八肋游仆虫第一类肽链释放因子eRFla多克隆抗体的制备、纯化及鉴定

将八肋游仆虫第一类肽链释放因子基因eRFla克隆入表达载体pRSETc,并在大肠杆茵B121(DE3)中进行诱导表达.表达形成的包含体经切胶回收,然后免疫大鼠制备抗血清.所获得的抗血清用Amersham Biosciences抗体纯化试剂盒进行纯化,SDS-PAGE电泳分析表明纯化后的抗体纯度很高.Western blotting印迹和ELISA鉴定结果表明抗体特异性好,效价为1 5 000.     

表皮生长因子受体Ⅲ型变异体(EGFRvⅢ) 多克隆抗体的制备及特异性

为了制备针对EGFRvⅢ的特异性多克隆抗体,在实验中利用耦联了血蓝蛋白的EGFRvⅢ特异性的PEP3合成短肽进行动物免疫,制备抗血清;构建了pET30a/E33XI原核表达载体,表达和纯化了融合蛋白His-E33(EGFRvⅢ的部分胞外区);以此融合蛋白作为抗原制备了抗体纯化柱,通过柱上纯化的方法从抗血清中提取针对EGFRvⅢ的特异性抗体.利用U87、U251、HeLa和HEK-293细胞进行免疫组化实验来检验抗体的特异性.     

抗人PD-1高亲和力抗血清的制备及其在PD-1免疫印迹分析中的应用

利用纯化的可溶性人PD-1胞外域(sPD-1)蛋白为免疫原免疫小鼠,获得抗血清.免疫印迹显示该抗血清与原核表达的sPD-1有特异性反应,但与某些菌体蛋白存在交叉反应.经菌体蛋白吸附法处理后,交叉反应明显减弱.以商业化抗人PD-1抗体和小鼠sPD-1抗血清分析经亲和层析富集的活化的Jurkat T细胞表达的PD-1,只有自制的抗血清鉴定到特异反应条带,其表观分子质量为49 ku.     

重组猪生长激素抗体的制备与纯化

为了获得重组猪生长激素抗体,对重组猪生长激素融合基因的真核表达产物进行免疫印迹(Western blot)检测,将利用DNA重组技术构建的重组质粒pPGH020在大肠杆菌(E.coli)BL21中进行诱导表达.表达产物经Ni+亲和层析柱纯化,获得纯化的重组猪生长激素融合蛋白.然后以此为抗原免疫新西兰大白兔,获得多克隆抗体,抗体再经硫铵沉淀、透析和亲和层析纯化.Western印迹结果表明,该纯化抗体显示了良好的免疫反应,其灵敏度比兔抗rpGH抗血清提高50倍,为猪生长激素融合基因在真核细胞的表达及功能鉴定奠定了基础.     

AtPCS1基因的原核表达及多抗血清的制备

利用RT-PCR扩增拟南芥螯合肽合成酶(AtPCS1)基因全序列,构建原核表达载体pET28a-AtPCS1并测序.将该载体转化大肠杆菌BL21,用0.75mmol/LIPTG诱导融合蛋白AtPCS1-His的表达,纯化该融合蛋白.用纯化的融合蛋白免疫小鼠,ELISA法测定抗血清的效价可达1:2000.抗血清与融合蛋白及拟南芥总蛋白的western-blot结果表明:制备的抗血清具有较高的活性和特异性.     

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.     

用多克隆抗体检测干扰素调节因子7的表达

干扰素调节因子7(IRF-7)是调节Ⅰ型干扰素依赖型先天免疫反应的关键因子,在真核细胞防御反应中发挥重要作用。本文在大肠杆菌中表达了重组IRF-7,利用亲和层析的方法进行了纯化,并制备了鼠抗IRF-7的多克隆抗体。所得到的抗血清能够在稀释27 000倍后成功用于免疫印迹检测。该抗血清不但能识别来源于大肠杆菌的抗原,还能检测真核细胞内转染后表达的IRF-7。使用该抗体证实,转染293T细胞后表达的IRF-7主要分布在细胞质内。所制备的抗血清可用于IRF-7的表达和功能研究。     

人细胞色素P450氧化还原酶的克隆表达和抗体的制备

细胞色素P450还原酶(CPR)是人细胞色素P450酶系的电子供给者,对后者活性的发挥起到重要作用.本研究将从人胚胎cDNA文库中得到的人CPR的编码基因,连接入pT7450表达载体中,在大肠杆菌BL21(DE3)中高效表达并纯化.每升发酵液可得到纯化蛋白49 mg,占总蛋白含量的10%,以细胞色素C为底物检测酶活性,比活为65 u/mg.利用纯化的CPR作为抗原,常规免疫纯系大耳家兔,获得高效价、高特异性的抗血清,抗体滴度为1∶200 000(ELISA法),纯化后抗体应用于不同组织中CPR含量的评价,证明重组CPR及其抗体可用于细胞色素P450的体外药物代谢及细胞色素P450与CPR作用机理的研究.     

