用于骨移植的hCbfa1重组腺病毒载体的构建

克隆hCbfa1的cDNA基因,构建其重组腺病毒载体,以用于研究hCbfa1腺病毒载体在人造骨移植中的作用.将hCbfa1的cDNA目的片段插入腺病毒柯氏质粒PAxCAwt中,再将此质粒与腺病毒DNA末端蛋白复合物共转染293个细胞,通过同源重组构建hCbfa1重组腺病毒载体.重组得到的阳性克隆经酶切、测序鉴定正确,包装纯化后,检测得病毒滴度为2×109PFU/mL,从而成功地构建了hCbfa1重组腺病毒载体,为下一步应用到人造骨移植的转基因治疗打下了基础.     

GGPPS基因干扰腺病毒载体的构建及鉴定

构建GGPPS基因干扰腺病毒质粒载体并加以鉴定.根据GGPPS基因序列设计GGPPS干扰序列引物,定向克隆至穿梭载体pshuttle-H1的BglⅡ和HindⅢ位点,PmeⅠ酶切线性化的pShuttle-H1-SiGGPPS干扰质粒,并与腺病毒载体(pAdEasy-1质粒)共同转化E.coli BJ5183感受态细菌,产生重组腺病毒载体.用PacⅠ酶切线性化的回收质粒,转染293A细胞包装腺病毒颗粒,在倒置显微镜下观察细胞CPE,用TCID50法测定病毒颗粒的浓度,并初步观察病毒感染PC12细胞对目的基因的干扰效率.经酶切鉴定、测序证实成功构建GGPPS基因干扰腺病毒载体.包装的腺病毒浓缩悬液滴度为1.995×107PFU/mL.GGPPS在人中具有保守性,该病毒能在人源的WRL-68细胞中成功表达,并且对GGPPS基因干扰效率达70%以上.     

重组腺病毒介导RNA干扰人DLK1基因表达的研究

构建抑制人DLK1基因表达的重组腺病毒载体,利用腺病毒载体介导的RNA干扰技术评价其在肝癌细胞株中的基因沉默效应.将针对人DLK1基因的RNAi寡核苷酸序列,连接到腺病毒穿梭质粒中,在含有腺病毒骨架质粒pAdEasy-1的大肠杆菌BJ5183内进行同源重组.重组腺病毒载体在HEK-293细胞中包装扩增,得到高滴度的重组腺病毒.通过绿色荧光蛋白示踪腺病毒的感染效果,并通过荧光实时RT-PCR,western blot的方法证实重组腺病毒能够显著抑制DLK1基因在肝癌细胞株中的表达.     

汉滩病毒囊膜糖蛋白g2基因重组腺病毒的构建与表达

获得汉滩病毒G2 基因 ,构建其重组腺病毒并在HEK2 93细胞中包装表达 ,为研究汉滩病毒基因疫苗提供了实验基础。设计引物采用PCR从含汉滩病毒 \|76 1 1 8株M基因的M5 6质粒扩增出糖蛋白G2 基因片段 ,并将其克隆入腺病毒载体Adeno XviralDNA ,筛选获得重组腺病毒DNA ,转染HEK2 93细胞 ,包装、扩增后得到汉滩病毒G2 基因重组腺病毒原种 ;并在感染细胞内初步表达 ,用ELISA检测表达产物。得到了含汉滩病毒G2 基因的重组腺病毒 ,其滴度约为 1 0 10 pfu/mL ,同时在感染的HEK2 93细胞中检测到汉滩病毒糖蛋白G2 的表达。含汉滩病毒糖蛋白G2 基因重组腺病毒的成功构建 ,为研究汉滩病毒基因疫苗提供了实验基础     

Ad-PML的构建及表达的实验研究

将PSG-CMV和PSG5-PML分别用EcoRI和BglII双酶切,构建PSG-PML载体.将质粒PSG-PML与含有型腺病毒右臂的质粒pPE3共转染至293细胞,产生含PML的重组腺病毒(Ad-PML).酶切、PCR、测序鉴定结果表明成功构建了PML的重组腺病毒,滴度达到5×107PFU/ml,并检测到PML蛋白的表达.     

汉坦病毒CTL多表位重组腺病毒的构建及免疫学分析

目的：构建含汉坦病毒M基因（编码糖蛋白G1）和部分S基因（编码核蛋白主要抗原区段）以及S基因的多个 CTL表位的多种重组腺病毒表达载体,并对这些重组腺病毒的免疫学特性进行研究。方法：构建含目的基因的重组腺病毒载体,并对其表达产物进行鉴定。利用纯化后的重组腺病毒免疫BALB/C小鼠,通过多种免疫学方法检测其免疫反应。结果：成功表达出可被汉坦病毒核蛋白特异性单抗(mAb)及糖蛋白G1 的特异性单抗所识别的融合蛋白;重组的腺病毒可以有效刺激针对汉坦病毒NP及GP的免疫应答;同时结果表明在CTL多表位间加入间隔序列AAY的重组腺病毒免疫小鼠能产生更高的细胞免疫应答。结论： 构建的含CTL多表位的重组腺病毒可以有效的提高嵌合基因刺激细胞免疫应答的能力。间隔序列AAY对于各CTL表位的分隔有效地提高了重组腺病毒的免疫效果。     

