重组人粒细胞集落刺激因子在Escherichia coli中的表达研究

根据人天然粒细胞集落刺激因子（Ｇ－ＣＳＦ）基因序列设计出引物,通过ＲＴ－ＰＣＲ从人外周血单个核细胞ｍＲＮＡ获得Ｇ－ＣＳＦｃＤＮＡ片段,将该片段与原核表达载体ｐＴ７构建成重组体,导入大肠杆菌后发现天然Ｇ－ＣＳＦｃＤＮＡ表达量并不理想,这可能是由于天然ｈＧ－ＣＳＦ５’端Ｇ＋Ｃ的比例过高,使转录后的ｍＲＡ很容易形成二级结构而影响翻译起始,因此在大肠杆菌中很难获得高效表达,根据密码子简并性原则,在不改变编码氨基酸顺序的前提下,通过重新设计ＰＣＲ引物,将ｈＧ－ＣＳＦ的前３个氨基酸密码子中的几个ＧＣ碱基作了改动,获得了新的ｃＤＮＡ突变体,将其与ＰＴ７的重组导入大肠杆菌,获得了高效表达。     

用复制酶基因cDNA探针检测表达病毒外壳蛋白基因马…

利用马铃薯卷叶病毒（ＰＬＲＶ）复制酶基因３＇端长约６００ｂｐ的ｃＤＮＡ，用缺口平移方法进行＾３２Ｐ同位素标记，制备成ｃＤＮＡ探针，通过核酸斑点杂交，对提纯的马铃薯卷叶病毒（ＰＬＲＶ）、病毒ＲＮＡ、ＰＬＲＶ感染的马铃薯汁液，表达ＰＬＲＶ ＣＰ基因的马铃薯叶片及块茎芽进行了检测。结果表明，＾３２Ｐ标记的复制酶基因ｃＤＮＡ探针特异性强、灵敏度高。可测得提纯病毒的最低含量为４０８ｎｇ／ｍｌ，病毒ＲＮＡ最低     

人锰超氧化物歧化酶cDNA基因的克隆

利用人胚胎肝组织提取总ＲＮＡ,从总ＲＮＡ 中分离纯化出ｍ ＲＮＡ,经逆转录得到ｃＤＮＡ 第一条链,并以之为模板和相应寡聚脱氧核苷酸为引物,进行ＰＣＲ 合成人锰超氧化物歧化酶ｃＤＮＡ 基因,将所得ｃＤＮＡ 克隆到质粒ｐＢｌｕｅｓｃｒｉｐｔＳＫ 上,转化大肠杆菌ＤＨ５α细胞,进行诱导表达筛选,将筛选的克隆进行ｃＤＮＡ 全序列测定     

牛α—s1酪蛋白基因3‘端DNA的PCR扩增,克隆及鉴定

本研究以牛血总ＤＮＡ为模板，用ＰＣＲ技术扩增牛α－ｓ１酪蛋白基因的５＇端３．６ｋｂＤＮＡ片段和３＇端１．５ｋｂＤＮＡ片段的调控区序列，得到了相应的特异性扩增片段．用Ｋｌｅｎｏｗ处理后，将两个扩增片段分别与经Ｓｍａｌ酶切的ｐＵＣ１８质粒载体用Ｔ４ＤＮＡ连接酶连接，转化大肠杆菌ＪＭ１０９．在含有Ｘ－ｇａｌ，ＩＰＴＧ和Ａｍｐ的选择培养基上培养．从白色菌落中提取质粒ＤＮＡ，进行限制酶切鉴定．结果得到一个插入有１．３ｋｂＤＮＡ片段的阳性克隆．对其进行部分序列分析表明，该片段为５＇端缺失约１７０ｂｐ的牛α－ｓ１酪蛋白基因３＇端及下游区序列．     

人胰岛素原乳腺表达载体的构建

通过ＰＣＲ方法扩增了人胰岛素（ＨＩ）基因１．３４ｋｂ的ＤＮＡ片段并克隆于ｐＵＣ１８的ＳｍａⅠ位点内。经ＤＮＡ序列分析表明，该片段为ＨＩ基因的氨基酸编码区及３＇端调控区序列。同时，应用ＰＣＲ方法对牛α－乳白蛋白（ＢαＬＡ）基因进行了扩增，得到了０．８４ｋｂＤＮＡ扩增片段。将其克隆于去除了ＥｃｏＲＩ位点的ｐＵＣ１８ＳｍａＩ位点内，经内切酶分析和ＤＮＡ测序证明该片段是ＢαＬＡ基因５＇调控区序列。ＥｃｏＲ     

dna Y基因产物提高人尿激酶原cDNA在大肠杆菌中的表达

对提高人水激酶原ｃＤＮＡ在大肠杆菌中的表达水平进行了研究。通过ＰＣＲ方法在其结构基因５‘端添加起密码子ＡＴＧ，用人工合成的不依赖于ρ因子的终止序列替换 ３’端非翻译区，得到完整的人尿激酶原结构基因。将其克隆在表达质粒ｐＫＫ２３３－２中，转化大肠杆菌Ｅ．ｃｏｌｉＪＡ２２１，人尿激酶原得到初步表达，但表达水平很低，原因可能是人尿激酶原ｃＤＮＡ富含真核细胞偏爱的密码子，而大肠杆菌中识别这些密码子的ｔＲＮ     

