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溶藻弧菌铁调蛋白基因的克隆、表达及其特征分析 总被引：1，自引：0，他引：1
铁为一切有机体所必需。对铁摄取与利用的调控在细菌生长和铁毒性中起重要作用。在革兰氏阴性细菌中,铁摄取调节蛋白(Fur)主管铁的代谢。溶藻弧菌是人和海洋动物共感染的一种主要病原弧菌。本研究克隆表达了溶藻弧菌的fur基因,并分析了其相关特征。该基因序列全长994 bp,其中包含450 bp的开放读码框,编码150个氨基酸残基的蛋白,推导的相对分子量为16.78 kD.在编码序列的上下游,RBS序列-、10序列-、35序列和二个潜在的转录终止子分别得到确定,但没有发现存在于大肠杆菌fur基因中的"铁盒子"。氨基酸序列的中段从G46位至D104位,为高度保守区域,其中包括含组氨酸丰富铁结合模型(H3-D-H-L-V-C-L-D-C-G)。SDS-PAGE分析表明,fur基因可在大肠杆菌中大量表达,带His-Tag的重组融和蛋白经镍亲和层析得到纯化,Western-blot分析结果显示,Fur蛋白具有免疫反应性。为进一步研究Fur蛋白的结构与功能打下基础。 相似文献
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从垂序商陆(Phytolacca americana)的重金属镉胁迫下商陆幼苗叶片的正反向抑制性消减杂交文库(SSH文库)中,获得商陆寡肽转运蛋白基因的EST序列.利用RACE的方法克隆到该基因的全序列cDNA(PaOPT,GenBank注册HQ647015).PaOPT基因在进化中的地位分析表明:它可能属于OPT3基因家族.利用实时荧光定量PCR的方法,分析PaOPT的组织表达特异性以及商陆种子成熟及萌发过程的表达模式.结果表明,该基因是一个在种子中特异性表达的基因,预测它对植物的生长发育有着重要的作用. 相似文献
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《青海科技》2017,(1)
植物脂质转运蛋白(Lipid Transfer Protein,LTP)是一类含量丰富并且由多基因家族编码的小分子碱性蛋白。本研究根据NCBI数据库中大麦LTP19的预测信息设计引物,以大麦品种Morex为参照,克隆青稞肚里黄的LTP19基因编码区域,对其核酸及蛋白序列进行生物信息学分析,并通过RT-PCR方法分析了青稞LTP19的组织表达情况。结果表明:青稞LTP19 c DNA片段长度为376 bp,与大麦LTP19基因同源性为99%;生物信息学分析表明青稞LTP19具有脂质转运蛋白家族共有特性,具有N端信号肽结构;半定量RT-PCR检测结果表明,LTP19在肚里黄各器官中表达趋势与Morex相似,茎和叶中表达量较高。 相似文献
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一株嗜热梭菌β-葡聚糖酶基因的克隆和表达 总被引：1，自引：0，他引：1
提取嗜热梭菌(Clostridium.sp)基因组DNA,首次通过PCR克隆了该菌的β-葡聚糖酶基因全长,结果表明:该基因全长1040bp,ORF为1003bp,编码334个氨基酸,计算分子量为37.8kD,等电点为7.67。经Blast分析,该序列与热纤梭菌同源性最高(99%),而与基因库中嗜热梭菌的同源性为94%,该基因已被GenBank接受(AY225318)。用BamHⅠ和XhoⅠ双酶切目的片段和表达载体pET鄄30a( )后相连接,构建重组表达载体pET鄄clo,并导入BL21细菌中表达.酶学特性表明:SDS鄄PAGE电泳在37kD左右有表达蛋白带,该工程菌最适酶活29.4U/mL,是出发菌的15倍,最适温度在80℃左右,最适pH在9左右。该工程菌可构建耐热性好、酶活高的杂合基因工程菌。 相似文献
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《科学中国人》2015,(26)
目的构建SLO原核表达质粒,重组蛋白的诱导表达并纯化。方法提取链霉菌溶血素O模板DNA,PCR法扩增slo基因。构建融合表达重组质粒p GEX-6p-1-slo和pet32a-tev-slo,将正确的重组质粒转化至大肠杆菌BL21和大肠杆菌BL21-DE3,使用异丙基硫代βD半乳糖苷(IPTG)诱导表达重组融合蛋白。采用亲和层析纯化重组蛋白,切除标签后,再通过亲和层析纯化,获取SLO重组蛋白。结果 PCR扩增出slo基因,基因片段(1700 bp)与理论一致。经SDSPAGE,蛋白质印迹显示重组蛋白相对分子质量为55 k Da,与数据库中的相对分子质量结果相符。结论成功构建了slo原核表达质粒,表达并纯化了SLO重组蛋白。 相似文献
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为探索水稻响应高盐低温逆境胁迫的分子调控机制,采用Affymetrix水稻表达谱芯片,分析研究了高盐低温胁迫下水稻转录因子基因的表达谱.检测到491个差异表达的水稻转录因子基因,属于56个转录因子家族.其中17个转录因子基因,高盐低温处理后表达均显著上调,4个转录因子基因,高盐低温下表达均显著下调,9个转录因子基因经高盐、低温处理后表达水平向相反方向变化.进一步采用实时荧光定量PCR,验证了6个转录因子基因在高盐低温胁迫条件下的差异表达变化,其结果和芯片杂交结果基本一致. 相似文献
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从拟南芥中克隆得到AtACO3基因的全长cDNA序列.半定量PCR实验结果表明,200 μmol/L CuCl2处理能明显抑制该基因的表达,而200 μmol/L ZnCl2处理1h则可显著促进其表达.为验证AtACO3蛋白N端的功能,构建AtACO3 5端1632 bp基因片段的原核表达载体pET30a-AtACO3-N,并转化大肠杆菌.SDS-聚丙烯酰胺凝胶电泳检测结果显示,该基因片段可以在大肠杆菌中稳定表达.进一步的抗性实验表明,异源表达基因AtACO3的N端序列能提高大肠杆菌的Zn2+耐受性. 相似文献
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Emad F. Eskander Ahmed A. Abd-Rabou Mervat S. Mohamed Shaymaa M. M. Yahya Olfat G. Shaker 《Indian journal of clinical biochemistry : IJCB》2012,27(4):344-350
