MTERF1基因真核表达载体的构建及其在C-33A细胞中的表达研究

采用PCR技术从pGEM-MTERF1重组克隆载体中扩增出人MTERF1基因cDNA的开放阅读框序列,经KpnⅠ和XhoⅠ双酶切后,以pcDNA3.1(+)为真核表达载体,构建重组质粒pcDNA3.1(+)-MTERF1;通过PCR、双酶切和DNA测序方法对重组子进行鉴定;将重组真核表达质粒pcDNA3.1(+)-MTERF1转染C-33A细胞,利用免疫印迹法检测MTERF1蛋白的表达。结果显示,重组质粒pcDNA3.1(+)-MTERF1经双酶切鉴定和菌落PCR鉴定均获得大小为1 200 bp的目的条带,DNA测序结果表明该序列与GenBank中人MTERF1基因序列完全相同,插入基因的大小和方向正确;免疫印迹结果表明,MTERF1蛋白的表达水平在转染pcDNA3.1(+)-MTERF1的C-33A细胞中表达高于转染pcDNA3.1(+)的C-33A细胞。通过对人MTERF1基因的真核表达载体的构建,为进一步研究人MTERF1基因的功能奠定了基础。     

小鼠白细胞介素-18的克隆及真核表达质粒的构建

克隆小鼠白细胞介素-18（mIL-18）,并与真核表达质粒pcDNA3.1/HisB重组,构建真核表达重组质粒.采用RT-PCR,从小鼠肝细胞中扩增IL-18的全长cDNA.经Hind Ⅲ,EcoR Ⅰ双酶切,将该cDNA片断插入表达载体pcDNA3.1/HisB.通过酶切、PCR及测序对重组体进行鉴定.经鉴定,重组质粒构建正确,成功构建了真核表达载体pcDNA3.1/mIL-18,为下一步进行IL-18的功能研究奠定了基础.     

HER-2基因特异性核酶设计与体外转录载体构建

目的 构建人表皮生长因子受体-2(HER-2)特异性核酶(ribozyme,RZ)及其底物基因的体外转录载体.方法 设计合成RZ基因,将该RZ克隆到真核表达载体pcDNA3.1( )中,测序鉴定.应用Trizol试剂提取MDA-MB-453细胞总RNA,RT-PCR扩增靶基因HER-2 cDNA片段,同样插入载体pcDNA3.1( ),测序鉴定.结果 经DNA测序分别证实合成的RZ基因序列和通过RT-PCR扩增的HER-2 cDNA序列克隆入pcDNA3.1( )中.结论 构建HER-2特异性RZ真核表达载体及含有HER-2 cDNA的载体,有助于进一步研究RZ对靶基因切割作用及雌激素受体阴性乳腺癌细胞MDA-MB-453中HER-2信号转导通路.     

一种新细胞因子基因真核表达载体的构建和鉴定

采用PCR方法,以人胎盘cDNA文库为模板,扩增出人B淋巴细胞刺激因子（hBLys）,经克隆测序及纯化后,再以此PCR产物为模板,用Nest-PCR方法进一步扩增得到B淋巴细胞刺激因子的胞膜外功能区域（hsBLyS）的DNA片段,纯化、克隆测定鉴定后,扩增并纯化粒,经酶切、纯化后克隆到真核表达质粒pcDNA3.1（＋）中,构成真核表达载体pcDNA3．1（＋）／hsBLyS。结果表明：用此方法制备得到的hsBLyS的DNA片段经测序鉴定与文献报道相符,构建的真核表达载体经鉴定也达到了预计的结果。     

bFGF真核表达载体构建及表达

旨在构建可在哺乳细胞中表达碱性成纤维细胞生长因子（basic Fibroblast Growth Factor,简称bFGF）的真核表达载体，以便用于研究bFGF基因治疗在骨组织工程中的应用，应用基因重组技术，将已经克隆的bFGF基因从PBR322－bFGF载体上亚克隆到真核表达载体pcDNA3．1上，构建的重组质粒经脂质体介导转染3T3细胞，36h后观察瞬时表达情况，酶切，PCR和DNA测序结果均证实了插入片段的正确性，免疫组织化学检测bFGF的表达分泌情况，结果显示部分经转染的细胞呈阳性（棕色颗粒）。结果表明已成功构建了bFGF真核表达载体pcDNA3.1-bFGF，并且bFGF能够在3T3细胞表达。     

