重组Bowman-Birk型大豆胰蛋白酶抑制剂的性质及抑制机理

利用大肠杆菌表达系统,成功表达并纯化获得了重组Bowman-Birk型大豆胰蛋白酶抑制剂(rBBTI).对比研究了天然Bowman-Birk型胰蛋白酶抑制剂(BBTI)和rBBTI的酶学性质和稳定性,以及分别抑制胰蛋白酶和糜蛋白酶的抑制动力学.结果表明:rBBTI和BBTI都有较好的热稳定性,pH对rBBTI的活性影响...     

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．     

固定化胰蛋白酶的制备及其性质研究

以EDTA-Sephadex G-50作载体,戊二醛为交联剂,对胰蛋白酶进行固定化.IR光谱结果显示:该项生化技术使胰蛋白酶得到了有效的固定,并且实现了较好的纯化,同时研究了固定化胰蛋白酶的一些性质,最适温度60℃,最适pH8.0,热稳定性,pH贮存稳定性以及对盐酸胍的稳定性均明显高于天然胰蛋白酶.     

毕赤酵母表达猪胃蛋白酶的研究

利用已有的猪胃蛋白酶原A cDNA,构建毕赤酵母pPIC3．5K—pA胞内表达载体．经纤维蛋白消化试验和干酪素琼脂平板法筛选,获得2株高表达的重组子,并研究了在不同pH、不同诱导时间、不同起始浓度和甲醇诱导浓度等条件下表达水平的高低．结果表明：在pH7、诱导72h、起始诱导浓度为OD600为2、甲醇诱导浓度为1％时,重组胃蛋白酶活力最高,达到6．46U／mL．     

不同来源SOD蛋白的稳定性比较

纯化制备了来源于猪血的天然SOD,由大肠杆菌体系表达的PTD-SOD1以及由酵母体系表达的重组SOD和PTD-SOD2三种基因重组SOD蛋白,并从pH稳定性、温度稳定性、水浴时间稳定性和外源试剂耐受性几个方面对它们进行了比较.结果表明,重组SOD的稳定性总体上是最强的,PTD-SOD2次之,PTD-SOD1最弱,而天然SOD仅比PTD-SOD1稍强.     

播娘蒿DsCOR基因的原核表达、蛋白纯化、及性质研究

利用PCR方法扩增DsCOR基因的蛋白编码序列,经BarnH Ⅰ、Sac Ⅰ酶切后连接入pET32a原核表达载体,构建的pET32a—DsCOR融合重组表达质粒转化大肠杆菌BL21进行原核表达．建立了“煮沸-镍离子亲和层析”的蛋白纯化方法．对纯化的DsCOR蛋白进行高温耐受性、pH耐受范围、紫外耐受性方面的研究．     

重组刺桐胰蛋白酶抑制剂的制备及活性鉴定

刺桐胰蛋白酶抑制剂(ETI)是一种Kunitz型胰蛋白酶抑制剂,能够抑制胰蛋白酶及组织型纤溶酶原激活剂的活性,因此ETI可以作为配体与固定相偶联用于亲和层析.利用化学合成及聚合酶链式反应(PCR)的方法获得ETI的编码基因,然后用NdeI/SacI酶切并插入载体pET25b ( )中构建重组表达质粒pET25b ( )/ETI .重组表达质粒转化大肠杆菌BL21(DE3)后,用IPTG诱导使重组ETI(rETI)过量表达形成包涵体.包涵体经过洗涤、复性及CM离子交换柱纯化,每升培养液可获得14 mgrETI ,其纯度超过92 %.活性测定表明,rETI对凝血酶及尿激酶的活性没有抑制作用,但能显著抑制胰蛋白酶、瑞替普酶及纤溶酶的活性,抑制常数Ki分别为5 .14×10-9,9 .4×10-8和2 .08×10-8mol/L.     

重组羧肽酶B在胰岛素原C肽制备工艺中的应用

用RT-PCR方法克隆了大鼠羧肽酶原B的cDNA编码序列,将其重组到原核表达质粒pT7-473中,并在大肠杆菌中以包涵体方式获得高表达。SDS-PAGE电泳及灰度扫描显示表达量达菌体总蛋白的35％,表达产物融合了表达质粒上的6His。在变性条件下,经Ni-NTA柱纯化,得到羧肽酶原B,在复性液中进行稀释复性后,胰蛋白酶切得具酶活性的羧肽酶B,然后经DEAE-FF分离纯化获得较纯的羧肽酶B(28．5mg／L),比活为13．5u／mg。用RP-HPLC分析胰蛋白酶 羧肽酶B酶解胰岛素原C肽三聚体的酶解产物,证明该重组的羧肽酶B可替代提取的羧肽酶B应用于胰岛素原C肽的制备工艺中。     

重组人组织型纤溶酶原激活剂缺失变体基因的克隆及工程菌的构建与鉴定

利用RT-PCR技术,从人脐带静脉上皮细胞中克隆人组织型纤溶酶原激活剂基因,将其接入TA克隆载体PCR2．1中,经DNA序列测定后,以该重组质粒DNA为模板,用PCR方法获得了人组织型纤溶酶原激活剂缺失变体(K2tPA)基因,将其转入pET29a,构建了重组表达质粒pET29a／K2tPA,并转化至大肠杆菌BL21(DE3)中,构成工程菌．经IPTG诱导表达,在40kDa处有一明显表达条带,表达量为约占菌体总蛋白的20％．该菌种在贮存与复苏及传代过程中具有良好的质粒稳定性和表达稳定性．     

土元溶栓蛋白的原核表达和活性检测

本文利用兼并引物成功克隆到土元类胰蛋白酶样丝氨酸蛋白酶基因,通过构建该基因的原核重组表达载体及IPTG体外诱导,包涵体蛋白复性,Ni 2+柱亲和层析纯化,最终获得了重组表达目的蛋白.经体外检测,该蛋白具有较好的溶栓活性,这为揭示土元溶栓作用的分子基础及其进一步的开发利用奠定了基础.     

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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