三聚氰胺多克隆抗体的制备及鉴定

用三聚氰胺偶联蛋白为抗原,按不同的时间规律及抗原剂量对成年兔进行免疫,获得特异性的三聚氰胺多克隆抗体.效价检验应用蛋白质电泳和间接ELISA方法.应用10%及12.5% SDS-PAGE,验证血清中IgG抗体的产生情况,再用间接酶联免疫吸附试验测得第4次免疫后抗体效价达到1.2×107.此法易于实施,且获得的血清样品具有较高的效价,为今后三聚氰胺多克隆抗体的分离纯化提供了可靠的实验依据.     

VMP1基因的克隆及其抗体的制备与鉴定

为了得到效价高、特异性强、能用于检测内源性 VMP1 蛋白的抗体,采用RT-PCR 法,从L929细胞株中克隆鼠VMP1基因,重组入pCMV5载体,用 PCR 扩增与其氨基酸 132-239 区域对应的DNA片断,将该片段连接到原核表达载体 pGST parallel 中,在大肠杆菌 BL21 菌株中大量表达,经谷胱甘肽-琼脂糖树脂纯化后,得到高纯度的 VMP1抗原.免疫新西兰兔,获得抗 VMP1 蛋白的兔抗血清,将血清纯化后即得到抗 VMP1 的多克隆抗体.用 Western blot 分析手段检测了该抗体的特异性及效价.在用该抗体检测外源性和内源性 VMP1 蛋白的试验中,证实该抗体效价高,特异性强,效果很理想.此方法可用于获得大量廉价的抗 VMP1 蛋白的高滴度和高特异性的抗体,为进一步研究 VMP1 在细胞内,尤其是在信号转导通路中的功能和作用奠定了基础.     

E-cadherin前体蛋白抗体的制备与鉴定

E-cadherin是一种重要的肿瘤转移抑制因子,它的功能异常能够导致肿瘤细胞的转移.细胞内成熟的E-cadherin由其前体蛋白剪切而成,目前还没有针对该前体蛋白的特异性抗体.为了得到能用于检测内源性E-cadherin前体蛋白(E-cadherin pre)的抗体,对E-cadherin基因用限制性内切酶PstⅠ和PvuⅡ进行双酶切,获取E-cadherin基因的N-端片段,将其重组入原核表达载体pGEX-4T-2中,在大肠杆菌BL21(DE3)菌株中表达重组蛋白,经谷胱甘肽-琼脂糖珠子纯化后,得到较高纯度的E-cadherin前体抗原.用该抗原免疫新西兰兔,获得抗血清,进一步纯化血清得到抗E-cadherinpre的多克隆抗体.用Western blot检测了外源性和内源性E-cadherin pre,发现该抗体效价高且特异性强;免疫荧光实验表明该抗体可以用于细胞染色,为深入研究E-cadherin pre在细胞内的功能奠定了基础.     

兔抗PEB1多克隆抗体的制备及鉴定

摘要：【目的】利用纯化的空肠弯曲菌表面蛋白PEB1,制备其多克隆抗体。【方法】用PEB1蛋白作为抗原免疫兔,分离血清得到兔抗PEB1多克隆抗体,用ELISA法检测抗体效价,并用Western-Blotting验证。【结果】PEB1蛋白免疫兔后,得到了兔抗PEB1多克隆抗体,间接ELASA法检测表明兔抗PEB 1血清效价达到1：10 000,Western印记检测表明,该抗体能与PEB1蛋白发生特异性结合,其特异性与敏感性均较好。【结论】获得了高效价的PEB1多克隆抗体。     

兔抗Myosin Ⅹ多克隆抗体的制备及初步应用

为制备兔抗肌球蛋白Ⅹ(myosinⅩ,MyoⅩ)多克隆抗体,合成了含有MyoⅩN端1 000～1 021个氨基酸的多肽,将纯化后的多肽与钥孔戚血蓝蛋白(keyhole limpethemocyanin,KLH)进行交联;通过背部皮下免疫新西兰大白兔获得免疫血清,并通过ELISA、免疫印迹、免疫荧光等分析对免疫血清进行了初步的验证.结果表明:经过4次免疫后可获得免疫血清,ELISA检测表明抗体终效价达到1∶51 200,抗原吸附实验进一步证明了此多克隆抗体的特异性.利用获得的多克隆抗体检测了多种组织中MyoⅩ的表达,发现兔抗MyoⅩ多克隆抗体可特异性识别多种组织中的MyoⅩ.免疫荧光染色表明该抗体可以显示MyoⅩ在COS-7细胞、NLT细胞和神经元中的分布.     

