脆壁克鲁维酵母KfPDI基因高表达菌株的构建

通过构建脆壁克鲁维酵母（Kluyveromyces fragilis）蛋白二硫化物异构酶基因KfPDI强表达单元并克隆到不同类型的酵母载体上转化酵母菌株,经筛选得到了带有不同拷贝数KfPDI基因的酵母转化子。酶活性测定结果表明这些转化子中的蛋白二硫化物异构酶表达水平不仅与其KfPDI基因拷贝数有关,而且与该基因是否整合入宿主染色体也密切相关。本文所得到的KfPDI基因高表达菌株有望作为增强外源蛋白正确折叠和分泌效率的基因工程宿主菌。     

脆壁克鲁维酵母KFade5，7基因的克隆及其5′非编码区结构与功能研究

通过补偿乳酸克鲁维酵母(Kluyveromyces lactis)KlADE5，7基因突变，从脆壁克鲁维酵母(Kluyveromyces fragilis)基因组文库中克隆了KfADE5，7基因。该基因全长4975bp，编码793个氨基酸，氨基酸序列与已知的酿酒酵母(Saccharomyces cerevisiae)ScADE5，7基因编码区有68．3％的同源度。利用KfADE5,7-lacZ融合基因，对5′非编码区进行缺失分析，发现了具有完整启动子功能的区域，在启动子上游还发现了两个增强转录和两个减弱转录的区域。     

脆壁克鲁维酵母KfADE5,7基因的克隆及其5''''非编码区结构与功能研究

通过补偿乳酸克鲁维酵母(Kluyveromyces lactis)KlADE5,7基因突变,从脆壁克鲁维酵母(Kluyveromyces fragilis)基因组文库中克隆了KfADE5,7基因.该基因全长4975bp,编码793个氨基酸,氨基酸序列与已知的酿酒酵母(Saccharomyces cerevisiae)ScADE5,7基因编码区有68.3%的同源度.利用KfADE5,7-lacZ融合基因,对5'非编码区进行缺失分析,发现了具有完整启动子功能的区域,在启动子上游还发现了两个增强转录和两个减弱转录的区域.     

脆壁克鲁维酵母蛋白二硫化物异构酶KfPDI的结构分析

利用ＤＮＡ限制性内切酶图谱分析将一个带有脆壁克鲁酵母蛋白二硫化物异构酶基因（ＫｆＰＤＩ）的１０ｋｂ初始克隆缩短为４ｋｂ的亚克隆,并测定了该基因的ＤＮＡ全序列。经分析发现：（１）该基因编码产物由５２０个氨基酸重印箕组成。（２）它与乳酸克鲁维酵母蛋一太化物异构酶在氨基酸序列上的同不原性为７８％。（３）民其他物种的ＰＤＩ基因在核苷酸和蛋白持水平上的同源性较弱,但都存在有两个高度保守的异构酶活性位点。     

WSSV-VAP1基因克隆及在毕赤酵母中的表达

根据WSSV-黏附蛋白(VAP1)基因序列设计了一对引物,在5‘引物和3‘引物中分别引入EcoRI和XbaI酶切位点。经PCR扩增,将VAP1基因克隆至穿梭载体pGAPZαA之GAP启动子下游位点,构建成含α-factor信号肽的重组表达载体,获得的重组质粒pGAPZαA-VAP1经线性化后转入毕赤氏酵母SMD1168菌株,构建成分泌型表达VAP1的酵母工程菌株。SDS-PAGE和Western-blot及结合活性检测分析表明,VAP1基因在该体系中得到成功表达,表达产物与对虾细胞膜具明显的结合活性。     

来源于芽孢杆菌(Bacillus circulans)的乳糖酶在毕赤酵母中的高效表达

根据毕赤酵母(Pichia pastoris)密码子选择偏好对来源于芽孢杆菌(Bacillus circulans)的乳糖酶基因lacO进行了分子改造.改造后的基因lacM按正确的阅读框架插入到毕赤酵母表达载体pPIC9上,构建得到重组毕赤酵母表达质粒.PCR检测、SDS-PAGE电泳分析和酶活力测定的结果表明,lacM基因已重组到酵母染色体上,并能正常分泌表达.与未改造的基因lacO的毕赤酵母重组子相比,酶蛋白表达量明显增加,约为改造前的3倍以上.     