The main objective of the current study is to examine the role of the statistical relation between BCL2 gene (Ala43Thr) single nucleotide polymorphism and growth hormone (GH1) levels in Egyptian HCV genotype-4 patients before and after treatment with pegylated interferon plus ribavirin. Eighty patients with HCV genotype-4 and 40 healthy volunteers as controls were enrolled in the prospective study. Gene polymorphism of BCL2 (Ala43Thr) using PCR-RFLP technique and GH1 concentrations using ELISA procedure were measured for all patients and controls. The present study resulted that Responder HCV genotype-4 Patients, with BCL2 43Ala genotype, have high significant increase in pre-treatment GH1 levels (>1 ng/ml); which represent normal levels, as compared to non-responders pre-treatment GH1 levels (<1 ng/ml); which represent low concentrations. We concluded that HCV genotype-4 patients who have normal GH1 concentrations and BCL-2 43Ala genotype can successfully achieve response to interferon based therapy. 相似文献
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Dielectrophoretic properties of DNA have been determined by measuring capacitance changes between planar microelectrodes. DNA sizes ranged from 100 bp to 48 kbp, DNA concentrations from below 0.1 to 70 μg∕ml. Dielectrophoretic spectra exhibited maximum response around 3 kHz and 3 MHz. The strongest response was found for very long DNA (above 10 kbp) and for short 100 bp fragments, which corresponds to the persistence length of DNA. The method allows for an uncomplicated, automatic acquisition of the dielectrophoretic properties of submicroscopical objects without the need for labeling protocols or optical accessibility. 相似文献
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Emad F. Eskander Ahmed A. Abd-Rabou Shaymaa M. M. Yahya Ashraf El Sherbini Mervat S. Mohamed Olfat G. Shaker 《Indian journal of clinical biochemistry : IJCB》2013,28(4):348-357
The prevalence of hepatitis C virus (HCV) infection varies across the world, with the highest percent of infections reported in Middle East, increasingly in Egypt. The current study aimed at examining the bio-statistical correlation and multiple regression analyses of pituitary growth hormone (GH) and liver activities among HCV genotype-4 patients treated with PEG-IFN-α plus RBV therapy. Herein, the current study was conducted on 100 HCV genotype-4 infected patients and 50 healthy controls. Patients received PEG-IFN-α/RBV for 24 weeks. Host RNA was isolated from patients’ sera for HCV genotyping and viral load determination. Moreover, the enzymatic activities of the liver, AFP, GH, PT, and CBC were performed in all volunteers. The present study resulted that the activities of the hepatic enzymes among HCV genotype-4 patients correlated together significantly. While, human GH showed a significant positive regression with pre-treatment ALT concentration in responders. Furthermore, multiple regression analysis for GH showed a significant positive correlation with pre-treatment ALT in HCV genotype-4 infected patients. We concluded that there were a putative significant relation between GH and pre-treatment ALT activity in HCV infection and response to IFN-based therapy. 相似文献
