HEK293F细胞瞬时表达PDGFR-β/Fc蛋白的条件优化

为优化HEK293F细胞瞬时转染条件,提高PDGFR-β的蛋白表达量,采用聚乙烯亚胺(Polyetherimide,PEI)为转染试剂,将质粒p XLG-PDGFR-β/Fc与PEI混匀聚合后加入到细胞悬液,37℃,φ=6%CO2,180 r/min悬浮振荡培养,培养7 d后收集样品,ELISA检测PDGFR-β蛋白的浓度。对细胞密度、DNA浓度和m(DNA):m(PEI)、聚合时间以及添加物(丙戊酸钠、葡萄糖和蛋白胨)进行优化。结果显示,HEK293F细胞瞬时转染的最佳条件为:细胞密度4×106cells/m L、DNA浓度2.0μg/106cells、m(DNA):m(PEI)为1:2、聚合时间5 min。同时,48 h内添加3 mmol/L丙戊酸钠,第3天补加葡萄糖和1 g/L的蛋白胨TN1可使PDGFR-β/Fc的蛋白表达量明显提高。实验表明,经过对HEK293F细胞瞬时表达PDGFR-β的转染条件的优化,蛋白表达量可达55 mg/L,为更大规模瞬时转染生产PDGFR-β/Fc蛋白奠定了基础。     

HIV-1病毒样颗粒真核表达系统的建立

目的 利用哺乳动物细胞表达HIV-1病毒样颗粒,为HIV-1病毒的生物学研究和进一步设计HIV-1中和优势表位预防性疫苗奠定基础.方法 以293T细胞作为宿主细胞,利用磷酸钙沉淀法将带有HIV-1病毒被膜蛋白gp160基因的pcDNA3.1/BaL gp160真核表达载体和带有HIV-1核心蛋白(gag)的pVRC3900真核表达载体共转染至293T细胞中,收集培养上清,经蔗糖不连续梯度离心,收集病毒样颗粒,经磷钨酸负染,透射电镜观察结果 .结果 以磷酸钙沉淀DNA转移技术,EGFP真核表达质粒为靶基因,293T细胞为宿主细胞,最佳转染效率达30%以上;将peDt NA3.1/BaL gp160(env)和pVRC3900(gag)真核表达质粒共转染,宿主细胞成功表达目的 蛋白,转染细胞培养上清经蔗糖密度梯度离心,电镜观察可见自行装配的HIV病毒样颗粒.结论 在真核细胞中表达HIV-1病毒样颗粒.     

脂质体转染人肝星状细胞和肝细胞条件优化

利用Lipofectamine 2000(Lipo)介导LacZ及EGFP基因转染人肝星状细胞系LX-2和人肝细胞系Chang Liver,探讨Lipo用量、质粒用量、转染时间、细胞初始接种密度及不同启动子驱动对转染效率的影响.结果显示,两种细胞均能得到高效转染,Chang Liver细胞转染效率(80%)高于LX-2细胞(60%).Lipo用量为每孔2μL,转染后6 h换液,LX-2细胞应用质粒DNA为每孔0.45μg,Chang Liver细胞应用质粒DNA为每孔0.30~0.45μg,此时转染效率最高.巨细胞病毒(CMV)启动子驱动LacZ转染效率与CMV早期增强子/鸡β肌动蛋白(CAG)启动子驱动增强型绿色荧光蛋白(EGFP)转染效率无显著差异;细胞初始接种密度对转染效率无显著影响.     

磷酸钙法将MMP-2，MMP-3双基因干扰载体转入HEK293T细胞的效率观察

检测用磷酸钙法能否将修饰后用于沉默MMP-2,MMP-3基因的慢病毒载体有效地转入到HEK293T细胞。利用磷酸钙法,MMP-2,MMP-3双基因干扰载体（MMP组）与对照质粒（对照组）被分别转染HEK293T细胞,在转染后的第24,48及72h,通过计算绿色荧光蛋白（GFP）阳性细胞数的百分比来判断转染效率。转染24h后,两组细胞均生长状况良好,荧光显微镜下观察,已有大量绿色荧光出现,计算MMP组转染效率为（91．5±6．2）％,对照组转染效率为（80．3±4．7）％;转染后48—72h,两组感染效率均未见明显下降。与对照相比,插入了MMP-2,MMP-3 shRNA模板序列的慢病毒载体没有降低磷酸钙法转染HEK293T细胞效率,反而有所增高。     

抗人PD-L1单克隆抗体的瞬转表达

利用HEK293 6E细胞瞬转表达抗人PD L1单克隆抗体. 将HEK293 6E细胞无血清悬浮培养, 筛选最佳生长培养基; 考察HEK293 6E细胞的筛选表达培养基、 DNA和PEI质量比及转染时细胞密度等转染条件, 并对收获的抗体进行SDS PAGE和ELISA鉴定. 结果表明: 在A无血清表达培养基中, 以2×106个/mL的细胞密度和m(DNA)∶m(PEI)=1的条件转染, 第4天收获抗体的产量最高, 为170.9 μg/mL; 收获PD-L1抗体的重链分子量约为50 000, 轻链分子量约为25 000.     

