产人胰岛素毕赤酵母工程菌的构建

根据人胰岛素的氨基酸序列和酵母偏爱的密码子,采用基因半合成技术合成人胰岛素基因.将合成的胰岛素基因克隆到pP IK 9质粒,构建了毕赤(P ich ia)酵母重组表达载体pP IN S319,用B g lⅡ线性化后用电转化法导入毕赤酵母G S ll5菌株,经过营养筛选和PCR复筛,得到含人胰岛素基因的毕赤酵母工程菌株.经SDS-PAGR和密度扫描分析表明,合成的人胰岛素基因能够在毕赤酵母中高效分泌性表达,表达量可达0.563 m g/mL,表达产物无需转肽过程,只经过胰蛋白酶和羧肽酶B简单处理即得到优质重组人胰岛素,其体内活力约为30 IU/m g.     

Monellin基因在酵母中的表达研究

本实验根据毕赤酵母偏爱密码子,人工合成了高甜度monellin基因,并构建了重组分泌型酵母表达载体pPIC9KM,通过电击转化获得了可高效分泌表达有甜味活性高甜度monellin的重组毕赤酵母GS115/pPIC9M.通过PCR检测证实monellin基因已经整合进酵母基因组,SDS-PAGE和Westerm blot免疫杂交知所表达的蛋白是目的蛋白,最后通过双盲测定证实表达的蛋白比同等重量的蔗糖甜约1000倍.     

RGD-拖丝蛋白基因的构建及其在毕赤酵母中的分泌表达

根据天然拖丝蛋白的高度重复性序列,引入与细胞黏附有关的精氨酸-甘氨酸-天冬氨酸(RGD)三肽序列,以毕赤酵母偏好的密码子化学合成RGD-拖丝蛋白基因单体,通过"头尾相连"的多聚化策略,倍加成16聚体与32聚体基因.分别将这两种多聚体与分泌型表达载体pPIC9K连接,转化毕赤酵母GS115,用G418筛选毕赤酵母重组菌.通过甲醇诱导表达,培养液上清的SDS-PAGE分析表明RGD-重组拖丝蛋白获得分泌型表达.     

日本曲霉酸性蛋白酶的分泌表达、性质及其在猪肉嫩化中的应用

为研究日本曲霉酸性蛋白酶的分泌表达、性质及其在猪肉嫩化中的应用。将日本曲霉来源的酸性蛋白酶基因(AjproA1)在毕赤酵母中高效表达,经5L发酵罐高密度发酵后测得该酸性蛋白酶酶活力为669.6U·mL^-1,蛋白质量浓度为6.64mg·mL^-1。氨基酸多重序列比对结果表明:该酸性蛋白酶属于天冬氨酸蛋白酶A1家族,与海枣曲霉来源的酸性蛋白酶同源性最高(73%),是一个新型的酸性蛋白酶。采用强阴离子交换层析柱对该酸性蛋白酶进行纯化得到电泳级纯酶,纯化后分析测得该重组酸性蛋白酶(AjproA1)的最适催化条件为pH值3.0,反应温度为45℃,在pH值为3.0~6.0及温度为40℃以下具有良好的稳定性。该酸性蛋白酶对不同蛋白质水解的分析结果表明,该酶底物水解特异性广泛,对酪蛋白组分中的κ-酪蛋白表现出最高水解活力。利用该酸性蛋白酶对猪肉进行处理,发现该酶能够有效降低猪肉剪切力,起到嫩化效果。研究结果旨在为新型酸性蛋白酶的开发及其在肉类嫩化中的应用提供理论参考。     

乙肝表面抗原在分泌型毕赤酵母中的表达及纯化

目的对分泌型毕赤酵母中表达乙肝表面抗原并进行亲和层析纯化。方法从已确诊的乙肝患者阳性血清中提取乙肝病毒DNA,PCR扩增乙肝病毒表面抗原编码基因S,将其插入含有仅分泌信号肽序列和6个组氨酸纯化标签的毕赤酵母表达载体pPICZα,成功构建了重组表达载体pPICZα—HBVs。电转化酵母菌株GS115,得到重组乙肝表面抗原S的酵母表达菌株。结果诱导培养基BMMY中甲醇终浓度为1％,诱导表达48h重组蛋白表达量及抗原性达到最高。非变性条件下亲和层析纯化后,薄层扫描分析得到纯度超过95％,表达量约为2mg／L的重组蛋白。结论Western-blot和ELISA分析表明,所得产物为乙肝表面抗原S蛋白,其纯度和产量足以免疫小鼠制备单克隆抗体。     

内切菊粉酶基因在毕赤酵母中的高水平表达研究

低聚果糖(Fructooligosaccharides,FOS)具有改善胃肠道环境、刺激和加强机体免疫反应、抑制肠道特定肿瘤的发生等生物活性.内切菊粉酶能够水解菊粉获得低聚果糖.为了实现内切菊粉酶的高表达,从无花果曲霉(Aspergillus ficuum ATCC16882)中克隆了内切菊粉酶编码基因INU2,并实现了在毕赤氏酵母中的重组分泌表达,摇瓶表达的酶活力为570.4U/mL.去除内切菊粉酶的内源性信号肽序列后,其重组表达水平显著提高,摇瓶表达的酶活力为1013.8U/mL,提高幅度77.7%.TLC分析表明:毕赤酵母重组表达的内切菊粉酶(不含内源性信号肽序列)能将2%长链菊粉水解为2～3个聚合度的低聚果糖.     