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对Beesia calthifolia等5种金莲花亚科植物的核糖体DNA中的内转录间隔区(ITS)序列及5．8S
rRNA基因的3′端序列进行了测定。这几种金莲花亚科植物的ITS-1的长度范围为225～232 bp,ITS-2
的长度范围为201～217 bp。B．calthifolia的ITS-1(227 bp)和ITS-2(215 bp)的长度及序列均与升麻
属及类叶升麻属植物相近,其5.8S rRNA基因的3′端序列也近乎与升麻属及类叶升麻属植物完全一致
(仅一个碱基缺失的差异),但其在上述几方面均与金莲花属植物相差甚远。以Ranunculus enysii作为
外类群,运用PAUP软件进行的系统发育分析表明:B．calthifolia,Cimicifuga acerina,C．brachycarpa
和Actaea asiatica形成一个单系群,并得到bootstrap分析的极强支持,B．calthifolia位于这一单系群的
基部。这一DNA序列分析结果与来自植物化学、孢粉学和细胞学的研究结果相吻合,更进一步支持铁破锣属是升麻族的自然成员,并可能是升麻族中一个最原始的类群。 相似文献
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BackgroundEndoglucanase, one of three type cellulases, can randomly cleave internal β-1,4-linkages in cellulose polymers. Thus, it could be applied in agricultural and industrial processes.ResultsA novel endoglucanase gene (JqCel5A) was cloned from Jonesia quinghaiensis and functionally expressed in Escherichia coli Rosetta (DE3). It contained 1722 bp and encoded a 573-residue polypeptide consisting of a catalytic domain of glycoside hydrolase family 5 (GH5) and a type 2 carbohydrate-binding module (CBM2), together with a predicted molecular mass of 61.79 kD. The purified JqCel5A displayed maximum activity at 55°C and pH 7.0, with 21.7 U/mg, 26.19 U/mg and 4.81 U/mg towards the substrate carboxymethyl cellulose, barley glucan and filter paper, respectively. Interestingly, JqCel5A exhibited high pH stability over a broad pH range of pH (3–11), and had good tolerance to a wide variety of deleterious chemicals including heavy metals and detergent. The catalytic mechanism of JqCel5A was also investigated by site mutagenesis and homology-modeling in this study.ConclusionsIt was believed that these properties might make JqCel5A to be potentially used in the suitable industrial catalytic condition, which has a broad pH fluctuation and/or chemical disturbance. 相似文献
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P. Bubber A. Sharma A. Chauhan D. D. Bansal 《Indian journal of clinical biochemistry : IJCB》2013,28(2):193-196
Growth retardation is one of the significant changes in chronic kidney disease (CKD) and is associated with increased morbidity and mortality. Disturbances in growth hormone (GH) are held responsible for several complications in CKD. GH is a protein based peptide hormone which directly or indirectly regulates renal functions to ensure homeostasis. We investigated the effects of growth hormone on plasminogen activators (PA) in rat kidney, PA and plasminogen activator inhibitor (PAI), glucose and fibrinogen in plasma and serum lipid profile. Rats were injected daily with 250 mU GH kg-1 body weight subcutaneously for one