米根霉L-乳酸脱氢酶基因的克隆及在大肠杆菌中的表达

文章以米根霉基因组DNA为模板,根据已公布的米根酶L-乳酸脱氢酶基因(ldnL)序列设计引物,PCR扩增得到含有ldnL的DNA片段;PCR产物T/A克隆后再双酶切,将ldnL片段连接到大肠杆菌表达载体pET17b,得到重组表达质粒pET17b-ldnL;pET17b-ldnL转化大肠杆菌BL21(DE3),摇瓶培养诱导表达后的菌体经SDS-PAGE电泳分析,结果表明克隆的ldnL基因在大肠杆菌中实现表达。     

粉尘螨Der f15的基因克隆与其表达载体的构建

先挑取纯培养的粉尘螨,提取总的RNA,后采用RT-PCR方法进行反转录出cDNA.由cDNA提取出目的基因进行片段扩增,产物连接入T载体(pMD18-T).经扩增后,用EcoR I和Xho I双酶切,将目的基因分别连接到pET28和pET32的表达载体上,得到重组质粒pET28和pET32,即粉尘螨的基因克隆与 表达载体构建成功.     

播娘蒿DsCOR基因的原核表达、蛋白纯化、及性质研究

利用PCR方法扩增DsCOR基因的蛋白编码序列，经BarnH Ⅰ、Sac Ⅰ酶切后连接入pET32a原核表达载体，构建的pET32a—DsCOR融合重组表达质粒转化大肠杆菌BL21进行原核表达．建立了“煮沸-镍离子亲和层析”的蛋白纯化方法．对纯化的DsCOR蛋白进行高温耐受性、pH耐受范围、紫外耐受性方面的研究．     

人酸性成纤维细胞生长因子的高效表达

构建能高效表达人酸性成纤维细胞生长因子（haFGF)的基因工程菌,优化产物分离纯化条件,并对纯化产物进行理化性质鉴定,采用PCR方法扩增haFGF cDNA,将该cDNA片段插入表达载体pET3c的表达框架中,筛选得到了重组质粒pET3c-haFGF,转化大肠杆菌BL21（DE3）,构建了表达菌件BL21（DE3）／pET3c-haFGF.IPTG诱导表达,通过离子交换,肝素亲和两步层析的方法分离纯化产物,用MTT法测定产物的活性,haFGF的表达量约为20％,经过两步层析纯化,获得了电泳纯的haFGF样品,纯化haFGF样品的比活性为ED50小于4.0ng/mL.BL21(DE3)/pET3c表达系统能高效表达haFGF,经过纯化得到了理化性质及生物活性与天然haFGF对照品一致的重组人酸性成纤维细胞生长因子。     

建立利用萤光素酶快速检测细胞活性的方法

运用PCR的方法,从萤火虫萤光素酶基因载体 pGL4.26 扩增萤火虫萤光素酶基因片段,将其插入连接于原核表达载体pET24a 中,构建重组表达载体pET24a-Luc.经酶切鉴定及序列分析后,将重组载体转化到表达菌株大肠杆菌BL21（DE3） 中,获得阳性重组菌BL21/pET24a-Luc.IPTG诱导蛋白高效表达并通过镍柱亲和层析纯化萤火虫萤光素酶.该目的蛋白活性用Bright-GloTM试剂进行验证并用于建立一种基于测量ATP含量的检测细胞生物活性的方法.与传统的细胞生物活性检测试剂盒MTT,CCK-8以及Alamar Blue比较,该方法具有反应迅速、活力高、灵敏度好、生产方便的优点,具有实际应用的潜力.     

exendin-4表达载体构建及其在大肠杆菌中分泌表达

目的:研究新型抗糖尿病药物exendin-4的分子作用机制,为exendin-4的大规模生产奠定基础.方法:采用重叠PCR法扩增出exendin-4的完整序列,并将其克隆至表达载体pET22b上,得到重组质粒pET-Exn4.重组质粒转化入大肠杆菌BL21(DE3)中,经IPTG诱导表达后,超声破碎表达产物,Tricine-SDS-PAGE分析exendin-4表达.结果:PCR、酶切鉴定和测序结果证明成功构建了重组表达载体pET-Exn4,Tricine-SDS-PAGE结果表明ex-endin-4基因在大肠杆菌中获得了分泌表达.结论:成功构建了exendin-4的重组表达载体并在大肠杆菌中实现分泌表达.     