甜蛋白Monellin在毕赤酵母中的分泌表达

在西非热带植物Dioscoreophyllum cumminsii中存在着一种由两条多肽构成的甜度极高的蛋白Monellin。本研究根据已知的Monellin晶体衍射分析结果和毕赤酵母(Pichia pastoris)基因偏爱密码子设计并合成了连接两条多肽的重组基因，插入到毕赤酵母分泌表达质粒pGAPZαA中。扩增后，电击穿孔转化毕赤酵母GS115，筛选高产菌株放大培养。以5升罐发酵培养，表达量为150mg／L。经纯化，可以获得大于130mg／L的具有高甜度的活性蛋白。     

一种新型广谱的原核型分泌表达载体的建立及人表皮生长因子的表达

通过Cyanobase藻类学数据库查询,从集胞藻6803转译后加工过程中的某些蛋白的分泌信号序列得到了可以用于分泌型表达的信号序列,进一步通过SignalP 3.0 Server分析软件预验证,从集胞藻6803得到4种不同蛋白的信号序列A、B、C、D,将这些信号序列插入pET-His-EGF表达载体hegf基因上游,得到了带有分泌型信号的pS-X系列分泌型表达载体,人表皮生长因子(hEGF)在其中的pS-A载体中实现了分泌型表达,hEGF分泌到周质空间的相对表达量约为1%,在其它3个载体中未见表达.获得的pS-A分泌型表达载体是一种新型分泌型表达载体,它利用蓝藻信号序列作为分泌表达的信号肽,实现了hEGF多肽在大肠杆菌中分泌表达,通过融合白亚细胞定位法处理得到的hEGF多肽在胞质、周质空间和培养基中表达的比例为85:33:25(density intensity/mm2),利用渗透休克处理方法得到的比例为50:37:25.由于A信号序列本身是集胞藻6803膜蛋白(ID:s110172/NCBI-GI:1001433)的分泌信号肽,说明pS-A载体是一种广谱的原核型分泌表达载体.这一结果为实现hEGF在螺旋藻中的分泌型表达奠定了基础.     

连续函数图象的业细计盒维数及其刻划

将分形理论中的一个重要概念计盒维数推广为精细计盒维数，讨论此定义的等价表示。并且给出了Ｒ＾ｎ上连续函数图象的精细计盒维数用函数空间刻划的充要条件。     

Fluoropyridine family: Bifunction as electrolyte solvent and additive to achieve dendrites-free lithium metal batteries

Electrolyte formulation with high stability towards both Li metal anode and high-voltage cathode is considered as one of key points for the high-energy density lithium metal batteries (LMBs).In our previous study,by adding only 2％ of 2-fluoropyridine (2-FP) as the additive in the carbonate and ether-based electrolyte formulations effectively suppressed Li dendrite growth.In this study,we further found that the main fluoropyridine (FP) family members can serve as not only the effective additive but also the excellent electrolyte solvent in the electrolyte formulations to enhance the performance of LMBs.For the 2-FP,when it was also used the electrolyte solvent and paired with single-salt lithium bis(trifluoromethylsulfonyl)imide (LiTFSI),the obtained electrolyte formulation of 1 M LiTFSI in pure 2-FP solvent not only allowed faster ion transport though solvation effect,but also possessed impressive oxidation stability window over 4.3 V.As a result,the high-voltage LiNi1/3Mn1/3Co1/3O2 (1.5 mA h cm-2)|Li metal battery with it exhibited a capacity retention of more than 80 ％ over a long-term cycle even at 0.45 mA cm-2 with a lean electrolyte (30 μL).Meanwhile,for another FP family member (i.e.,3-FP) as the electrolyte additive,the 4.3 V LMBs with the carbonate-based electrolyte containing only 1 ％ of 3-FP maintained 83.9 ％ of initial capacity after 200 cycles at 0.75 mA cm-2.Density functional theory (DFT) calculations and experiments confirmed that three typical FPs,i.e.,2-FP,3-FP and 4-FP can not only regulate the initial Li nucleation process,but more importantly also induce a protective layer,leading to a uniform and dendrites-free Li deposition.This bifunction of the FP family member as either electrolyte solvent or additive in the electrolyte formulations should be promising for the achieving of dendrites-free high-energy density LMBs.     

