绿色荧光蛋白融合人核糖核酸酶抑制因子逆转录病毒载体pLNCX-EGFP-C1-hri的构建及鉴定

构建表达重组绿色荧光蛋白融合人核糖核酸酶抑制因子的逆转录病毒载体pLNCX-EGFP-c1-hri,为探讨人核糖核酸酶抑制因子抗肿瘤作用机制打下基础.用亚克隆法,将目的片段egfp-c1-hri从表达载体pEGFP-C1-hri克隆到pLNCX上,用酶切筛选得到阳性克隆后,用脂质体法将其转染到小鼠黑色素瘤细胞B16中,用800 mg/L G418筛选2周后,在荧光显微镜下检测其表达.双酶切鉴定得到pLNCX-EGFP-C1-hri阳性克隆,荧光显微镜显示绿色荧光在B16细胞质中高效表达.成功构建逆转录病毒表达载体pLNCX-EGFP-C1-hri.     

携带TIMP-1基因逆转录病毒载体的构建及其实验研究

构建携带组织金属蛋白酶抑制物ＴＩＭＰ－１基因的逆转录病毒载体，并用于感染胰腺癌ＳＷ１９９０细胞系，观察其侵袭、转移能力的变化．利用常规分子克隆技术，通过对逆转录病毒表达载体ＰＭＮＳＭ（Ｆ６）质粒的去磷酸化、粘端连接，构建ＴＩＭＰ－１基因逆转录病毒表达载体ＰＭＮＳＭ／ｈＴＩＭＰ；经限制性内切酶酶切、电泳鉴定其结构正确后，用电穿孔法对体外培养的ＰＡ１３７包装细胞进行基因转移，Ｇ４１８筛选阳性克隆，收集上清，并用于感染胰腺癌ＳＷ１９９０细胞系的研究．构建获得ＴＩＭＰ－１基因逆转录病毒表达载体ＰＭＮＳＭ／ｈＴＩＭＰ，有效滴度为１０４ＣＦＵ／ｍＬ～１０６ＣＦＵ／ｍＬ．本研究构建获得的ＴＩＭＰ－１基因逆转录病毒表达载体，在感染胰腺癌ＳＷ１９９０细胞系，观察其侵袭、转移能力的研究中得到有效应用     

绿色荧光蛋白标记的NTE活性域表达载体的构建及表达 

利用含有特定限制性内切酶识别位点的引物，通过聚合酶链式反应扩增出人神经病靶标酯酶活性域的编码序列，经T载体克隆测序正确后，双酶切回收特异片段定向插入到增强型绿色荧光表达载体pEGFPN3中，通过酶切反应鉴定，构建了绿色荧光蛋白标记的神经病靶标酯酶活性域的融合表达载体pNESTEGFP。采用脂质体转染的方法将其转染到人神经瘤母瘤细胞SHSY5Y中，用荧光显微镜观察发现神经病靶标酯酶活性域分布于细胞质中，而且没有导致内质网膜的聚集，表明表达载体成功构建和表达。     

绿色荧光蛋白标记的NTE活性域表达载体的构建及表达

利用含有特定限制性内切酶识别位点的引物,通过聚合酶链式反应扩增出人神经病靶标酯酶活性域的编码序列,经T载体克隆测序正确后,双酶切回收特异片段定向插入到增强型绿色荧光表达载体pEGFP-N3中,通过酶切反应鉴定,构建了绿色荧光蛋白标记的神经病靶标酯酶活性域的融合表达载体pNEST-EGFP.采用脂质体转染的方法将其转染到人神经瘤母瘤细胞SH-SY5Y中,用荧光显微镜观察发现神经病靶标酯酶活性域分布于细胞质中,而且没有导致内质网膜的聚集,表明表达载体成功构建和表达.     

