构建的FIt3L胞外域原核表达载体在大肠杆菌中的表达

目的:构建含组氨酸标签的FMS样酪氨酸激酶3配体(Fms-like tyrosine kinase 3 ligand,FIt3L)胞外域蛋白的原核表达载体,并在大肠杆菌中进行表达.方法:依据FIt3L基因序列设计引物,以RT-PCR的方法从小鼠脾脏总RNA中克隆FIt3L基因并构建其胞外域原核表达载体,测序鉴定后在大肠杆菌BL21(DE3)菌株中诱导表达,用免疫印迹鉴定.结果:克隆到FIt3L编码区全长序列,经DNA测序证明与已报道的序列相同;构建羧基端带有His6标签的FIt3L胞外域蛋白的原核表达载体,转化大肠杆菌后SDS-PAGE分析显示在BL21(ED3)中获得较高表达;免疫印迹分析表明IPTG诱导后表达的FIt3L胞外域蛋白的相对分子质量(Mr)为19 000,与理论值相符,此蛋白与抗His6标签的抗体发生特异性反应.结论:成功克隆FIt3L基因,构建其胞外域蛋白的原核表达载体,并在大肠杆菌中获得表达.     

广西巴马小型猪卵泡抑素(Follistatin)克隆及原核表达

目的通过RT-PCR扩增获得广西巴马小型猪Follistatin的cDNA全序列,经克隆测序分析其cDNA序列结构,进而构建pET-Fo原核表达质粒,使用IPTG诱导表达获得其融合蛋白,为将来进一步研究Follistatin对广西巴马小型猪肌肉生长和繁殖性能的影响奠定基础。方法从广西大学巴马小型猪卵巢中提取总RNA,并以其为模板,应用逆转录-聚合酶链式反应(RT-PCR),在合成的特异性引物引导下,扩增获得广西巴马小型猪卵泡抑素(Follistatin)cDNA的全序列,长度为1 035 bp。将PCR产物克隆到pMD18-T载体后进行序列测定及分析。结果广西巴马小型猪卵泡抑素cDNA序列与GenBank中已报道的家猪卵泡抑素同源性高达99.4%。同时,将目的基因插入到原核表达载体pET 32a 多克隆位点中,构建了Follistatin的原核表达载体,并转化于大肠杆菌BL21。转化菌经异丙基硫代半乳糖苷(IPTG)诱导,变性聚丙烯酰胺凝胶电泳(SDS-PAGE)和蛋白质免疫印迹(Western blotting)分析。结论实验已成功地表达了Follistatin的融合蛋白(约58.4 ku)。     

构建的Flt3L胞外域原核表达载体在大肠杆菌中的表达

目的：构建含组氨酸标签的FMS样酪氨酸激酶3配体（Fms—like tyrosine kinase 3 ligand,Fh3L）胞外域蛋白的原核表达载体,并在大肠杆菌中进行表达。方法：依据Fh3L基因序列设计引物,以RT—PCR的方法从小鼠脾脏总RNA中克隆Fh3L基因并构建其胞外域原核表达载体,测序鉴定后在大肠杆菌BL21（DE3）菌株中诱导表达,用免疫印迹鉴定。结果：克隆到Flt3L编码区全长序列,经DNA测序证明与已报道的序列相同;构建羧基端带有His6标签的Flt3L胞外域蛋白的原核表达载体,转化大肠杆菌后SDS-PAGE分析显示在BL21（ED3）中获得较高表达;免疫印迹分析表明IPTG诱导后表达的Flt3L胞外域蛋白的相对分子质量（Mr）为19000,与理论值相符,此蛋白与抗His6标签的抗体发生特异性反应。结论：成功克隆Flt3L基因,构建其胞外域蛋白的原核表达载体,并在大肠杆菌中获得表达。     

人Delta-like4ext-26-217蛋白原核表达载体的构建及表达

构建人Delta-like4ext-26-217原核表达载体并表达。采用PCR方法扩增hDll4ext-26-217多核苷酸序列,克隆入pMD18T载体。测序正确后,将其亚克隆入pET32a( )原核表达载体,获得pET32a( )-hDll4ext-26-217载体。以该载体转化E.coli菌株BL21,IPTG诱导其表达。采用SDS-PAGE、Western Blot方法鉴定目的蛋白的表达。结果表明,采用SDS-PAGE,Western Blot等方法均可检测到目的蛋白TRX/hDll4ext -26-217,分子量约42.2 KD,主要以包涵体形式大量存在。以上结果为Detla-like4功能的进一步研究奠定了基础。     

