斜带石斑鱼神经坏死病毒主衣壳蛋白的原核表达与纯化

将含有斜带石斑鱼Epinephelus coioides神经坏死病毒(orange-spotted grouper nervous necrosis virus,OGNNV)主衣壳蛋白(major capsid protein, MCP)基因的重组表达质粒载体pET32a-MCP转化到大肠杆菌BL21(DE3)中进行融合表达,经SDS-PAGE分析和Western-blot鉴定,证实了重组大肠杆菌融合表达了斜带石斑鱼神经坏死病毒主衣壳蛋白.表达的融合蛋白主要以不可溶的包涵体形式存在,提取的包涵体中融合蛋白含量占60%以上,经柱层析纯化蛋白,纯化度达90%以上.     

HLA-A*0207重链胞外区原核表达载体的构建及表达

目的：克隆HLA-A*0207(A2)重链基因，构建在羧基端融合生物索化酶BirA底物肽(BirA substrate peptide，BSP)的A2重链胞外区原核表达载体，在大肠杆菌中进行表达。方法：以RT-PCR方法从HLA-A2^ 供白细胞中克隆A2基因并进行DNA测序，并以PCR方法构建在羧基端融合BSP的A2重链胞外区表达载体，在大肠杆菌B121(ED3)中进行表达。结果：从31名HLA-A2^ 供白细胞中克隆到的基因经DNA测序显示，只有从供2得到的基因是HLA-A*0207。将编码该基因编码重链胞外区1-275的序列和编码BSP的序列融合，构建融合蛋白表达载体，并以测序验证。融合蛋白在B121(ED3)中获得高效表达，产物相对分子质量为35000，约占菌体总蛋白的30％，主要存在于包涵体中，对包涵体进行洗涤后得到纯度为80％的重组蛋白。结论：成功克隆HLA-A*0207基因，构建了其胞外区和BSP融合蛋白表达载体并在大肠杆菌中获得高效表达。     

高频等位基因HLA-A*1101重链胞外域-BSP融合蛋白原核表达载体的构建及表达鉴定

目的:构建HLA-A*1101重链胞外域羧基端融合生物素化酶B irA底物肽(BSP)的融合蛋白(HLA-A11-BSP)原核表达载体,并在大肠杆菌中表达该融合蛋白。方法:以RT-PCR法扩增并克隆HLA-A*1101重链基因的cDNA,以PCR方法构建HLA-A11-BSP的表达载体,在大肠杆菌BL21(DE3)中诱导表达,并以免疫印迹法进行鉴定。结果:从HLA-A2阴性的供者外周血单个核细胞中克隆到HLA-A*1101重链基因的cDNA,以此cDNA为模板,将编码HLA-A*1101重链胞外域1~276序列与编码BSP的序列融合,构建HLA-A11-BSP融合蛋白表达载体,重组质粒经测序验证。融合蛋白在BL21(DE3)中诱导后获得高效表达,约占菌体总蛋白的20%;其相对分子质量约为35 000,与理论值一致。免疫印迹分析显示表达产物主要存在于包涵体中,上清液中几乎无任何产物存在。结论:成功构建表达HLA-A11-BSP融合蛋白的原核表达载体,该融合蛋白在大肠杆菌中以包涵体形式获得高水平表达。     

食管癌相关基因2(ECRG2)包涵体的溶解与纯化

采用生物工程的手段,用插有食管癌相关基因2(ECRG2)开放阅读框(ORF)的原核表达质粒pEGX-4T-1转化大肠杆菌E.coli,诱导表达了重组的GST-ECRG2融合蛋白;收集GST-ECRG2融合蛋白包涵体,经SKL溶解后在PBS中透析复性,用GST亲和层析柱纯化,从而获得一定数量的高纯度重组ECRG2蛋白.     

重组嵌合生长抑素工程菌的构建、表达与免疫

将动物生长抑素(SS)与大肠杆菌不耐热肠毒素B亚单位(LTB)融合基因插入p ET28a系统,构建重组质粒pET28a-A4030-2-LTB并转化到大肠杆菌BL21(DE3)感受态中,获得了大肠杆菌表达菌株BL21(DE3)p ET28a-A4030-2-LTB,然后使用SDS-PAGE检测该融合蛋白的表达并优化其发酵与包涵体洗涤条件,接着采用亲和层析法进行纯化,并用Western Blotting技术检测该融合蛋白的免疫原性.研究结果表明,在37℃,加入1 mmol/L的IPTG培养3 h后,融合蛋白的表达量最高且稳定表达;在包涵体洗涤的过程中,使用含有φ=1%的Triton X-100、2 mol/L尿素和0.5 mol/L NaCl的缓冲液洗涤包涵体沉淀,其效果最佳,用Western Blotting方法检测出该蛋白能发生抗原抗体反应,并且将重组蛋白免疫7只小鼠后,其中有6只小鼠产生了抗SS抗体,表达产物具有免疫原性.此研究为进一步开发生长抑素基因工程疫苗提供了可靠的实验依据.     

