短小芽孢杆菌木聚糖酶的同源建模及分子动力学模拟

克隆并测序来源于短小芽孢杆菌的木聚糖酶基因,测定其编码产物的理化件质并鉴定其表达产物为常温酶蛋白.利用同源建模构建出具有较高精度的三级结构模型,并进行优化和评估.利用分子动力学模拟木聚糖酶基因对热不稳定的分子机制,了解影响该酶热稳定性的因素,寻找对热不稳定性的区域.通过与嗜热木聚糖酶比较发现,两者在3个区域的分子动力学...     

酯酶基因Est2的异源表达及酶学性质研究

对来自α/β水解酶超家族的酯酶基因Est2进行了克隆、异源表达及纯化,然后对其酶学性质进行了研究.研究结果显示:酯酶Est2的最适底物为对硝基苯乙酸酯(p-NPC2),最适反应温度为50℃,最适pH为8.0;Est2具有较好的pH稳定性,在pH 5.0～10.0范围内,其相对酶活性保持在50％以上;在40℃条件下具有最...     

拟南芥耐干旱相关转录因子DREB1A基因的克隆及序列分析

设计引物利用PCR技术从拟南芥中克隆与植物耐旱相关序列DRE相结合的转录因子DREB1A的基因,得到扩增谱带后,进行TA克隆,经蓝白斑筛选获得pDREB重组质粒,经PCR验证和序列测定确认扩增所得片段为DREB1A基因,并对该基因序列进行了分析.     

化学与酶促相结合合成并克隆核糖体失活蛋白Gelonin基因

利用已知Gelonin的氨基酸序，逆向推算出整个基因的碱基序列，根据E.coli的密码子偏爱性和后续基因克隆的需要，通过沉默突变设计相应的酶切位点和碱基序列，将整个基因分为四段，每个片段约175－220pb，每一个片段中的互补链从5′末端用化学合成100－120的碱基单链，其中两个链的3′末端有20个互补碱基。利用T4DNA聚合酶酶促添补成双链DNA，用分子克隆技术，分别构建重组子，然后再构建成含整个Gelonin基因的表达载体pET-gel进行表面，经诱导后，获得了一个28000的重组蛋白，并主要以可溶形式存在。     

棒曲霉Ac－22β－木聚糖酶合成的调控

棒曲霉（Ａｓｐｅｒｇｉｌｌｕｓｃｌａｖａｔｕｓ）Ａｃ－２２β－木聚糖酶的合成被木糖、木聚糖和含木糖苷的物质所诱导，而受葡萄糖、果糖、甘露糖和蔗糖等易利用的碳源阻遏，但它可产生部分组成型木聚糖酶，蛋白质合成抑制剂环已亚胺及呼吸抑制剂叠氮化钠、碘乙酸强烈抑制β－木聚糖酶的合成．     

棒状杆菌亮氨酸操縱子在大肠杆菌中的克隆和表达

以质粒pTZ22为载体,用HindШ限制酶切割谷氨酸棒状杆菌染色体DNA进行克隆。分离到带有亮氨酸操纵子的4.5kb片段。用Southern切口移位分子杂交检测,证明在4.5kb的片段中既包含有leuB基因,而且对leuA基因也表现同源性。     

Functional characterization of cadmium-responsive garlic gene AsMT2b： A new member of metallothionein family

METALLOTHIONEINS(MT)ARE LOW-MOLECULAR-WEIGHT AND CYSTEINE-RICH PROTEINS.THE FIRST MT PROTEIN WAS ISOLATED FROM EQUINE RENAL CORTEX IN1957[1].GENES EN-CODING MTS OCCUR IN DIVERSE ORGANISMS SUCH AS ANIMALS,HIGHER PLANTS,EUKARYOTIC MICROORGANISMS AND IN SOME…     

Rational design of glycerol dehydratase： Swapping the genes encoding the subunits of glycerol dehydratase to improve enzymatic properties

1,3-propanediol (1,3-PD) is an important material for chemical industry,and there has been always much interest in the production of 1,3-PD using all possible routes. The genes encoding glyc-erol dehydratase (GDHt) from Citrobacter freundii,Klebsiella pneumoniae and metagenome were cloned and expressed in E. coli. All glycerol dehy-dratases but the one from metagenome could be detected to show enzyme activities. In order to im-prove the enzymatic properties of GDHts,the genes encoding α and β-γ subunits were cloned,and the enzyme characteristics were evolved by rational de-sign based on their 3D structures which were con-structed by homology modeling. Six heteroenzymes were obtained by swapping the α subunit genes of these three different-source-derived GDHts. The pH,thermal stability and Vmax of some heteroenzymes were dramatically improved by 2―5 times compared with the wild one (GDHtKP). The GDHt cloned from metagenome,originally proved to be with no enzyme activity,was converted into active enzyme by swap-ping its subunits with other different GDHts. In addi-tion,the effect of subtle 3D structural changes on the properties of the enzyme was also observed.     

血吸虫膜相关抗原基因的克隆

目的:筛选日本血吸虫(Schistosoma japonicum,S.japonicum)新的疫苗候选抗原基因.方法:以S.japonicum成虫DNA为模板,PCR扩增相关基因,然后将其克隆XpBS-T载体,通过菌落PCR对产物进行检测,对阳性克隆子测序,与GeneBank中的已知相关序列进行比对分析.结果:菌落PCR获得一条与PCR产物一致的DNA片段,序列测定结果表明PCR产物与S.japonicum 22.6 kDa抗原分子核苷酸序列具有高度同源性.其目的基因大小636 bp,具有一个189 bp的ORF,能编码63个氨基酸.ORF不是全长的阅读框,只获得部分编码区.表达产物可含有一个包含9个氨基酸残基的潜在螺旋跨膜片段(25～33位)和一个酪氨酸激酶磷酸化位点(38～39位).讨论:成功克隆出一个与S.japonicum 22.6kDa抗原编码基因有高度同源性的基因.预测该基因为膜相关蛋白基因,可编码血吸虫体表膜相关蛋白.在生理机能上,该膜相关蛋白可能是信号传递分子.     

沼泽红假单胞菌GroE基因高保守区DNA片段的克隆,测序和特性分析

利用原核微生物GroE基因特定区域高保守性设计了简并引物，以Rhodopseudomonas palustris Y6的染色体为模板进行PCR扩增，得到片段594bp的产物，将该DNA片段连接到pUC-T载体多克隆位点上，转化大肠杆菌DH5α菌株，得到阳性重组子。经Southem 杂交验证，已克隆的594bpDNA片段来自Rhodopseudomonas palustris测序结果分析表明该片段是细菌groE基因高保守区。