week. Growth hormone treatment increased PA activity significantly in rat kidneys as compared to controls. No changes were seen in PA, PAI and fibrinogen levels in the plasma of two groups of rats. There was significant decrease in plasma glucose, total cholesterol and LDL-cholesterol levels in serum of treated group resulting in the decrease of HDL/LDL and total cholesterol/cholesterol ratios. However, triglycerides and VLDL showed significant higher activity in the serum of treated group as compared to controls. Our data suggests that GH administration might improve renal function by increasing PA activity in kidney as well as by reducing the cholesterol content in blood. GH may be effective in improving growth failure as it helps to maintain the normal homeostatic balance. 相似文献
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研究了国产防己科蝙蝠葛族tirb．Menispermeae9属20种和外类群青牛胆族trib．Tinosporeae 2属3种植物完整的ITS(包括5.8S rDNA)序列。trib．Menispermeae的ITS长527～601 bp，排序后长667bp。当gap处理为missing时具281个有信息位点。PAUP软件分析结果表明：①trib．Menispermeae是一个单系类群，该分支得到hootstrap l00％的支持；②确定了存疑种Pachygone valida的系统学位置，该种是Coc—culus属的成员；③Sinomenium和Menispermum两属有很近的系统学关系，组成族内稳定的一支，它们的ITS序列同源性极高，ITS1比族内其它属长41～73bp；④Stephania和Cyclea也是系统发育关系很近的两个类群。前者具两个主要分支，其IIS1、ITS2的G+C含量差异较大，在种类组成上，该两大支与传统上Stephania属内处理的2个亚属——千金藤亚属subgen．Stephania和山乌龟亚属subgen．Tuberiphania基本一致；Cyclea属内种间的ITS序列差异小，同源性极高。 相似文献
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Kirtimaan Syal Anand Srinivasan Dibyajyoti Banerjee 《Indian journal of clinical biochemistry : IJCB》2015,30(3):323-328
Diabetes and tuberculosis are world’s most deadly epidemics. People suffering from diabetes are susceptible to tuberculosis. Molecular link between the two is largely unknown. It is known that Vitamin A receptor (RXR) heterodimerizes with Vitamin D receptor (VDR) and Peroxisome proliferator-activator receptor-γ (PPARγ) to regulate Tryptophan-aspartate containing coat protein (TACO) expression and fatty acid metabolism respectively, so it would be interesting to check the expression of these genes in diabetes mellitus (DM) patients which might explain the susceptibility of diabetics to tuberculosis. In this study, we checked the expression of RXR, VDR, TACO and Interferon-γ (IFNγ) genes in type-2 DM patients for understanding the link between the two diseases. We observed down regulation of RXR gene and corresponding up regulation of TACO gene expression. We have not observed significant change in expression of VDR and IFNγ genes in type-2 DM patients. Repression of RXR gene could hamper VDR-RXR heterodimer formation and thus would up regulate TACO gene expression which may predispose the type-2 DM patients to tuberculosis. Also, decrease in RXR-PPARγ heterodimer could be involved in DM. 相似文献
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世界制造业已进入了一个巨大的变革时期,传统的相对稳定的市场变成动态多变的市场,速度已成为知识经济时代市场焦点。通过分析国内制造企业的现状及存在的问题,提出了应从人员,技术和组织三方面入手,提高企业快速反应能力,从而增强制造企业竞争力的观点。 相似文献