酵母Elp3在毕赤酵母中的分泌表达及活性测定

酵母Elp3是Elongator复合物的催化亚基,可参与组蛋白乙酰化修饰与基因转录延伸.生物信息学分析及有关研究表明Elp3除组蛋白乙酰转移酶(Histone acetyltranferase,HAT)活性外,可能还拥有组蛋白去甲基化酶活性.本文以yElp3的pYES2yElp3质粒为模板,PCR获得yElp3基因全长,插入改造过的毕赤酵母表达载体pPIC9KH,转化巴斯德毕赤酵母GS115菌株.经表型鉴定、PCR分析及G418筛选获Mut+型多拷贝整合菌,经0.5%的甲醇诱导和Ni-NTA纯化,得到目的蛋白并对其进行体外酶活测定.SDS-PAGE分析表明yElp3在毕赤酵母中实现了高效分泌表达,Western印迹证实得到的蛋白为yElp3.OPA法测得纯化蛋白拥有较好的HAT活性,具有生物活性的Elp3蛋白的获得为体外测定其第二结构域是否有催化活性奠定了基础.     

两种杂合抗菌肽在毕赤酵母中的表达及活性测定

参照毕赤巴斯德酵母(Pichia pastoris)的偏好密码子,改造克隆杂合抗菌肽CA(1～7)M(4～11)和CB(1～7)M(4～11)基因,将改造后的基因克隆到pPICZα-A 载体中,构建分泌型表达载体pPICZα-A-CAM和pPICZα-A-CBM,转化宿主菌Pichia pastoris X-33，在甲醇的诱导下进行表达.实验结果表明,杂合抗菌肽获得表达,两种表达产物具有明显的抗菌活性.此外，本文还比较了二者的抗菌谱，分析了杂合肽的性质，优化了酵母电转化条件.     

南极假丝酵母脂肪酶B在毕赤酵母中的分泌型表达及酶学性质初探

将南极假丝酵母脂肪酶B(CALB)基因克隆到毕赤酵母表达载体pPICZαA(含pAOX1启动子)和pGAPZαA(含pGAP启动子)中,转入毕赤酵母表达宿主KM71H,经三丁酸甘油酯平板活性筛选获得高效表达的酵母工程菌株.分泌型表达CLAB酵母工程菌的酶活较高,经摇瓶发酵96h后,CLAB活力达到11.1U/mL.经发酵罐高密度发酵84h后,CLAB活力达到179.2U/mL,蛋白质量浓度为1.36mg/mL.体积分数12%SDS-PAGE凝胶电泳显示重组CALB相对分子量为36kDa.该酶最适温度为40℃,最适pH值为8.0.在pH7.5~9.5较稳定,经55℃保温1h,残余酶活还有48.5%.5mmol/LMg2+、Ni+、Co2+和体积分数0.10%SDS对CALB酶活影响很小,而Zn2+强烈抑制酶催化反应,体积分数0.05%TritonX-100对CALB稍有激活作用.     

米曲霉碱性蛋白酶基因的克隆表达及水解特性

米曲霉碱性蛋白酶对植物蛋白具有高效的水解能力和较好的脱苦作用.为了便于大量获得该酶以利于工业上的应用,文中通过RT-PCR克隆获得米曲霉碱性蛋白酶基因,将之转化到毕赤酵母KM71菌株中并成功表达,之后用该酶进行牛胰岛素氧化链B链及大豆和花生蛋白的水解试验.结果显示:在10L发酵罐中诱导表达时,发酵液中重组碱性蛋白酶的酶活达4100U/mL;该蛋白酶最适反应pH值和温度分别为8.5 ～9.5和50℃,在pH值为6.0 ～10.0和温度低于40℃时具有较好的稳定性;该酶具有广泛的肽键选择性;该酶对大豆和花生蛋白显示出了强于部分商品化蛋白酶的水解能力.以上结果表明:该重组蛋白酶具有较大的开发利用潜力.     

An Index mechanism for multi-scale view in spatial databases

There are numerous geometric objects stored in the spatial databases. An importance function in a spatial database is that users can browse the geometric objects as a map efficiently. Thus the spatial database should display the geometric objects users concern about swiftly onto the display window. This process includes two operations:retrieve data from database and then draw them onto screen. Accordingly, to improve the efficiency, we should try to reduce time of both retrieving object and displaying them. The former can be achieved with the aid of spatial index such as R-tree, the latter require to simplify the objects. Simplification means that objects are shown with sufficient but not with unnecessary detail which depend on the scale of browse. So the major problem is how to retrieve data at different detail level efficiently. This paper introduces the implementation of a multi-scale index in the spatial database SISP (Spatial Information Shared Platform) which is generalized from R-tree. The difference between the generalization and the R-tree lies on two facets: One is that every node and geometric object in the generalization is assigned with a importance value which denote the importance of them, and every vertex in the objects are assigned with a importance value,too. The importance value can be use to decide which data should be retrieve from disk in a query. The other difference is that geometric objects in the generalization are divided into one or more sub-blocks, and vertexes are total ordered by their importance value. With the help of the generalized R-tree, one can easily retrieve data at different detail levels.Some experiments are performed on real-life data to evaluate the performance of solutions that separately use normal spatial index and multi-scale spatial index. The results show that the solution using multi-scale index in SISP is satisfying.     