低分子量单链尿激酶基因在酵母中的表达

Ｍ１３ｓｃｕＰＡ３２ｋ克隆载体经ＨｉｎｄⅢ酶切得到ｓｃｕＰＡ３２ｋ的基因，与分泌型酵母表达载体ｐＧＬ重组得到正向插入的ｓｃｕＰＡ３２ｋ酵母表达载体．用改进的酵母完整细胞快速高效转化法转化ＡＢ１０３酵母菌株，转化效率达到１．８×１０５个转化子／μｇ载体ＤＮＡ，并在直接测活培养板上筛选出阳性转化子．用纤维蛋白平板溶圈测活法检测结果表明阳性转化子能分泌纤溶酶原激活剂．     

小鼠B7—1cDNA克隆,测序,表达及其抗瘤效应的初步探讨

用反转录ＰＣＲ的方法,从ＢＡＬＢ／ｃ小鼠脾细胞中扩增出Ｂ７－１ｃＤＮＡ后,插入ｐｃＤＮＡ３质粒中构建成小鼠Ｂ７－１ｃＤＮＡ的真核表达载体ｐＣＤ－ｍＢ７．１,经酶切鉴定和序列分析证实此表达载体中插入的Ｂ７－１ｃＤＮＡ的序列与献报道一致。     

HGVNS3cDNA片段的表达及其免疫原性的研究

一段长度为１３２７ｂｐ的庚型肝炎病毒ｃＤＮＡ在大肠杆菌ＢＬ２１（ＤＥ３）ＡＬ菌株中得到表达。此ｃＤＮＡ被插入到噬粒ｐＧＥＭＤ中,位于编码大肠杆菌ＲＮＡ聚合酶σ＾７０－因子氨基端８个氨基酸残基的ＤＮＡ序列的下游,     

人核蛋白P68 cDNA基因克隆及全序列测定

靠人合成４个混合聚核苷酸探针，从一个人盘λｇｔ１１ ｃＤＮＡ库筛选到含有ｐ６８ ｃＤＮＡ基因的阳性噬菌斑。经次克隆得到含ｐ６８ ｃＤＮＡ基因的ｐＢＲＢｔ７－ｐ６８质粒。用末端终止法对此ｃＤＮＡ基因进行了全序列测定。分析结构证明得到的ｐ６８ｃＤＮＡ基因含有２２８７３个苷酸，具有一个可阅读框架，共编码６１４个氨基酸。其５＇端非编码区含有１３０个碱基。３＇端非编码区含有３１５个碱基，比已发表的３＇端非     

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.     

An Analysis of Catherine＇s Character——Wuthering Heights

Wuthering Heights, Emily Bronte＇s only novel, was published in December of 1847 under the pseudonym Ellis Bell. The book did not gain immediate success, but it is now thought one of the finest novels in the English language. Catherine is the key character of this masterpiece, because everybody and everything center on her though she had a short life. We can understand this masterpiece better if we know Catherine well.     

Lower Paleozoic oil relationships within Williston Basin, Canada

The Williston Basin is a significant petroleum province, containing oil production zones that include the Middle Cambrian to Lower Ordovician, Upper Ordovician, Middle Devonian, Upper Devonian and Mississippian and within the Jurassic and Cretaceous. The oils of the Williston Basin exhibit a wide range of geochemical characteristics defined as "oil families", although the geochemical signature of the Cambrian Deadwood Formation and Lower Ordovician Winnipeg reservoired oils does not match any "oil family". Despite their close stratigraphic proximity, it is evident that the oils of the Lower Palaeozoic within the Williston Basin are distinct. This suggests the presence of a new "oil family" within the Williston Basin. Diagnostic geochemical signatures occur in the gasoline range chromatograms, within saturate fraction gas chromatograms and biomarker fingerprints. However, some of the established criteria and cross-plots that are currently used to segregate oils into distinct genetic families within the basin do not always meet with success, particularly when applied to the Lower Palaeozoic oils of the Deadwood and Winnipeg Formation.     

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.     

Design and analysis of generic LBS application developing platform

Location based services is promising due to its novel working style and contents.A software platform is proposed to provide application programs of typical location based services and support new applications developing efficiently. The analysis shows that this scheme is easy implemented, low cost and adapt to all kinds of mobile nework system.     

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