植酸酶基因表达与调控机制研究(Ⅱ)──番茄幼苗cDNA文库构建与植酸酶基因筛选

采用异硫氰酸胍法从发芽３～４ｄ的番茄幼苗中提取出总ＲＮＡ,用Ｏｌｉｇｏ（ｄＴ）纤维素亲和层析分离纯化ｍＲＮＡ．以ｍＲＮＡ为模板,Ｏｌｉｇｏ（ｄＴ）为引物用逆转录法合成双链ｃＤＮＡ．将ｃＤＮＡ与ＥｃｏＲＩ接头连接后克隆到表达载体λｇｔ１１的单一ＥｃｏＲＩ位点,经体外包装后感染宿主菌Ｙ１０９０,成功地构建了完整的番茄幼苗ｃＤＮＡ文库．用颜色筛选法筛选重组子,然后用植酸酶抗体做探针进行免疫筛选,得到两个阳性克隆．挑取阳性克隆噬菌斑扩增后纯化ＤＮＡ,经琼脂糖凝胶电泳鉴定,显示确有外源ＤＮＡ存在．该插入片断序列测定正在进行．     

小鼠4.5S RNA逆转座子cDNA的克隆及其表达载体的构建

利用ＲＴ－ＰＣＲ方法,从小鼠肝脏组织总ＲＮＡ中扩增出４．５Ｓ ＲＮＡ的ｃＤＮＡ．此ｃＤＮＡ被克隆到ｐＧＥＭ３Ｚｆ（＋）质粒,酶切鉴定并测序。然后将该序列插入以虫荧光素酶基因作了报导基因的表达载体ｐＳＶｌｕｃ２０的ＰｖｕⅡ位点,构建;了４．５Ｓ ＲＮＡ逆转座子的表达载体ｐＳＶｌｕｃ２０－４．５Ｓ,并对４．５Ｓ ＲＮＡ逆转座子的生物学功能进行了研究。     

人血小板生成因子（TPO）的cDNA克隆在小板生成效应

用ＲＴ－ＰＣＲ方法，从人胎肝总ＲＮＡ中扩增得到约１．１ｋｂ人ＴＰＯ ｃＤＮＡ编码顺序，并进行分子克隆，将其中一个克隆（ｐＴＰＯ４７）亚克隆到Ｍ１３载体，测定全部ＤＮＡ顺序。它的碱基顺序与已报道的顺序完全相同，有一个１０５９ｂｐ的开放阅读框架可编码３５３个氨基酸。ｐＴＰＯ４７亚克隆到哺乳动物表达载体ｐＣＥＰ４，在哺乳动物２９３细胞系瞬时表达后，进行Ｎｏｒｔｈｅｒｎ杂交，结果显示有约１．４ｋｂ的转录产     

人血小板生成因子（TPO）的cDNA克隆及促血小板生成效应

用ＲＴ－ＰＣＲ方法，从人胎肝总ＲＮＡ中扩增得到约１．１ｋｂ人ＴＰＯｃＤＮＡ编码顺序，并进行分子克隆．将其中一个克隆（ｐＴＰＯ４７）亚克隆到Ｍ１３载体，测定全部ＤＮＡ顺序．它的碱基顺序与已报道的顺序完全相同，有一个１０５９ｂＰ的开放阅读框架可编码３５３个氨基酸．ｐＴＰＯ４７亚克隆到哺乳动物表达载体ｐＣＥＰ４，在哺乳动物２９３细胞系瞬时表达后，进行Ｎｏｒｔｈｅｒｎ杂交，结果显示有约１．４ｋｂ的转录产物．瞬时表达培养液腹腔注射小鼠后，可提高血小板计数４２．３％．血小板生成因子的克隆对进一步研究血小板生成调节以及该因子作用的分子机理具有重要意义．     

丙型肝炎病毒核酸疫苗的研究

从丙型肝炎病人的阳性血清中提取出丙型肝炎病毒ＲＮＡ，通过ＲＴ－ＰＣＲ的方法获得丙型肝炎病毒Ｃ区基因片段。并将此片段克隆到真核细胞表达载体ｐｃＤＮＡ３．１（＋），得到重组质粒ｐｃＤＮＡ－ＨＣＶ／Ｃ，再将其通过肌肉注射免疫ＢＡＬＢ／小鼠后，小鼠产生了抗ＨＣＶ／Ｃ区抗体。     

