiency of Sec incorporation in physiological conditions is assumed to be 87%.     

Cloning, expression and antigenicity of the L. donovani reductase

The protozoan parasite Leishmania undergoes a morphological and biochemical transformation from the promastigote to the amastigote form during its life cycle, which is reflected in the expression of stage-specific proteins. One of these proteins shows homology to a superfamily of reductase proteins. We have cloned the reductase gene from L donovani and have shown that it differs in only one nucleotide from the L. major homologue, resulting in one amino acid change. A cytosine (C) to guanine (G) transposition in the coding sequence leads to a nonconserved substitution of asparagine (N) for lysine (K). Only 2 of 22 plasma samples from patients with visceral leishmaniasis were found to have detectable anti-reductase antibodies and peripheral blood mononuclear cells (PBMC) from one of three individuals previously infected with visceral leishmaniasis proliferated in the presence of recombinant reductase protein. Interestingly, 6 of 10 PBMC isolated from Danish controls proliferated in the presence of the reductase protein. Intracellular IFNgamma was found in a significant percentage of cells in all the tested PBMC cultures from Danes, whereas IL4 was only found in a small proportion of cells, or not at all. The results indicate the presence of cross-reacting CD45R0 memory T-cells in individuals not exposed to Leishmania. Several previous studies have shown that T-cells from nonexposed individuals often respond to crude Leishmania antigen preparations. The present study suggests that this reactivity is partly caused by T-cells recognising L. donovani reductase.