广西巴马小型猪生长激素(pGH)基因全序列克隆及序列分析

目的克隆广西巴马小型猪生长激素(pGH)基因全序列,并与已发表的其它品种猪的生长激素(pGH)基因全序列进行比较,探讨该基因在广西巴马小型猪中可能存在的变异。方法以广西大学巴马小型猪基因组DNA为模板,扩增巴马小型猪生长激素基因的全序列,将该扩增片段亚克隆到pMD18-T载体后测序。再将测序结果在NCBI进行BLAST。结果测序结果证明克隆得到了巴马小型猪GH基因,全长为2 007 bp。同源性比较发现,巴马小型猪GH基因全序列与GenBank中发表的五指山猪、太湖猪及普通猪的GH基因全序列的同源性分别为99.4%、98.3%、93.9%。     

草鱼生长激素基因的克隆及原核表达研究

应用RT-PCR技术从草鱼脑垂体总RNA中克隆草鱼生长激素cDNA(cGH)，全长669bp，含开放阅读框633bp，编码210个氨基酸，分子量为23．6kDa，等电点为6．28；与已报道的草鱼GH有12个碱基、3个氨基酸残基的差异，同源性为98％．将草鱼cDNA定向插入原核表达载体pGEX-4T-1，构建了重组草鱼GH基因质粒pGEX-GcGH，经IPTG诱导，pGEX-GcGH在大肠杆菌中可表达49．6kDa的融合蛋白．     

牛泡沫病毒BFV3026 gag基因的克隆及其在大肠杆菌中的表达

从牛泡沫病毒BFV3026感染的胎牛肺细胞中提取Hirt DNA,PCR扩增BFV3026　gag基因。序列分析确证后，将其克隆于原核表达载体pET32A，重组质粒转化大肠杆菌BL21（DE3），经IPTG诱导，使Gag蛋白得以高效表达。SDS－PAGE及Western blot证实：表达蛋白占菌体总蛋白的40%，本工作的完成为Gag蛋白功能的研究奠定基础。     

Abd-Bcr/Abl原核表达载体的构建和表达

构建Bcr/Abl-Abd原核表达载体,并表达、观察对白血病细胞K562的影响.以慢性白血病PGD210质粒为模板,应用聚合酶链反应(PCR)扩增出Abd编码区序列,克隆入pMD18t载体,经酶切测序鉴定正确.再通过酶切重组克隆入原核表达载体pET32a,构建pET32a-Abd重组表达质粒,经酶切、测序鉴定,序列、读码框均正确.通过转化E.coli BL21(DE3),经IPTG诱导表达,纯化.结果PCR扩增出特异性的501bp的目的片断,以此构建的重组质粒pET32a-Abd能够在上清中表达约36.6 kd的目的蛋白,Westem blot鉴定为特异表达.证明成功构建了原核表达载体pE732a-Abd,并表达在上清中,表达蛋白约占菌体总蛋白的6%,纯化后达到74.3%,Westem blot检测为特异性表达,为进一步研究该蛋白对慢性白血病细胞的作用奠定了分子基础.     

编码拟南芥含BAG结构域蛋白的原核表达载体构建

生物信息学分析显示,拟南芥基因At5g62390编码一种钙调素结合蛋白,在其钙调素结合结构域有一个BAG(Bcl-2-associated athnogene)结构域存在,与钙调素结构域部分重叠.为了从实验上进一步研究该蛋白的钙调素结合特征及BAG结构域在钙调素结合中可能的调节作用,通过聚合酶链式反应(PCR)扩增不同结构域编码区的cDNA序列,构建到原核表达载体pET32-a中.测序分析表明:目的序列已正确克隆到表达载体上,为进一步表达目的蛋白及其不同结构域用于生化功能鉴定打下了基础.     

小鼠附睾特异性辅脂酶的原核表达及其抗体的制备

目的:利用原核表达系统重组表达小鼠附睾特异性辅脂酶(meClps),并制备抗meClps抗体。方法:通过生物信息分析meClps蛋白结构,Oligo 6设计引物,PCR法从小鼠附睾cDNA中扩增meClps全长序列并克隆到pMD19-T载体中。将含meClps序列pMD19-T载体通过PCR扩增成熟肽区段序列,重组到pET-21b原核表达载体上,经菌落PCR及测序鉴定后,转化到表达菌株E.coliBL21(DE3),经IPTG诱导蛋白表达后用Tricine-SDS-PAGE和Western blotting检测重组meClps的表达。包涵体沉淀经浓度为2 mol/L尿素洗涤,Tricine-SDS-PAGE分离、染色、脱色,切下目的条带用生理盐水浸泡、磨碎与弗氏佐剂混合后免疫新西兰大白兔获取免疫血清,Western blotting检测抗体与meClps反应。结果:在原核表达系统成功重组表达meClps,目的蛋白主要以包涵体形式存在,制备了高效价的兔抗meClps抗体。结论:成功建立meClps的原核表达系统和获得兔抗meClps抗体。     

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.     