pEGFP-C1-MCH真核表达载体的构建及其在HEK293 细胞系中的表达

目的:构建pEGFP-C1-MCH真核表达载体,并将其转染入HEK293细胞中,筛选阳性细胞克隆,为研究MCH基因在能量代谢中的功能及机制提供细胞模型.方法:提取脑组织总RNA,反转录为cDNA,参照Genbank中提供的序列设计引物扩增MCH基因全长.再将该基因全长cDNA克隆至质粒pEGFP-C1,经菌落PCR筛选及双酶切和DNA测序鉴定,成功构建了含有目的基因MCH的重组质粒pEGFP-C1-MCH.并利用脂质体2000介导其转染HEK293细胞,用荧光显微镜和RT-PCR检测EGFP和MCH在细胞中的表达.结果:克隆的pEGFP-C1-MCH质粒序列中的MCH与Gen Bank相符;细胞转染72 h后,转染成功的细胞在荧光显微镜下表达较强的绿色荧光,MCH基因稳定表达.结论:pEGFP-C1-MCH真核表达载体的构建及其在HEK293细胞中的稳定表达,为研究MCH基因在能量代谢中的功能及作用机制提供了实验模型.     

人脑微血管内皮细胞ACTG1基因真核表达载体的构建及表达

为了构建人脑微血管内皮细胞ACTG1基因的真核表达载体及在HEK293细胞中的表达,本研究采用RT-PCR方法,以单增李斯特菌侵染的人脑微血管内皮细胞提取的总RNA为模板,扩增ACTG1基因片段,TA克隆后经Sal I和Eco R I双酶切、测序等鉴定重组质粒,重组质粒再经双酶切,与真核表达载体p ACGFP连接,酶切鉴定后,Lipofactamine 2000转染HEK293细胞,荧光显微镜检测绿色荧光;同时采用Western-blotting对表达产物进行了鉴定。结果表明:PCR扩增得到1128 bp的ACTG1基因片段,ACTG1-p ACGFP真核表达载体构建成功,转染24 h后,在荧光显微镜下可见绿色荧光,Western-blotting检测分析,其分子量约为68 k Da的融合蛋白,与预期的大小相符合,表明重组蛋白表达成功,本研究为单增李斯特菌与脑微血管内皮细胞c DNA文库中相互作用蛋白的筛选和鉴定奠定了基础。     

EGFP和ALR融合基因表达载体构建及其在HeLa细胞中的表达

从人血液中提取总DNA,利用PCR技术扩增人肝细胞再生增生因子基因,将其插入表达载体pEGFP—C1的多克隆位点中,构建增强绿色荧光蛋白（enhanced green fluorescence protein gene,EGFP）和人肝细胞再生增强因子（human augmenter of liver regeneration gene,ALR）融合基因表达载体pEGFP／ALR,并将其转染Hela细胞系,用含G418的DMEM／F12培养液筛选转基因细胞,然后利用PCR和聚丙烯酰胺凝胶电泳检测转基因细胞中ALR基因的存在及其表达,用荧光显微镜检测EGFP基因的表达．结果显示：得到了构建正确的EGFP和ALR融合基因表达载体;在转基因细胞中,PCR扩增得到1．7Kb的ALR条带,蛋白电泳得到57KD大小条带．与ALR和EGFP融合蛋白大小相符;荧光显微镜下观察到发绿色荧光的Hela细胞．在转基因细胞中,EGFP和ALR同时存在并表达,绿色荧光蛋白可作为报告基因指示目的基因的表达,从而简化了目的基因繁琐的检测手段．     

猪脑心肌炎病毒VR-129 B株VP2重组蛋白的构建表达及其免疫活性分析

为获得大量猪脑心肌炎VP2基因及蛋白研究的细胞模型,构建pDC315-EMCV-VP2真核表达载体,且将其转染到293T细胞,筛选出阳性质粒进行克隆。EMCV VR-129B株VP2基因序列参照GenBank(登录号:X74312),利用RT—PCR方法扩增VP2的全基因序列,将其与质粒pDC315经NheI和XhoI双酶切后连接,构建pDC315-EMCV-VP2重组表达质粒,并将其转染至293T细胞。使用荧光定量PCR方法观察pDC315-EMCV-VP2真核表达载体在细胞中的表达情况。双酶切PCR结果获得大小为780 bp的基因片段,测序结果显示与GenBank上已经公布的同名基因(登录号:X74312)序列片段同源性为100%,重组质粒构建成功。实时荧光定量PCR(QPCR)结果显示,重组质粒转染组与空质粒组之间相比,EMCV-VP2基因的表达量显著上调(P0.001);在荧光显微镜下观察转染细胞,转染成功部分出现较亮绿色荧光,EMCV-VP2基因表达稳定。从转染细胞中提取重组蛋白与EMCV阳性血清和阴性血清进行ELisa反应,具有较好免疫活性。     