HEK293 PEAK瞬转快速表达抗体蛋白平台的建立

将HEK293 PEAK细胞用Gibco FreeStyleTM293表达培养液进行无血清悬浮驯化培养,获得无血清悬浮培养的HEK293 PEAK细胞,并经有限稀释法获得细胞亚克隆,建立抗体瞬转表达的工程细胞株.用转染试剂PEI将质粒转入该细胞株中表达抗体.通过优化质粒DNA与转染试剂PEI的比例,质粒与转染试剂的比例为1∶2时,转染效率最佳,并对瞬转表达抗体进行鉴定和活性分析,确认获得具有生物活性的抗体(Herceptin),抗体分子量约150 kD,结果表明已成功建立了以无血清悬浮培养的HEK293 PEAK细胞为宿主细胞的瞬转快速表达抗体蛋白平台.     

人乳头瘤病毒假病毒制备方法的优化

人乳头瘤病毒假病毒可广泛应用于中和抗体鉴定和疫苗免疫性评估等研究.本研究对多质粒共转染293FT细胞生产HPV假病毒的方法进行了深入优化,结果显示不同的HPV结构蛋白表达质粒转染比例对于假病毒生产效率有显著的影响,HPV主要结构蛋白L1的表达水平是影响生产效率的主要因素,L2表达质粒的用量过高或过低均不利于假病毒的生产,而报告质粒的用量对假病毒生产效率的影响相对较小.综合比较显示,在该体系中L1和L2表达质粒和报告质粒以合适比例(如1:1/10:1/2)进行共转染可获得更为理想的生产效率.本研究同时也在293FT细胞中对磷酸钙转染方法进行了优化,通过磷酸钙与质粒用量的交叉配比试验获得了较优的转染条件.通过优化,建立了更为高效的HPV假病毒大量制备方法,在HPV16、HPV18、HPV6、HPV11假病毒生产中的应用结果显示较原方法可显著提高生产效率.本研究为HPV假病毒中和实验的规模化应用提供了有利条件.     

构建逆转录病毒PBabepuro-Bcl-2表达质粒及稳定转染细胞株

目的:构建pBabepuro-Bcl-2逆转录病毒表达质粒,鉴定表达,在小鼠胚胎成纤维细胞NIH3T3中建立过表达Bcl-2稳定转染细胞。方法:以人上皮细胞的cDNA为模板,利用X-tremeGENE HP(Roche)转染293T细胞,蛋白质免疫印迹法在蛋白水平检测表达,利用逆转录病毒感染NIH3T3细胞。结果:成功构建了逆转录病毒表达质粒pBabepuro-BCl-2,蛋白水平检测证实其在293T细胞可以有效地表达,成功建立了过表达Bcl-2的NIH3T3稳定转染细胞。结论:通过构建pBabepuro表达质粒及在NIH3T3建立过表达Bcl-2稳定转染细胞,为进一步研究Bcl-2的功能调控奠定了基础。     

293T细胞转染表达ARG1及其对砷代谢的影响

研究含有ARGI基因的真核表达载体PcDNA3.1-IE-ARG1转染真核细胞后,在细胞中的表达及对细胞砷代谢的影响。由脂质体介导将PcDNA3.1-IE-ARG1转染入293T中,用real-timePCR方法检测ARG1基因的表达,用原子吸收分光光度法测定24h砷染毒后细胞内砷含量及细胞排砷量。重组质粒成功转入293T细胞中且表达良好;与转染前细胞比较,转染重组质粒的细胞在不同浓度砷染毒下细胞内砷含量明显降低(P0.05),并且细胞的排砷量也高于转染前的;此外,转染表达ARGI基因的细胞在砷染毒48h后其内GSH及GST的含量高于转染前细胞。这些表明,ARG1基因在真核细胞中相对高表达后在砷代谢排出中发挥了一定的作用。     

PEI介导pCEP4/EGFP瞬时转染293-F细胞条件优化

为构建真核表达载体pCEP4/EGFP,PEI介导瞬时转染293-F细胞,观察转染效率,优化转染条件.通过NotI和Hind III酶切位点将增强型绿色荧光蛋白基因(EGFP)插入真核表达载体p CEP4中,并转化Trans5α感受态细胞,氨苄青霉素筛选培养,挑取阳性克隆扩增后提取质粒,酶切鉴定正确后,PEI介导转染293-F细胞,流式细胞仪检测转染效率.实验结果表明,成功构建了真核表达载体pCEP4/EGFP,当N/P(PEI/DNA)为25,悬浮培养模式下转染293-F细胞,48 h转染效率为77.57%,重组载体p CEP4瞬时转染293-F细胞,可获得理想转染效果.     

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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