乙肝病毒融合表面抗原SA—28基因在酵母中的表达

利用基因工程技术，先将乙肝病毒表面抗原Ｓ－ｐｒｅＳ１融合基因ＳＡ－２８置于酵母杂合启动子ＡＤＨ２－ＳＵＣ２的控制下，然后将ＳＡ－２８基因的表达单元插入高稳定质粒ｐＨＣｌ１的ＢａｍＨＩ位点，构建成表达质粒ＹＦＤ１５０和ＹＦＤ１５０－ｏ并将其转化酿酒酵母Ｙ１９。对转化子表达ＳＡ－２８基因的研究表明：表达受葡萄糖浓度调控；表达产物具有Ｓ和ｐｒｅＳ１双重抗原性，并形成密度为１．２０－１．２２ｇ／ｍｌ的颗粒     

Application of multivariate curve resolution to the analysis of yeast genome-wide screens

In this work, the application of Multivariate Curve Resolution to the analysis of yeast genome-wide screens obtained by means of DNA microarray technology is shown. In order to perform the analysis of this type of data, two algorithms based on Alternating Least Squares (MCR-ALS) and on its maximum likelihood weighted projection (MCR-WALS) variant are compared. The utilization of the modified weighted alternating least (WALS) squares algorithm is motivated by the rather poor quality, uncertainties and experimental noise associated to DNA microarray data. Moreover, a large number of missing values are usually present in these data sets and the weighted WALS approach allowed circumventing this problem. Two different experimental datasets were used for this comparison. In the first dataset, gene expression values in budding yeast were monitored in-response to glucose limitation. In the second dataset, the changes in the gene expression caused by the daunorubicin drug were monitored as a function of time. Results obtained by application of Multivariate Curve Resolution in the two cases allowed a good recovery of the evolving gene expression profiles and the identification of metabolic pathways and individual genes involved in these gene expression changes.     

导入四碳二羧酸转移酶基因对费氏中华根瘤菌共和固氮效率的影响

将来自苜蓿中华根瘤菌（Ｓｉｎｏｒｈｉｚｏｂｉｕｍ ｍｅｌｉｌｏｔｉ）的四碳二羧酸转移酶基因（ｄｃｔＡＢＤ）经ｐＩＪ２９２５克隆到广宿主、稳定性质粒ｐＴＲ１０２上,获得诱导型表达的重组质粒ｐＨＮ２０２。在此基础上再引入来自ｐＤＢ３０所含的发光酶基因（ｌｕｘＡＢ）作分子标记,以ｐＴＲ１０２为基础建成带有ｄｃｔＡＢＤ和ｌｕｘＡＢ的重组质粒ｐＨＮ２０５。＃? ｄｇ ｕｓ ｓｇ ｒｕｖ ｗｇｋ ｌｆｔｑ,ｕ     

Adaptation as a genome-wide autoregulatory principle in the stress response of yeast

The gene expression response of yeast to various types of stresses/perturbations shows a common functional and dynamical pattern for the vast majority of genes, characterised by a quick transient peak (affecting primarily short genes) followed by a return to the pre-stimulus level. Kinetically, this process of adaptation following the transient excursion can be modelled using a genome-wide autoregulatory mechanism by means of which yeast aims at maintaining a preferential concentration in its mRNA levels. The resulting feedback system explains well the different time constants observable in the transient response, while being in agreement with all the known experimental dynamical features. For example, it suggests that a very rapid transient can be induced also by a slowly varying concentration of the gene products.     