新基因hSSB1逆转录病毒载体的构建、鉴定和稳定株的筛选

 hSSB1 (Human Single strand DNA binding protein) 是参与细胞DNA损伤应答的一个重要信号分子。根据GenBank 提供的hSSB1基因序列扩增其cDNA序列,插入到pBABE逆转录病毒载体中, 连接后的质粒转化后经过双酶切,PCR扩增及测序来鉴定pBABE-hSSB1阳性克隆。将阳性表达的pBABE-hSSB1和包装质粒转染到HEK293T细胞中,产生病毒液。将包装好的病毒感染细胞并用嘌呤霉素(puromycin)筛选稳定表达hSSB1的细胞株。重组pBABE-hSSB1质粒经双酶切,PCR扩增鉴定及DNA测序分析等方法证实克隆成功。Western blotting检测发现转染重组质粒pBABE-hSSB1的细胞株中hSSB1蛋白的表达水平高于对照组。该研究成功构建了针对hSSB1基因的逆转录病毒载体(pBABE-hSSB1),并得到了稳定高表达hSSB1的细胞株, 为深入研究其功能奠定了基础。     

Fractalkine基因真核表达质粒的构建及其在小鼠肝癌细胞中的表达

为克隆小鼠趋化因子Fractalkine(．FK)基因，构建真核表达质粒，并在小鼠肝癌细胞中表达，用以进行肿瘤的基因治疗，用RT-PCR法，从小鼠乳腺癌细胞D2F2扩增FK的cDNA，插入pCR2．1 TOPO载体，测序证实后，将其亚克隆至质粒pIRES中构建FK真核表达载体；用脂质体将重组质粒转染小鼠肝癌MM45 T．Li细胞，经G418筛选获得抗性细胞克隆，用RT-PCR和免疫化学方法鉴定转染细胞中FK基因的表达．结果表明：经限制性内切酶酶切图谱分析和DNA序列测定证实目的基因已插入重组质粒，RT-PCR和免疫化学方法证明转基因MM45T．Li细胞克隆中存在小鼠FK基因的表达。     

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.     

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.     

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-     

Density Functional Theory for the Response of Periodic Systems to Electric Fields Based on the Vector Potential Approach

正The electronic and nuclear(structural/vibrational)response of 1D-3D nanoscale systems to electric fields gives rise to a host of optical,mechanical,spectral,etc.properties that are of high theoretical and applied interest.Due to the computational difficulty of treating such large systems it is convenient to model them as infinite and periodic(at least,in first approximation).The fundamental theoretical/computational problem in doing so is that     

Call for Papers

<正>"The Journal of Shanghai Normal University:Mathematics"is published by Shanghai Normal University as regular issues of The Journal of Shanghai Normal University each year from 2014 in English.The editors-in-chief of the issues are professors Yuhao Cong and Maoan Han.The Journal of Shanghai Normal University was started in 1958 with     

Journal of Donghua University ( English Edition) Contents of Volume 31

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Brief Introduction to “Journal of Jilin University( Science Edition) ”

<正>"Journal of Jilin University(Science Edition)"is a comprehensive academic journal in the fields of science sponsored by Jilin University and administrated by the Ministry of Education of the Peoples Republic of China.The journal started publication in 1955.The original name at starting publication was"Journal of Natural Science of Northeast People University",which was changed into"Acta Scientiarum Naturalium Universitatis Jilinensis"in 1958 owing to the name change of the university.The present journal name has