棉花脱氢抗坏血酸还原酶基因的克隆、原核表达与纯化

为进一步探讨脱氢抗坏血酸还原酶的生物学功能,克隆棉花的脱氢抗坏血酸还原酶基因,对该基因进行了原核表达,并对重组蛋白进行纯化和分析。通过RT-PCR方法扩增脱氢抗坏血酸还原酶基因全长,利用BamH I和Xhol I酶切位点将其克隆至组氨酸(histidine,His)融合蛋白表达载体pET28a中,转化大肠杆菌BL21(DE3)后,经异丙基硫代--βD-半乳糖苷(isopropy--βD-5-thiogalactoside,IPTG)诱导表达,用SDS-PAGE鉴定表达产物,并用亲和层析柱纯化重组表达的pET28a-DHAR蛋白。结果表明:重组体PET28a-DHAR经测序和酶切鉴定证实构建成功。导入大肠杆菌BL21进行表达,SDS-PAGE分析目的蛋白高效表达,相对分子量为26 kD左右,并获得了纯化的His-DHAR融合蛋白。     

IGF-Ⅱ在原核生物中的表达

胰岛素生长因子IGF-Ⅰ和IGF-Ⅱ在机体的许多组织中都有表达,具有促进细胞生长和分裂等的多种生物学功能。在本研究中,我们采用RT-PCR法克隆和扩增到了IGF-Ⅱ基因,通过双酶切、胶回收纯化、连接后得到了pET21b-IGF-Ⅱ重组质粒载体,然后将其转化DH5α中,经IPTG诱导后表达的重组蛋白经纯化,并用Western blot检测,显示重组质粒pET21b-IGF-Ⅱ编码一个约25kD的外源蛋白。     

家蚕抗菌肽CM4和人可溶性BAFF融合基因的克隆、表达及活性鉴定

采用PCR法从含有人可溶性BAFF基因的pET30a( )扩增得到人sBAFF基因,通过rPCR将抗菌肽Cecropin CM4基因的2条单核苷酸链进行扩增得到CM4基因,再采用over-lap PCR法通过linker将hsBAFF与Cecropin CM4融合基因相连接.经纯化和鉴定后,定向插入到原核表达载体pET30a( )中,然后转化E.coliBL21(DE3),通过实验确定了表达该融合基因的最佳诱导条件:IPTG终浓度为1.0 mmol/L,诱导时间为5 h.温度为30℃,其表达量占全菌蛋白的40%.表达产物经SDS-PAGE分析,得到相对分子质量约为22 000的重组蛋白并且存在超声裂解后的上清中.重组蛋白经Western blot检测,结果显示重组蛋白可被鼠抗人可溶性BAFF的抗体识别.采用分子筛Sephadex G-75对重组融合蛋白进行纯化,并经SDS-PAGE对其鉴定.通过对其生物学功能的检测得知,纯化后的重组融合蛋白对大肠杆菌K12D31和真菌有明显的抑菌能力.     

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.     

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。     

A computer generator for randomly layered structures

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

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.     

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.     

海南岛地体及其毗邻陆缘晚中生代—新生代古地磁研究和构造演化

本文叙述了对海南岛及其毗邻大陆边缘白垩纪到第四纪地层岩石进行古地磁研究的全部工作过程。通过分析岩石中剩余磁矢量的磁偏角及磁倾角的变化,提出海南岛白垩纪以来经历的构造演化模式如下:早期伴随顺时针旋转而向南迁移,后期伴随逆时针转动并向北运移。联系该地区及邻区的地质、地球物理资料,对海南岛上述的构造地体运动提出以下认识:北部湾内早期有一拉张作用,主要是该作用使湾内地壳显著伸长减薄,形成北部湾盆地。从而导致了海南岛的早期构造运动,而海南岛后期的构造运动则主要是受南海海底扩张的影响。海南地体运动规律的阐明对于了解北部湾油气盆地的形成演化有重要的理论和实际意义。     

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.     

《Chinese Science Bulletin》SUBJECT INDEX TO VOLUME 59(2014)

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