产气肠杆菌α-乙酰乳酸脱羧酶基因的克隆和表达

根据已知的α-乙酰乳酸脱羧酶（α-acetolactatedecarboxylase，ALDC，EC．4．1．1．5）基因序列，利用PCR方法从产气大肠杆菌（Enterobacter aerogenes）中克隆到0．8kb的DNA片段，经DNA序列分析表明该DNA片段为ALDC基因．将该片段插入到原核表达载体pBV220中，获得表达质粒pBVEDAD，转化大肠杆菌DH5α（Escherichia coli），经热诱导后，通过SDS-PAGE分析和酶活测定，结果表明ALDC获得了高效表达.重组细胞表达的ALDC酶活是出发菌产气肠杆菌（Eaerogenes）的1100倍．DH5α（pBVEDAD）在无选择压力下37℃连续培养60代，在42℃下热诱导5h未见质粒丢失．     

TIR突变的hbFGF在原核系统中高效的表达

目的：为获得非融合高表达hbFGF的大肠杆菌表达系统；方法：设计PCR引物，将hbFGF的5’端前10个密码子突变，优选大肠杆菌较偏好的密码子并降低G C含量，通过双酶切法将突变后的hbFGF克隆人pBV220质粒载体中，筛选出重组子，再通过温控法对重组菌体进行诱导，分析表达情况，并进一步纯化和测活；结果：bFGF在该系统中高水平表达，表达量超过30％，其中绝大部分形成包涵体，经过包涵体变复性等纯化步骤后测活性，证明有生物活性；结论：TIR区域的基因突变对hbFGF在pBV220中高效表达起到关键作用。     

重组人截短型IL—6表达载体的构建及表达

旨在建立人截短型IL-6原核高效表达系统。用PCR方法获得截短的人IL-6基因（rhIL-6cDNA）,将其插入克隆载体pUC18中,经证实后再克隆入表达载体pBV220中,鉴定阳性表达载体。结果建立了截短型IL-6基因的克隆载体,经测序证实完全正确;构建了截短型IL-6的原核表达质粒pBV220/rhIL-6并获得高效表达;初步纯化的表达蛋白的比活性为5×10     

大鼠CuZn—SOD cDNA的克隆及高效表达

采用PCR技术,扩增大鼠CuZn-SOD cDNA,双酶切后与表达载体pBV220连接,构建出表达质粒pBV-RCS,并转化大肠杆菌DH5α,经金属离子和42℃诱导,pBV-RCS得到高表达,经SDS－PAGE及薄层扫描测定表达蛋白占菌体总蛋白的49％,是目前国内外SOD高表达菌株之一^[1,2],表达蛋白以包涵体形式存在于菌体内,经超声波破壁后,再经透析复性和稀释复性,表达蛋白具有特异性SOD酶活性（1298U/mL)。α     

人类扭转蛋白A的基因克隆、表达与蛋白质纯化(Ⅰ)——DYT1基因克隆与原核表达

通过基因工程方法，用大肠杆菌原核表达系统表达人扭转蛋白A．从该蛋白编码序列中设计引物，以人肝cDNA文库作模板扩增到编码该蛋白的基因片段．将所得片段与pMDl8-T载体连接，转化到JM109大肠杆菌中，从转化平板上挑出菌落，用碱裂解法提取质粒，通过PCR和酶切分析，筛选到阳性克隆，测序结果与文献报道结果一致．提取质粒，用BamHI和XhoI酶切，回收目的片段，分别克隆到原核表达载体pET28a（4-）和pGEX-6P-1中，转化JM109受体菌，从JM109受体菌中提出质粒，再转化到BL21（DE3）菌中，筛选出阳性克隆，构建了人扭转蛋白A原核表达载体．IPTG诱导该工程菌，培养并离心收集．SDS-PAGE结果表明该蛋白在两种载体中均得到了高效表达．     

枯草芽孢杆菌葡萄糖脱氢酶基因的克隆及在大肠杆菌中的高效表达

利用PCR技术扩增得到来源于枯草芽孢杆菌的葡萄糖脱氢酶基因片段，并构建重组质粒pUC-T-GDH，然后将基因片段克隆于大肠杆菌表达载体pBV220中．得到表达质粒pBV-GDH．葡萄糖脱氢酶在含有表达质粒的基因工程菌株中得以诱导表达，聚丙烯酰胺凝胶电泳及薄层扫描结果表明，经诱导表达的葡萄糖脱氢酶约占基因工程菌株总蛋白的45％．葡萄糖脱氢酶活力测试表明，基因工程菌株无细胞抽提液中葡萄糖脱氢酶的活力为7.8U／毫克，约为对照组的30倍．实验结果初步说明，广泛应用于工业化生产的葡萄糖脱氢酶可以在基因工程菌株中得以大量表达并维持较高活力．     