小鼠肥大细胞蛋白酶4的原核表达和多克隆抗体制备

文章构建小鼠肥大细胞蛋白酶4（mMCP-4）原核表达体系,获得原核表达蛋白,并制备兔抗mMCP-4多克隆抗体,提取小鼠脾细胞总 RNA ,运用 RT-PCR技术获得目的基因 mMCP-4,构建重组原核表达载体pET28a-mMCP-4,将其转化到大肠杆菌BL21中诱导表达。mMCP-4融合蛋白经Ni亲和层析法纯化后,作为抗原免疫兔子获得mMCP-4多克隆抗体。采用ELISA法测抗体效价,western blot检测抗体特异性。结果表明,构建重组表达质粒pET28a-mMCP-4,经大规模原核表达获得大量mMCP-4融合蛋白,制备的兔抗血清效价达1∶128000,并表现出较好特异性。研究结果为进一步研究mMCP-4生物学功能奠定了基础。     

左氧氟沙星人工抗原的制备及其抗体鉴定

用N-羟基琥珀酰亚胺法把左氧氟沙星与载体蛋白牛血清白蛋白偶联制备免疫抗原,用氯甲酸异丁酯法将左氧氟沙星与鸡卵清蛋白偶联,制备包被检测抗原,采用紫外扫描法对合成的抗原进行鉴定.用免疫抗原免疫大白兔获得抗左氧氟沙星多克隆抗体血清,经间接ELISA试验检测特异性抗体效价,测得效价达1:212以上.通过紫外扫描及间接ELISA试验,结果表明,制备的抗原和抗体获得成功.     

VMP1基因的克隆及其抗体的制备与鉴定

为了得到效价高、特异性强、能用于检测内源性VMP1蛋白的抗体，采用RT-PCR法，从L929细胞株中克隆鼠VMP1基因，重组入pCMVS载体，用PCR扩增与其氨基酸132-239区域对应的DNA片断，将该片段连接到原核表达载体pGST parallel中，在大肠杆菌BL21菌株中大量表达，经谷胱甘肽-琼脂糖树脂纯化后，得到高纯度的VMP1抗原．免疫新西兰兔，获得抗VMP1蛋白的兔抗血清，将血清纯化后即得到抗VMP1的多克隆抗体．用Western blot分析手段检测了该抗体的特异性及效价．在用该抗体检测外源性和内源性VMP1蛋白的试验中，证实该抗体效价高，特异性强，效果很理想．此方法可用于获得大量廉价的抗VMP1蛋白的高滴度和高特异性的抗体，为进一步研究VMP1在细胞内，尤其是在信号转导通路中的功能和作用奠定了基础．     

兔抗人多发性骨髓瘤细胞多克隆抗体的制备与鉴定

制备人多发性骨髓瘤全细胞兔多克隆抗体。用人多发性骨髓瘤细胞系ARH-77全细胞和福氏完全佐剂通过背部皮内多点注射首次免疫新西兰大白兔,第14 d用ARH-77全细胞和福氏不完全佐剂同样剂量加强免疫,第28 d和38 d时再次免疫。第45 d颈动脉采血80 mL,4℃静置过夜,收集血清。然后采用亲和层析系统纯化血清中的多克隆抗体。用酶联免疫吸附试验(ELISA)检测纯化多克隆抗体的效价;免疫印迹法和荧光免疫细胞化学检测纯化多克隆抗体的特异性。结果:ELISA检测多克隆抗体滴度为1∶20 000,免疫印迹检测结果显示该抗体具有较高特异性,荧光免疫细胞化学检测多克隆抗体能够有效的结合细胞表面抗原。实验获得了高效价的特异性的兔抗人骨髓瘤细胞多克隆抗体,为进一步研究多发性骨髓瘤诊断和治疗奠定了基础。     

绿色荧光蛋白GFP多克隆抗体的制备及在多种细胞中的特异性鉴定

绿色荧光蛋白是当前在生物及生物医学研究当中应用最为广泛的一种荧光标记蛋白.为开发一种高灵敏度、高特异性以及简单经济的GFP多克隆抗体,并使之适用于在跨物种细胞里表达的GFP蛋白进行检测,本文利用gfp基因cDNA全序列分别构建了带有6×His和GST标签的原核表达载体pET28A-gfp和p GEX-2T-gfp,并将其转入大肠杆菌(Escherichia coli)BL21(DE3)细胞进行诱导表达,而后进行聚丙烯酰胺凝胶电泳(SDS-PAGE)表达鉴定,分别获得了相对分子质量为2.6×10~4和5.2×10~4的重组6×His/GST-GFP融合蛋白.将亲和纯化的6×His-GFP融合蛋白免疫新西兰大白兔,采集第5次免疫后血清用ELISA测定效价为1﹕32,000.抗血清依次经Protein A亲和纯化和GST-GFP抗原抗体纯化后,用免疫印迹实验(Western blot)方法检测抗体特异性,结果表明制备的多克隆抗体能很好地识别在莱茵衣藻(Chlamydomonas reinhardtii)、HEK293细胞和大肠杆菌中表达的GFP,对继续开展GFP相关研究具有重要意义.     

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-