中蜂囊状幼虫病病毒部分结构蛋白基因的克隆及序列分析

根据小ＲＮＡ 病毒科（ Ｐｉｃｏｒｎａｖｉｒｉｄａｅ） 中病毒ＲＮＡ 所具有的结构特征, 采用ｍＲＮＡｃａｐｔｕｒｅ ｋｉｔ 提取纯化中蜂囊状幼虫病病毒（Ｃｈｉｎｅｓｅｓｃａｂｒｏｏｄ ｖｉｒｕｓ ＣＳＢＶ） 的ＲＮＡ, 并以之为ｃＤＮＡ合成的模板． 依据小ＲＮＡ病毒科中的脊髓灰质炎病毒结构蛋白基因序列设计了一对引物ＶＰ５和ＶＰ３ , 通过ＰＣＲ 扩增获得预期大小约为１ １００ ｂｐ的ＤＮＡ 片段, 将此片段克隆到ｐＧＥＭＴｅａｓｙ载体上并直接测序． 序列分析表明, 该片段为中蜂囊状幼虫病病毒部分结构蛋白基因, 与意蜂幼虫囊状病病毒结构蛋白基因序列的同源性为８６－８ ％ , 与之对应氨基酸序列的同源性高达９３－４ ％ ． 该病毒株为一种新型的蜜蜂囊状幼虫病病毒株     

蜂毒肽前体蛋白cDNA的克隆及序列分析

从蜜蜂（Ａｐｉｓｍｅｌｌｉｆｅｒａ）毒腺中提取总ＲＮＡ,通过ＲＴ－ＰＣＲ方法扩增得到了蜂毒肽前体蛋白的ｃＤＮＡ。扩增产物克隆到ｐＴ７Ｂｌｕ－Ｔ载体,再进一步将插入片段酶切连接到ＰＵＣ１１８载体上,构建了与β－半乳糖苷酶部分序列相融合的蜂毒肽前体蛋白表达体并转化大肠杆菌ＪＭ１０９。     

人粒细胞集落刺激因子cDNA的克隆与表达

用脂多糖诱导正常人单核细胞使产生粒细胞集落刺激因子。从诱导细胞中提取出总ＲＮＡ，以特上物经逆转录ＰＣＲ扩增出粒细胞集落刺激因子ｍＲＮＡ和ｃＤＮＡ，将其插入经改造的分泌型表达载体ｐＩＮ－ｏｍｐ Ａ，并转化受体菌Ｅ．ｃｏｌｉ。克隆的ｃＤＮＡ通过限制性酶双酶切的３＇端、５＇端及中间序列探针分子杂交鉴定，结果与预期一致。表达质粒在大肠杆菌哟ＩＰＲＴＧ诱导，粒细胞集落刺激因子被分泌到细菌的周质中。产物提取后     

缺铁玉米根cDNA文库的构建及蛋白和cDNA差异比较

采用异硫氰酸胍酚氯仿法分离铁胁迫玉米根总ＲＮＡ,然后采用寡聚ｄＴ 纤维素层析柱法纯化Ｐｏｌｙ（Ａ） ＋ ＲＮＡ．用含有ＸｈｏＩ位点的ｌｉｎｋｅｒｐｒｉｍｅｒ 锚定合成ｃＤＮＡ 第一条链后,采用替代法合成第二条链,接着连接上ＥｃｏＲＩＡｄａｐｔｅｒ 以便定向重组到载体上．ｃＤＮＡ 经过ＸｈｏＩ消化和分级分离后,带有ＸｈｏＩ和ＥｃｏＲＩ粘性末端的ｃＤＮＡ 定向重组到λＺＡＰ表达载体的ＸｈｏＩ和ＥｃｏＲＩ位点上．经体外包装,重组的噬菌体转导宿主菌ＸＬ１Ｂｌｕｅ ＭＲＦ,最终获得滴度为４－５ ×１０５ ｐｆｕ／ｕｇ 的λＺＡＰ 表达ｃＤＮＡ 文库．通过差异筛选法和质膜双向电泳验证了缺铁和加铁条件下ｃＤＮＡ 和质膜蛋白的差异     

胞内型磷脂酶A2基因亚克隆与基因表达

目的 研究花生四稀酸在细胞中的释放及前列腺素的合成,构建与花生四稀酸释放相关的表达系统。即胞内型磷脂酶Ａ２（ｃｙｔｏｓｏｌｉｃ ｐｈｏｓｐｈｏｌｉｐａｓｅ ｃＰＬＡ２）ｃＤＮＡ－ｐＲＣ／ＣＭＶ。方法 从克隆载体ｐＭＴ２用Ｓａｌ１制备ｃＰＡＬ２ｃＤＮＡ;平末端连接ｃＰＡＬ２－ｐＲＣ／ＣＭＶ;转染鼠巨噬细胞及筛选正向阳性克隆;ｎｏｒｔｈｅｒｎ ｂｌｏｔ检测ｃＰＬＡ２ ｃＤＮＡ基因表达。结果 人ｃＰＬＡ     

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.     

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     

An affordable accurate evaluation of the binding conformations predicted by molecular docking studies in bio-system

Franck-Condon factors bridge the gap between theoretical modeling and experimental observations for molecular electronic spectroscopy and electron transfer.Under the displaced harmonic oscillator approximation,multidimensional Franck-Condon factors are decomposed into a product of many one-dimensional(1D)Franck-Condon(FC)factors,and each 1D-FC factor is associated with one Huang-Rhys factor that determines the leading contribution of     

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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