甲/戊型肝炎病毒重组抗原基因的克隆及表达

 将甲肝病毒vp1编码基因(aa 24~171)和戊型肝炎病毒ORF2基因(aa 431~615)通过一段编码柔性甘氨酸链(Gly4 Ser Gly4)的基因片断连接,获得编码HAV/HEV融合蛋白基因(AEAg342).将该融合基因插入质粒pBV220,构建表达质粒pBV220-AEAg342.将pBV220-AEAg342转化入大肠杆菌BL21中进行表达,表达量约占菌体总量的20%,经离子交换层析纯化,目的蛋白纯度达90%以上.Western blot证实该蛋白能与甲肝及戊肝患者阳性血清发生特异性反映,为进一步开发双价疫苗和联合诊断试剂奠定了一定基础.     

hIL-10在大肠杆菌中的表达及其生物学活性鉴定

构建了含有人白细胞介素-hIL-10（Human Interleukin10，hIL-10）基因的重组质粒，在大肠杆菌中的高效表达，并对其生物学活性进行了鉴定．用RT-PCR扩增hIL-10 cDNA，并插入原核表达载体PET-32b．将重组质粒转入BL21（DE3）感受态细胞，在37℃用IPTG诱导hIL-10表达．表达的重组蛋白经复性纯化后，用夹心ELISA法检测其对外周血单核细胞（PBMC）合成IFN-γ的抑制作用．重组质粒PET-32b／hIL-10的DNA序列分析显示，克隆的DNA序列和文献报道的hIL-10 cDNA序列一致．SDS-PAGE表明，重组蛋白相对分子质量为18000．活性测定结果显示，重组蛋白能显著抑制PBMC合成IFN-γ．这表明构建的PET-32b／hIL-10可以在大肠杆菌中高效表达，经复性和纯化后，获得了具有高纯度和活性的hIL-10     

盐藻rbcS基因克隆及其原核表达

1,5-二磷酸核酮糖羧化酶／加氧酶（Ribulose1,5-bisphosphate carboxylase／oxygenase,E,C．4,1．1．39简称RuBisCO）是光合作用中的关键酶,作者应用RT-PCR技术从一种极其耐盐的生物-杜氏盐藻的总RNA中克隆RuBisCO的小亚基（rbcS）的cDNA,它的开放读码框为570bp,编码190个氨基酸,将此cDNA定向克隆于原核表达载体pET-30a（＋）中,构建重组质粒pET-rbcS,经IPTG诱导,在大肠杆菌株BL21中表达．SDS-PAGE电泳表明,rbcS融合蛋白分子量约为26kDa．     

大肠杆菌ppsA,pckA基因的克隆与串联表达

磷酸烯醇丙酮酸合成酶（ppsA)和磷酸烯醇丙酮酸羧激酶（pckA）是糖代谢中心途径中生成磷酸烯醇丙酮酸（PEP）的2个关键酶，在大肠杆菌中分别由ppsA和pckA基因编码，首先用PCR方法从大肠杆菌K12菌株基因组DNA扩增得到了ppsA和pckA基因，并将ppsA,pckA以单独或串联的方式克隆到大肠杆菌表达质粒pλPR上，构建成的质粒pλPR-ppsA,pλPR-pckA,pλPR-ppsA-PckA导入E.coli P2392菌株中表达，结果表明，ppsA,pckA基因的单独表达使宿主细胞的ppsA和pckA酶活力分别提高到5．2和2．5倍，串联表达使宿主细胞PAt和PckA酶活力分别提高到3．1和2．3倍，还使得以PEP为底物合成的3－脱氧α－阿拉伯 酮糖－7－磷酸（DAHP）产量提高到2．1倍，2个基因串联表达比单个基因表达更有利于提高DAHP的产量。     

Markedness and its Interpretation in Language Use

Language markedness is a common phenomenon in languages, and is reflected from hearing, vision and sense, i.e. the variation in the three aspects such as phonology, morphology and semantics. This paper focuses on the interpretation of markedness in language use following the three perspectives, i.e. pragmatic interpretation, psychological interpretation and cognitive interpretation, with an aim to define the function of markedness.     

Features of Women's language

Language is a means of verbal communication. People use language to communicate with each other. In the society, no two speakers are exactly alike in the way of speaking. Some differences are due to age, gender, statue and personality. Above all, gender is one of the obvious reasons. The writer of this paper tries to describe the features of women＇s language from these perspectives： pronunciation, intonation, diction, subjects, grammar and discourse. From the discussion of the features of women＇s language, more attention should be paid to language use in social context. What＇s more, the linguistic phenomena in a speaking community can be understood more thoroughly.     

