大肠杆菌酒石酸脱氢酶β亚基的克隆、表达、纯化及包涵体的复性

利用PCR技术从大肠杆菌BL21中获取酒石酸脱氢酶β亚基基因(TtdB),并将之克隆到质粒pUC18上,转化大肠杆菌DH5α细胞.经测序证明序列无误后,将之与表达载体pTrcHisC连接,在大肠杆菌BL21中经IPTG诱导表达,通过SDS-PAGE和双波长扫描分析,确定酒石酸脱氢酶β亚基在大肠杆菌中表达时以包涵体形式存在.目的蛋白用TALON金属亲和树脂纯化,通过分步透析逐步去除变性剂的方法复性.复性产率可达90%.     

重组人TRAIL胞外片段的原核表达系统选择及蛋白复性研究

采用PCR技术从人肝脏cDNA文库扩增出编码114～281位氨基酸的TRAIL基因片段,将之克隆至原核表达载体pET-30a、pQE-30和pJLA503中,上述重组载体经IPTG诱导表达后,SDS-PAGE鉴定显示pET-30a系统表达效率最高,目的蛋白占菌体总蛋白含量的40%左右;采用透析法或梯度稀释法对TRAIL胞外区片段包涵体进行复性,非还原SDS-PAGE的结果显示梯度稀释法明显好于透析法;纯化后的TRAIL胞外区片段蛋白具有明显的生物学活性,效果与标准品相似;通过添加不同的折叠促进剂对复性效率影响的研究,确立了TRAIL胞外区片段适宜的复性方法,复性效率达75%～80%.     

人胰蛋白酶原-2在大肠杆菌中的表达、纯化与活性测定

胰蛋白酶原-2是急性胰腺炎相关的敏感而特异的标志物,并且与多种肿瘤转移的过程密切相关．对胰蛋白酶原-2基因5’端4个氨基酸的密码子碱基进行简并突变,并在其3’端连接6个组氨酸编码序列构建含重组质粒pBVTg2的大肠杆菌工程菌-pBvTg2／DH5α．重组大肠杆菌经温度诱导后获得了高效表达,重组蛋白占总菌体蛋白的20％,并以包涵体形式存在．包涵体经尿素缓冲液裂解后,通过Ni-NTA亲和树脂一步分离纯化．纯化蛋白经SDS-PAGE分析及利用anti—histidine mAb进行Western blotting分析,以及蛋白N末端测序分析,结果表明与目的蛋白特征相符合．纯化的重组人胰蛋白酶原-2经复性后能与天然人胰蛋白酶原-2的单抗antihTrypsinogen2 mAb特异结合,复性后的蛋白在肠激酶作用后测得具有74BAEEU／mg的胰蛋白酶比活力,为进行胰蛋白酶原-2单克隆抗体的制备及其检测和应用奠定了基础．     

一种简便快速工艺制备重组人胸腺肽α1

将构建的含有胸腺肽α₁融合蛋白的编码基因插入表达质粒pET28a(+)中，并在大肠杆菌BL21中进行表达，该菌经IPTG诱导融合蛋白高表达后，离心收集菌体，超声波破碎，包含体裂解，目标蛋白质的体外变性复性后，经DEAE-Sepharose Fast Flow阴离子交换层析纯化，每升菌液中可获得260mg融合蛋白。经重组肠激酶酶切反应、Ni-Sepharose亲和层析，Sephadex G-25柱纯化后，得到了纯度达97%的重组人胸腺肽α₁，该纯化工艺简单快速，经三步层析即可得到纯度达97%的重组人胸腺肽α₁，且产量达82mg/L，为大批量用细菌表达重组人胸腺肽α₁及纯化提供了参考。     

重组人干扰素α2b和胸腺肽αl融合蛋白的分离纯化

考察了大肠杆菌E.coli表达的重组人干扰素α2b和胸腺肽αl融合蛋白包涵体的变性、复性及融合蛋白的分离纯化过程。实验结果表明：包涵体经7mol/L盐酸胍,10mmol/L DTT变性;1mol/L尿素,2mmol/L还原型谷胱甘肽,0.2mmol/L氧化型谷胱甘肽脉冲加样稀释复性;金属螯合层析收集0.3mol/L咪唑洗脱峰,冷干后经重组肠激酶30℃酶切24h、Sephadex G50 柱纯化,可得到纯度达90%的重组人干扰素α2b和胸腺肽αl融合蛋白,且最终目的融合蛋白产量达68mg/g干菌体。     