Characterization of Ordovician carbonate reservoirs, southeastern Saskatchewan, Canada

The discovery of the prolific Ordovician Red River reservoirs in 1995 in southeastern Saskatchewan was the catalyst for extensive exploration activity which resulted in the discovery of more than 15 new Red River pools. The best yields of Red River production to date have been from dolomite reservoirs. Understanding the processes of dolomitization is, therefore, crucial for the prediction of the connectivity, spatial distribution and heterogeneity of dolomite reservoirs.The Red River reservoirs in the Midale area consist of 3～4 thin dolomitized zones, with a total thickness of about 20 m, which occur at the top of the Yeoman Formation. Two types of replacement dolomite were recognized in the Red River reservoir: dolomitized burrow infills and dolomitized host matrix. The spatial distribution of dolomite suggests that burrowing organisms played an important role in facilitating the fluid flow in the backfilled sediments. This resulted in penecontemporaneous dolomitization of burrow infills by normal seawater. The dolomite in the host matrix is interpreted as having occurred at shallow burial by evaporitic seawater during precipitation of Lake Almar anhydrite that immediately overlies the Yeoman Formation. However, the low δ18O values of dolomited burrow infills (-5.9‰～ -7.8‰, PDB) and matrix dolomites (-6.6‰～ -8.1‰, avg. -7.4‰ PDB) compared to the estimated values for the late Ordovician marine dolomite could be attributed to modification and alteration of dolomite at higher temperatures during deeper burial, which could also be responsible for its 87Sr/86Sr ratios (0.7084～0.7088) that are higher than suggested for the late Ordovician seawaters (0.7078～0.7080). The trace amounts of saddle dolomite cement in the Red River carbonates are probably related to "cannibalization" of earlier replacement dolomite during the chemical compaction.     

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.     

A computer generator for randomly layered structures

AcomputergeneratorforrandomlylayeredstructuresYUJia shun1,2,HEZhen hua2(1.TheInstituteofGeologicalandNuclearSciences,NewZealand;2.StateKeyLaboratoryofOilandGasReservoirGeologyandExploitation,ChengduUniversityofTechnology,China)Abstract:Analgorithmisintrod…     

An Index mechanism for multi-scale view in spatial databases

There are numerous geometric objects stored in the spatial databases. An importance function in a spatial database is that users can browse the geometric objects as a map efficiently. Thus the spatial database should display the geometric objects users concern about swiftly onto the display window. This process includes two operations:retrieve data from database and then draw them onto screen. Accordingly, to improve the efficiency, we should try to reduce time of both retrieving object and displaying them. The former can be achieved with the aid of spatial index such as R-tree, the latter require to simplify the objects. Simplification means that objects are shown with sufficient but not with unnecessary detail which depend on the scale of browse. So the major problem is how to retrieve data at different detail level efficiently. This paper introduces the implementation of a multi-scale index in the spatial database SISP (Spatial Information Shared Platform) which is generalized from R-tree. The difference between the generalization and the R-tree lies on two facets: One is that every node and geometric object in the generalization is assigned with a importance value which denote the importance of them, and every vertex in the objects are assigned with a importance value,too. The importance value can be use to decide which data should be retrieve from disk in a query. The other difference is that geometric objects in the generalization are divided into one or more sub-blocks, and vertexes are total ordered by their importance value. With the help of the generalized R-tree, one can easily retrieve data at different detail levels.Some experiments are performed on real-life data to evaluate the performance of solutions that separately use normal spatial index and multi-scale spatial index. The results show that the solution using multi-scale index in SISP is satisfying.     

Jane Eyre——A Feminist In The 19th Century

In the 19th century the society was controlled by men, and women were just appendants of them, they had not any rights and freedom. But Jane was an exception, she showed some characteristics of early feminist. Jane showed her characteristics of feminism in three aspects: rebellion, equality, and independence. These characteristics were helpful to her success, and feminism is the only way out for women of that time.     

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-