Characterization of Ordovician carbonate reservoirs, southeastern Saskatchewan, Canada

The discovery of the prolific Ordovician Red River reservoirs in 1995 in southeastern Saskatchewan was the catalyst for extensive exploration activity which resulted in the discovery of more than 15 new Red River pools. The best yields of Red River production to date have been from dolomite reservoirs. Understanding the processes of dolomitization is, therefore, crucial for the prediction of the connectivity, spatial distribution and heterogeneity of dolomite reservoirs.The Red River reservoirs in the Midale area consist of 3～4 thin dolomitized zones, with a total thickness of about 20 m, which occur at the top of the Yeoman Formation. Two types of replacement dolomite were recognized in the Red River reservoir: dolomitized burrow infills and dolomitized host matrix. The spatial distribution of dolomite suggests that burrowing organisms played an important role in facilitating the fluid flow in the backfilled sediments. This resulted in penecontemporaneous dolomitization of burrow infills by normal seawater. The dolomite in the host matrix is interpreted as having occurred at shallow burial by evaporitic seawater during precipitation of Lake Almar anhydrite that immediately overlies the Yeoman Formation. However, the low δ18O values of dolomited burrow infills (-5.9‰～ -7.8‰, PDB) and matrix dolomites (-6.6‰～ -8.1‰, avg. -7.4‰ PDB) compared to the estimated values for the late Ordovician marine dolomite could be attributed to modification and alteration of dolomite at higher temperatures during deeper burial, which could also be responsible for its 87Sr/86Sr ratios (0.7084～0.7088) that are higher than suggested for the late Ordovician seawaters (0.7078～0.7080). The trace amounts of saddle dolomite cement in the Red River carbonates are probably related to "cannibalization" of earlier replacement dolomite during the chemical compaction.     

低渗透非均质砂岩油藏启动压力梯度研究

理论推导与室内实验相结合,建立了低渗透非均质砂岩油藏启动压力梯度确定方法。首先借助油藏流场与电场相似的原理,推导了非均质砂岩油藏启动压力梯度计算公式。其次基于稳定流实验方法,建立了非均质砂岩油藏启动压力梯度测试方法。结果表明:低渗透非均质砂岩油藏的启动压力梯度确定遵循两个等效原则。平面非均质油藏的启动压力梯度等于各级渗透率段的启动压力梯度关于长度的加权平均;纵向非均质油藏的启动压力梯度等于各渗透率层的启动压力梯度关于渗透率与渗流面积乘积的加权平均。研究成果可用于有效指导低渗透非均质砂岩油藏的合理井距确定,促进该类油藏的高效开发。     

On Tragic Consciousness In The Works By Ernest Hemingway

As an American modern novelist who were famous in the literary world, Hemingway was not a person who always followed the trend but a sharp observer. At the same time, he was a tragedy maestro, he paid great attention on existence, fate and end-result. The dramatis personae's tragedy of his works was an extreme limit by all means tragedy on the meaning of fearless challenge that failed. The beauty of tragedy was not produced on the destruction of life, but now this kind of value was in the impact activity. They performed for the reader about the tragedy on challenging for the limit and the death.     

A computer generator for randomly layered structures

AcomputergeneratorforrandomlylayeredstructuresYUJia shun1,2,HEZhen hua2(1.TheInstituteofGeologicalandNuclearSciences,NewZealand;2.StateKeyLaboratoryofOilandGasReservoirGeologyandExploitation,ChengduUniversityofTechnology,China)Abstract:Analgorithmisintrod…     

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.     

Jane Eyre——A Feminist In The 19th Century

In the 19th century the society was controlled by men, and women were just appendants of them, they had not any rights and freedom. But Jane was an exception, she showed some characteristics of early feminist. Jane showed her characteristics of feminism in three aspects: rebellion, equality, and independence. These characteristics were helpful to her success, and feminism is the only way out for women of that time.     

Achieving Unusual States of Matter Under High Pressure

正The periodicity of the elements and the non-reactivity of the inner-shell electrons are two related principles of chemistry,rooted in the atomic shell structure.Within compounds,Group I elements,for example,invariably assume the+1 oxidation state,and their chemical properties differ completely from those of the p-block elements.These general rules govern our understanding of chemical structures and reactions.Using first principles calcula-     

Exchange-Correlation and Electronic Excitation Energies from Pairing Matrix Fluctuations

We have developed an adiabatic connection to formulate the ground-state exchange-correlation energy in terms of pairing matrix linear fluctuations.This formulation of the exchange-correlation energy opens a new channel for density functional approximations based on the many-body perturbation theory.We illustrate the potential of such approaches with an approximation based on the particle-particle Random Phase Approximation(pp-RPA).This re-