靶向hTERT的shRNA腺病毒载体的构建及其对MCF-7细胞hTERT表达的影响

设计针对人端粒酶逆转录酶催化亚基(hTERT)的干扰靶序列GGAACACCAAGAAGTTCATCT,构建siRNA表达质粒PgenesilhTERT;随后将shRNA表达框克隆到入门载体pENTRTM1A,构建重组质粒pENTR/U6-hTERT-polyA;使用同源重组方式在体外将该表达框重组到腺病毒载体pAd/PL-DEST,得到重组腺病毒质粒pAd/U6 –TERT-polyA;线性化的重组腺病毒质粒在HEK 293细胞内包装为具有感染能力的病毒颗粒rAd-hTERT.同时构建不针对任何基因的shRNA阴性对照rAd-HK,空病毒对照rAd-blank和只含EGFP的rAd -EGFP.四种病毒经酶切、电泳分析表明插入序列正确,腺病毒载体构建成功.Western blotting测定转染后各组hTERT蛋白的表达情况,证实转染rAd-hTERT 48 h后,hTERT的表达明显被抑制.实验表明基于Gateway技术的腺病毒介导的shRNA载体构建成功,rAd-hTERT可有效抑制乳腺癌MCF-7细胞hTERT基因的表达.本研究为针对端粒酶的基因治疗奠定了实验基础.     

利用Gateway技术构建天山雪莲cDNA表达文库

以天山雪莲为材料,利用gateway技术构建天山雪莲cDNA表达文库。从经低温处理后的天山雪莲叶片中提取并分离mRNA,合成双链cDNA;cDNA经BP重组反应重组到入门载体pDONR222上,构建得到天山雪莲en-try cDNA文库;天山雪莲entry cDNA再经LR重组反应将雪莲cDNA重组到植物表达载体pLEELA上,从而构建了天山雪莲cDNA表达文库。结果表明:cDNA入门文库滴度达到1.2×107cfu/mL,文库总容量为4.8×107cfu。表达文库的滴度为6.9×106cfu/mL,文库总容量为2.76×107cfu,插入片段平均大小1000 bp。阳性克隆率100%。天山雪莲cDNA表达文库的建立为进一步高通量筛选天山雪莲抗寒基因奠定了基础。     

HDAC1重组腺病毒载体的构建及鉴定

利用Ad-Easy腺病毒表达系统构建含有人源性组蛋白去乙酰化酶1(HDAC1)基因的重组腺病毒,并检测其在脐带间充质干细胞(h UC-MSCs)中的表达及对h UC-MSCs增殖的影响.RT-PCR扩增人HDAC1 ORF序列,构建p Ad Track-CMV-HDAC1重组腺病毒穿梭质粒,Pme I单切后将其转化进入E.coli BJ5183与腺病毒骨架质粒同源重组,Pac I单切线性化后转染至HEK293细胞中扩增和包装p Ad-HDAC1,采用空斑法鉴定病毒滴度;p Ad-HDAC1感染h UC-MSCs,倒置荧光显微镜下观察荧光表达,qRT-PCR和Western Blot分别检测细胞中HDAC1在mRNA水平和蛋白水平的表达,CCK-8实验检测HDAC1高表达对h UC-MSCs增殖的影响.结果显示成功扩增人HDAC1 ORF序列并构建p Ad-HDAC1重组腺病毒;病毒感染后h UC-MSCs中HDAC1 mRNA和蛋白表达水平明显增高,并且促进h UC-MSCs的增殖.成功构建HDAC1高表达的腺病毒,可有效提高h UC-MSCs中HDAC1的表达并促进细胞增殖.     

cnksr2基因干扰腺病毒的构建及鉴定

构建小鼠cnksr2基因干扰腺病毒质粒载体并加以鉴定.根据小鼠cnksr2基因序列设计cnksr2干扰序列引物,定向克隆至穿梭载体pshuttle-H1的BglⅡ和HindⅢ位点,PmeⅠ酶切线性化的pShuttle-H1-Sicnksr2干扰质粒,并与腺病毒载体(pAdEasy-1质粒)共同转化E.coli BJ5183感受态细菌,产生重组腺病毒载体.用PacⅠ酶切线性化的回收质粒,转染293A细胞,包装腺病毒颗粒,在倒置显微镜下观察细胞病理性变化(Cell pathological effect,CPE),用TCID50法测定病毒颗粒的浓度,并初步观察病毒感染PC12细胞对目的基因的干扰效率.经酶切鉴定、测序证实成功构建小鼠cnksr2基因干扰腺病毒载体.包装的腺病毒浓缩悬液滴度为3.98×107PFU/mL.cnksr2在大鼠及小鼠中具有保守性,该病毒能在大鼠来源的PC12细胞中成功表达,并且对cnksr2基因干扰效率达50%以上.结论:成功构建了小鼠cnksr2基因的干扰腺病毒载体.     

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.     

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.     

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