丙二酸盐克罗诺杆菌PspA包涵体蛋白的表达、复性及纯化方法优化

为研究丙二酸盐克罗诺杆菌(Cronobacter malonaticus)中噬菌体休克蛋白A(phage shock protein A,PspA)的结构与功能，文章构建了PspA融合蛋白表达载体，但重组蛋白以包涵体形式大量表达；为获取较高浓度的可溶性蛋白进行后续探究，使用尿素裂解液使蛋白变性后，经多种方法复性GST-PspA包涵体蛋白，使其重新恢复生物学活性。研究结果表明，4℃低温条件下包涵体蛋白经尿素梯度稀释透析以控制复性速率，防止蛋白分子快速聚集，可以实现高效复性和纯化GST-PspA融合蛋白。为进一步探索Psp系统中各蛋白的功能及相互作用的研究提供一定的参考。     

小鼠H-2D~b-BSP融合基因的构建与表达

目的:构建小鼠H-2Db-BSP融合基因,并诱导其在大肠杆菌中表达.方法:采用RT-PCR方法从C57BL/6小鼠淋巴细胞中扩增出H-2Db链的胞外段基因,经双酶切置换已构建的HLA-A*0201-BSP重组体中的HLA-A*0201胞外段序列,使H-2Db与BirA酶底物肽(BSP)序列融合,构建H-2Db-BSP融合基因表达载体,转化大肠杆菌BL21(DE3)菌株,筛选重组子,并经IPTG诱导融合蛋白表达.结果:构建的H-2Db-BSP融合基因插入正确且序列与GenBank一致;经1mmol/L浓度的IPTG诱导后4h蛋白表达达到高峰,且融合蛋白以包涵体形式存在.结论:成功构建H-2Db-BSP融合基因,并诱导其在大肠杆菌得以表达,为进一步构建H-2Db四聚体,研究T细胞应答提供了物质基础.     

抗栓肽和尿激酶原（B链）嵌合体的构建与表达

通过基因工程重组技术,将抗栓肽（decorsin）基因与尿激酶的B链即scu-PA(B)基因用柔性Linker((Gly₄Ser)₃)融合在一起,构建新的嵌合体基因dscuPA(B),在大肠杆菌Rosetta(DE3)plysS中通过IPTG诱导表达, 并考察了重组质粒在Rosetta(DE3)plysS在传代中的分离稳定性。该融合蛋白在大肠杆菌中是以包涵体的形式存在,对包涵体进行变性及复性后,并通过Zn²⁺螯合柱和SP阳离子交换柱进行纯化。质谱分析表明分子量为33.735kD,与理论值相符。目的蛋白质的纯度可达90%。纤维蛋白平板法测得嵌合分子的比活为90000IU/mg,与scuPA-32k的比活相近。体外血小板聚集实验表明融合蛋白有较强的抑制血小板聚集的功能,抑制常数IC50为0.31μmol/L。以上这些结果表明,该融合蛋白不但具有较强的溶栓功能,而且具有抗栓功能,可能是一种具有很好发展前景的双功能溶栓抗栓药物。     

海洋放线菌S.areniocola CNS-205腺苷化结构域基因的克隆、表达和纯化

海洋放线菌S.arenicola CNS-205是首次报道的专属海洋性放线菌属Salinispora的代表菌株之一.选取海洋放线菌S.arenicola CNS-205非核糖体多肽合成酶氨基酸腺苷化结构域基因sare 0357,对其进行生物信息学分析后进行原核重组质粒的构建和表达及蛋白纯化.分别构建了pET-28a-sare 0357和pGEX-KG-sare 0357重组质粒,并转化E.coli BL21(DE3)进行不同诱导条件的诱导表达,sare 0357基因均以包涵体的形式表达.收集Sare0357重组蛋白包涵体,选取6mol/L的盐酸胍对其进行变性并将变性后的重组蛋白进行镍柱纯化.得到了可溶性的Sare0357融合蛋白.     

AiiA融合蛋白包涵体变性和复性的研究

通过IPTG诱导含有pGEXaiiA-B15质粒的工程菌使其大量表达AiiA融合蛋白.由于所表达的融合蛋白多为不可溶的包涵体,须经分离、变性溶解,再经过一个合适的复性过程才能实现变性蛋白的正确折叠,得到具有生物活性的蛋白.通过含N-十二烷基肌氨酸钠的缓冲液A溶液及尿素等变性剂使包涵体溶解,磷酸盐缓冲液透析复性.SDS-PAGE电泳表明包涵体已由不可溶转化成可溶的蛋白.抑菌试验证明复性后的AiiA蛋白对胡萝卜欧文氏软腐病菌引起的马铃薯软腐病有较明显的抑制作用.     

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.     

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-     

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