Perspherical Approaches for Electrons in Atoms and Charged Excitons in Quantum Wells

Instead of following Fock’s expansion,we solve the Schrodinger equation for some quantum mechanical manybody systems such as electrons in atoms and charged excitons in quantum wells in a similar way in hyperspherical coordinates by expanding the wave functions into orthonormal complete basis sets of the hyperspherical hannonics(HHs)of hyperangles and generalized Laguerre polynomials(GLPs)of the hyperradius.This leads the equation to     

Advances in broadband wireless communications under high-mobility scenarios

Future mobile communication systems aim at providing very high data transmission rates, even in high-mobility scenarios such as high-speed wheel-track trains, maglev trains, highway vehicles, airplanes, guided missiles or spacecraft. A particularly important commercial application is the strong and increasing worldwide demand for high- speed broadband wireless communications （up to 574.8 km/ h test speeds or 380 km/h commercial speeds） in railways, providing data, voice and video services for applications such as onboard entertainment services to passengers, train control, train dispatch, train sensor status handling and sur- veillance. In such high-mobility scenarios, there are a number of communication challenges, including fast hand- over, location updating, high-speed channel modeling, estimation and equalization, anti-Doppler spreading tech- niques, fast power control, and dedicated network architec- ture. Because signal transmission in very high-speed scenarios will inevitably experience serious deterioration, it is imperative to develop key broadband mobile communi- cation techniques for such high-speed vehicles.     

Wuhan University Journal of Natural Sciences TOTAL CONTENTS

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Scale-based spatial data model for GIS

Being the primary media of geographical information and the elementary objects manipulated, almost all of maps adopt the layer-based model to represent geographic information in the existent GIS. However, it is difficult to extend the map represented in layer-based model. Furthermore, in Web-Based GIS, It is slow to transmit the spatial data for map viewing. In this paper, for solving the questions above, we have proposed a new method for representing the spatial data. That is scale-based model. In this model we represent maps in three levels: scale-view, block, and spatial object, and organize the maps in a set of map layers, named Scale-View, which associates some given scales.Lastly, a prototype Web-Based GIS using the proposed spatial data representation is described briefly.     

英文诗歌中的喻类修辞

为了有助于提高英文爱好者的个人情操、文化素养和学习兴趣,以及专业人员对英语学习和研究进行多途径的探索,本文通过一些精选诗歌的引证和分析,着重论述了喻类修辞法在英文诗歌中的运用其及效果。     

Man Is Unable to Change the Miserable Life——Analysis of the Arrangement of Four Characters in The Glass Menagerie

Tennessee Williams is considered as one of the most important American playwrights since World War II.The Glass Menagerie is his first successful drama,which describes a tragic situation of family and means to say that Man is unable to change the miserable life,no matter whatever means he try,This essay focuses on the analysis of the arrangement of the four main characters:Laura.Amanda,Jim and Tom to reveal the theme.Laura is fragile.Amanda is brave.Jim is vital.Tom is sensible.And all of them develop and try the different means to struggle against life,but fail tragically.With the evidence,the paper comes to conclusion naturally that Man is unable to change the miserable life,and he dooms to fail.     

Characterization of root-nodulating bacteria on Retama raetam in arid Tunisian soils

The aim of this study is to investigate the diversity of Retama raetam root-nodule bacteria isolated from arid regions of Tunisia. Twelve isolates, chosen as representative for different 16S rRNA gene patterns, were characterized by 16S rRNA gene sequencing and phenotypic analysis. Isolates were assigned to Sinorhizobium, Rhizobium and Agrobacterium. Symbiotic properties of Sinorhizobium and Rhizobium isolates showed a large diversity in their capacity to infect their host plant and fix atmospheric nitrogen. Strain RK 22 identified as Rhizobium was the most effective isolate.     

An Affordable Accurate Evaluation of the Binding Conformations Predicted by Molecular Docking Studies in Bio-System

正Recently,docking has been widely used to predict the binding-modes of protein-inhibitors,when the crystal complexes structure was absent.Most docking algorithms are able to generate a large number of probable conformations,it,however,is difficult to effectively evaluate these docking poses and identify the most reasonable bindingmode.In the present study,on the basis of the crystallographic data of human 3-hydroxy-3-methylglutaryl coenzyme     

WIMAX与WI-FI技术分析及联合组网

介绍了WiMAX与Wi-Fi两种无线宽带接入技术,并对两者之间的关系及相互之间的影响做了对比及分析,并对WiMAX的关键技术进行了详细说明,最后对两者的联合组网方式做了简单的探讨。