人Kai-1基因真核表达载体的构建及其体外表达

克隆人Kai-1基因,构建其真核表达载体,并在体外细胞系中进行表达验证。提取人正常肝细胞系HL7702的总RNA,通过RT-PCR其反转录为c DNA;以该c DNA为模板,通过PCR扩增出Kai-1基因的编码区,将该目的片段纯化后亚克隆入真核表达载体pc DNA3.1myc-his(-)中,利用菌落PCR及DNA测序分别对Kai-1基因编码区的大小及序列进行鉴定。将所构建的重组质粒通过脂质体瞬时转染Hela细胞,48 h后裂解细胞,利用Western blot检测有无目的蛋白的表达。测序证实所克隆的Kai-1编码区c DNA正确地插入pc DNA3.1myc-his(-)中,经Western blot检测证实其在Hela细胞中得到表达。成功克隆了人Kai-1 c DNA,构建了其真核表达载体,并在Hela细胞中得到有效表达,为进一步研究Kai-1的功能奠定了基础。     

大鼠csrp2基因真核表达质粒的构建及表达

获得大鼠csrp2,基因片段并构建真核表达载体.RT-PCR法从大鼠心脏组织中提取总 DNA,并扩增出相应大小的csrp2 DNA片段,测序正确后克隆入真核表达载体pcDNA 3.1(-),重组质粒酶切鉴定正确后以阳离子聚合物转染Cos-7细胞,分别以RT-PCR 方法检测mRN-表达和间接免疫荧光技术检测目的蛋白的表达.结果构建了重组质粒pcDNA 3.1(-)-csrp2,RTPCR结果证明csrp2,可在cos-7细胞中转录,用间接免疫荧光检测,表达有CRP2蛋白的细胞着染.成功构建了大鼠csrp2基因的真核表达载体pcDNA 3.1(-)-csrp2,csrp2,基因可以在Cos-7细胞中表达.     

人类8型疱疹病毒K1基因真核表达载体的构建

为构建人类8型疱疹病毒(Human herpesvirus 8,HHV-8)K1基因的真核表达载体,采集卡波氏肉瘤(Kaposi's sarcoma,KS)患者血清,采用柱式病毒DNA抽提试剂盒抽提血清中的病毒DNA,采用套式PCR技术检测HHV-8的特异性片断KS330233以判断是否得到了HHV-8 DNA,采用PCR技术扩增HHV8 K1基因并纯化回收,酶切、连接入真核表达载体pcDNA3.1,转化大肠杆菌DH5α,挑取阳性克隆pcDNA3.1/HHV8-K1扩增后,提取质粒,进行双酶切和DNA测序鉴定.结果显示采集了经典型KS血清3例,提取了血清HHV-8DNA,扩增了特异性片断KS330233,测序结果正确提示我们得到了HHV8 DNA,扩增了HHV8 K1基因,构建了pcDNA3.1/HHV8-K1真核表达载体,经限制性内切酶酶切鉴定及测序分析证实了其序列的正确性.由此可知:成功构建了pcDNA3.1/HHV8-K1真核表达载体,为进一步研究该基因的功能奠定良好的实验基础.     

胰岛自身抗原GAD65片段和胰岛素B链基因共表达DNA疫苗的构建与表达

构建两种胰岛自身抗原谷氨酸脱羧酶（ glutamic acid decarboxylase, GAD） 65片段与胰岛素B链基因共表达DNA疫苗,并检测其在体外COS-7细胞中的表达。PCR方法从GAD65质粒中扩增出GAD190-385和GAD490-570两个片段的cDNA,overlap法再分别与信号肽基因进行拼接,将拼接后的融合基因SGAD190-515和SGAD490-570分别与胰岛素B链基因依次克隆入双启动子真核表达载体pBudCFA．1中。重组质粒经酶切和测序鉴定后,脂质体体外转染COS-7细胞,Western blot方法检测目的基因在该细胞中的表达。结果表明核酸序列测定克隆的融合基因和胰岛素B链基因序列与报告序列一致,开放阅读框正确,Western blot显示转染了DNA疫苗的COS-7细胞中均可检测两个目的基因的表达。两种GAD65片段与胰岛素B链基因共表达DNA疫苗均成功构建,为自身免疫糖尿病的免疫干预研究奠定了实验基础。     

EGFR基因C端结构域真核表达载体的构建

构建EGFR基因C端结构域的真核表达载体.应用PCR技术,从含EGFR基因C端结构域的大肠杆菌DH 5α中扩增其序列,亚克隆到真核表达载体pcDNA3.1( )中,经酶切及测序进行验证.PCR扩增片段与预期结果相符,真核表达载体构建成功,测序结果与GenBank公布的基因一致.成功地构建了EGFR基因C端两个结构域的真核表达载体.     

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-