重组人胸腺肽α1在大肠杆菌中的表达纯化和鉴定

用PCR将化学合成的人胸腺肽α1基因扩增并克隆到pMD18-T载体中,经过KpnI／SacI酶切、连接将胸腺肽基因插入到表达载体pET32b中硫氧还蛋白下游．将重组质粒转化至表达宿主BL21(DE3)中,经IPTG诱导,Zn—Sepharose亲和层析纯化以及复性处理,从每升菌液中可获得35mg硫氧还蛋白-胸腺肽α1融合蛋白．经凝血因子Xa酶切、Zn—Sepharose亲和层析以及反相高效液相色谱纯化获得3．8mg重组人胸腺肽．小鼠胸腺细胞凋亡实验表明,重组人胸腺肽能显地抑制地赛米松诱导的小鼠胸腺细胞的凋亡,且该抑制作用与重组人胸腺肽的浓度呈剂量依赖关系．     

重组人截短型IL-6在大肠杆菌DH5α中的高效表达与纯化

为研究重组人截短型白细胞介素6(rhIL-6)工程菌高效表达的影响因素及纯化方法的优化.观察不同培养温度对重组人截短型IL-6工程菌生长密度和IL-6表达的影响;复性蛋白终浓度对复性效率的影响;细菌诱导培养后,经破菌-洗包-溶包初步纯化后,再经柱层析纯化IL-6.其结果为30℃培养后42℃诱导培养5h的IL-6表达效率最高,达32.8%;复性蛋白终浓度在0.5 g/L以下时,蛋白复性效率最佳,比活性为4.42×108 μmol/min*mg-1;进一步柱层析后,IL-6的纯度达96.5%,得率可达46.5%.最后得出培养和纯化工艺简便易行,可获得高纯度、高比活性的截短型rhIL-6的结论.     

人Sox2基因的克隆表达和纯化

应用RT-PCR方法从人胚胎十细胞中扩增出Sox2基因,构建pET28b-Sox2表达载体.用IPTG诱导转化pET28b-Sox2表达载体的大肠杆菌BL21(DE3).并优化表达条件为37℃ 1PTG0.8 mmol/L诱导4 h.以Ni-NTA亲和层析法纯化Sox2重组蛋白,对变性蛋白进行柱上和透析复性,复性后蛋白得率为0.7 mg/g湿菌重.     

锌指蛋白Zbed3的表达与分离纯化

将编码小鼠锌指蛋白Zbed3的基因克隆到大肠杆菌表达载体pET28c,构建了含组氨酸标签的表达质粒pET28c-Zbed3,并将其转入大肠杆菌BL21-CodonPlus(DE3)-RP;在20℃条件下,采用100μmol/L的异丙基硫代--βD半乳糖苷(IPTG)诱导、培养20 h而获得含Zbed3的菌体;菌体裂解液经超滤浓缩后采用镍(螯合)柱分离纯化得到锌指蛋白Zbed3(浓度达60 mg/L);同时,采用30%醋酸溶解包涵体,用0.5 mol/L的Tris-HCl(pH=8.8)缓冲液中和而使Zbed3在生理pH环境中复性.结果表明,Zbed3复性后在离心上清液中的回收率达到80%,其复性蛋白质与表达产物为可溶性(天然态)Zbed3的圆二色性光谱特征相一致,以此验证了复性后Zbed3空间构象的正确性.     

盐藻磷酸果糖激酶DsPFK基因的原核表达及酶活测定

利用原核表达载体pET-32a构建盐藻磷酸果糖激酶DsPFK基因的表达载体pET-32a-DsPFK.转化大肠杆菌BL21(DE3),通过不同温度及不同浓度IPTG诱导后获得以包涵体形式表达的DsPFK重组蛋白,经亲和层析获得大小44.4kDa的纯化蛋白,通过柱上复性的方法测定重组DsPFK蛋白的酶活为950U.     

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     

On the Thermodynamic Limit for Responses to Static Electromagnetic Fields

For molecular systems,the quantum-mechanical treatment of their responses to static electromagnetic fields usually employs a scalar-potential treatment of the electric field and a vector-potential treatment of the magnetic field.Although the potential for each field separately is associated with the choice of an(unphysical)origin,the precise choice of the origin for the electrostatic field has little consequences for the results.This is different for the     

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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