Gene networks reconstruction and time-series prediction from microarray data using recurrent neural fuzzy networks

Reverse engineering problems concerning the reconstruction and identification of gene regulatory networks through gene expression data are central issues in computational molecular biology and have become the focus of much research in the last few years. An approach has been proposed for inferring the complex causal relationships among genes from microarray experimental data, which is based on a novel neural fuzzy recurrent network. The method derives information on the gene interactions in a highly interpretable form (fuzzy rules) and takes into account the dynamical aspects of gene regulation through its recurrent structure. To determine the efficiency of the proposed approach, microarray data from two experiments relating to Saccharomyces cerevisiae and Escherichia coli have been used and experiments concerning gene expression time course prediction have been conducted. The interactions that have been retrieved among a set of genes known to be highly regulated during the yeast cell-cycle are validated by previous biological studies. The method surpasses other computational techniques, which have attempted genetic network reconstruction, by being able to recover significantly more biologically valid relationships among genes     

海洋哈维氏弧菌溶血素蛋白在酵母菌细胞表面上的展示及其活性的测定

为制备以酒精酵母为载体的基因工程疫苗,参照其Genbank中基因序列设计一对引物,PCR扩增得到预期长度的产物,双酶切插入用于酒精酵母表面展示的穿梭质粒载体pYD1,转化大肠杆菌TOP10,提取阳性质粒转化酒精酵母菌株EBY100,诱导表达后,用免疫荧光和流式细胞仪检测外源蛋白的表达,结果测得最佳诱导时间为36～48h,诱导培养后约30.0%左右的酵母细胞表达外源蛋白;同时以EBY100和转空质粒的酵母菌株为对照,测得诱导培养后的阳性酵母转化株的细胞对牙鲆鱼血细胞具有溶血活性,以此酵母活细胞分设高中低三个浓度组,腹腔注射养殖牙鲆幼鱼,检测其毒性,结果表明此重组酵母对牙鲆是安全的.该研究为下一步鱼用活载体疫苗免疫效果的研究奠定了基础.     

Electrophoretic cell manipulation and electrochemical gene-function analysis based on a yeast two-hybrid system in a microfluidic device

A novel microfluidic device with an array of analytical chambers was developed in order to perform single-cell-based gene-function analysis. A series of analytical processes was carried out using the device, including electrophoretic manipulation of single cells and electrochemical measurement of gene function. A poly(dimethylsiloxane) microstructure with a microfluidic channel (150 microm in width, 10 microm in height) and an analytical chamber (100 x 20 x 10 microm (3)) were fabricated and aligned on a glass substrate with an array of Au microelectrodes. Two microelectrodes positioned in the analytical chamber were employed as a working electrode for the electrophoretic manipulation of cells and electrochemical measurements. A yeast strain ( Saccharomyces cerevisiae Y190) carrying the beta-galactosidase reporter gene was used to demonstrate that the device could detect the enzyme. Target cells flowing through the main channel were introduced into the chamber by electrophoresis using the ground electrode laid on the main channel. When the cell was treated with 17beta-estradiol, gene expression was triggered to produce beta-galactosidase, catalyzing the hydrolysis of p-aminophenyl-beta- D-galactopyranoside to form p-aminophenol (PAP). The enzymatically generated PAP was detected by cyclic voltammetry and amperometry at the single-cell level in the chamber of the device. Generator-collector mode amperometry was also applied to amplify the current response originating from gene expression in the trapped single cells. After electrochemical measurement, the trapped cells were easily released from the chamber using electrophoretic force.     

The use of myristic acid as a ligand of polyethylenimine/DNA nanoparticles for targeted gene therapy of glioblastoma

To establish a gene delivery system for brain targeting, a low molecular weight polyethylenimine (PEI(10?K)) was modified with myristic acid (MC), and complexed with DNA, yielding MC-PEI(10?K)/DNA nanoparticles successfully. The nanoparticles were observed to be successfully taken up by the brains of mice. The transfection efficiency of the nanoparticles was then investigated, and both the in?vitro and in?vivo gene expression of MC-PEI(10?K)/DNA nanoparticles is significantly higher than that of unmodified PEI(10?K)/DNA nanoparticles. The anti-glioblastoma effect of MC-PEI(10?K)/pORF-hTRAIL was demonstrated by the survival time of intracranial U87 glioblastoma-bearing mice. The median survival time of the MC-PEI(10?K)/pORF-hTRAIL group (28 days) was significantly longer than that of the PEI(10?K)/pORF-hTRAIL group (24 days), the MC-PEI(10?K)/pGL(3) group (21 days) and the saline group (22 days). Therefore, our results suggested that MC-PEI(10?K) could be potentially used for brain-targeted gene delivery and in the treatment of glioblastoma.