Daxx-C抗血清的制备与纯化及初步应用

将含有Daxx-C端的融合蛋白6His-DM626-740纯化物免疫小鼠,分析抗血清效价并将其纯化,为进一步分析Daxx在人乳头瘤病毒16型(HPV16)阳性宫颈癌组织中的分布奠定基础。将6His-DM626-740纯化物以皮下多点注射和腹腔注射方式免疫接种4只8周龄雌性BALB/c小鼠,初次免疫3周后,加强免疫1次,以后每周加强免疫1次;共免疫4次。每次免疫前及末次免疫后7天取血,ELISA测定抗体效价。饱和硫酸铵盐析沉淀法初步纯化抗血清,并用Western blot检测之。培养Caski细胞,利用间接免疫荧光试验观察Daxx在细胞中的分布与定位。纯化物免疫小鼠后,制备的Daxx-C多克隆抗体效价为1:6400。纯化的抗血清能够与IPTG诱导表达的6His-DM626-740及其纯化物发生结合反应。在Caski细胞,显绿色信号的Daxx主要分布于胞浆,少数分布于胞核且在核膜附近荧光较强,呈区域性聚集。6His-DM626-740纯化物具有良好的免疫原性;纯化的抗血清可用来检测细胞中的Daxx。     

兔抗Red蛋白抗血清的纯化及MC3T3细胞中Red蛋白表达时相的分析

为了纯化兔抗Red蛋白的3种多克隆抗体,并用其研究Red蛋白在真核细胞中的表达时相,首先于30℃培养了不含H的基因的空载体（pDH2）大肠菌,经彻底裂菌后用丙酮沉淀仝菌蛋白,用其在不同时间吸附3种待纯化的Red抗体,然后用间接ELISA法及Western-blot法检测r3种抗体的效价和特异性。最后州纯化后的3种Red抗体检测三顺反子表达载体pCMV-glblx的3种蛋白表达产物在MC3T3细胞中的表达时相。EUSA及Western-blot检测结果表明：3种抗血清经纯化后特异性好且效价没有受到影响,随后用纯化的抗体成功地研究了Red基因在真核细胞中的表达时相,此结果为Red重组系统在真核生物基因功能的研究中提供了必要的条件。     

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.     

Lower Paleozoic oil relationships within Williston Basin, Canada

The Williston Basin is a significant petroleum province, containing oil production zones that include the Middle Cambrian to Lower Ordovician, Upper Ordovician, Middle Devonian, Upper Devonian and Mississippian and within the Jurassic and Cretaceous. The oils of the Williston Basin exhibit a wide range of geochemical characteristics defined as "oil families", although the geochemical signature of the Cambrian Deadwood Formation and Lower Ordovician Winnipeg reservoired oils does not match any "oil family". Despite their close stratigraphic proximity, it is evident that the oils of the Lower Palaeozoic within the Williston Basin are distinct. This suggests the presence of a new "oil family" within the Williston Basin. Diagnostic geochemical signatures occur in the gasoline range chromatograms, within saturate fraction gas chromatograms and biomarker fingerprints. However, some of the established criteria and cross-plots that are currently used to segregate oils into distinct genetic families within the basin do not always meet with success, particularly when applied to the Lower Palaeozoic oils of the Deadwood and Winnipeg Formation.     

An Analysis of Catherine＇s Character——Wuthering Heights

Wuthering Heights, Emily Bronte＇s only novel, was published in December of 1847 under the pseudonym Ellis Bell. The book did not gain immediate success, but it is now thought one of the finest novels in the English language. Catherine is the key character of this masterpiece, because everybody and everything center on her though she had a short life. We can understand this masterpiece better if we know Catherine well.     

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.     

Design and analysis of generic LBS application developing platform

Location based services is promising due to its novel working style and contents.A software platform is proposed to provide application programs of typical location based services and support new applications developing efficiently. The analysis shows that this scheme is easy implemented, low cost and adapt to all kinds of mobile nework system.     

橡胶颗粒沥青混合料低温抗裂性试验研究

以AC-13级配为基础,将橡胶颗粒代替部分集料掺入混合料中,以低温弯曲试验为评价方法对不同橡胶颗粒掺量下沥青混合料的低温抗裂性进行研究,并引入应变能密度值对混合料的低温抗裂性进行综合评价.试验结果表明:橡胶颗粒沥青混合料试件的破坏微应变均超过2 300,满足冬寒区的技术指标;无论是否掺加橡胶颗粒,随着温度的下降,沥青混合料破坏时的最大弯拉强度增大,弯拉应变降低,劲度模量增大;弯曲应变能密度在胶粒掺量为1%左右时具有较大的弯曲应变能密度值,此时橡胶颗粒沥青混合料具有较好的低温抗裂性.     

A computer generator for randomly layered structures

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

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。     

Quantitative trait loci （QTLs） for quality traits related to protein and starch in wheat

Quality traits in wheat （Triticum aestirum L.） were studied by quantitative trait locus （QTL） analysis in a recombinant inbred line （RIL） population, a set of 131 lines derived from Chuan 35050 × Shannong 483 cross （ChSh）. Grains from RILs were assayed for 21 quality traits related to protein and starch. A total of 35 putative QTLs for 19 traits with a single QTL explaining 7.99-40.52% of phenotypic variations were detected on 10 chromosomes, 1D, 2A, 2D, 3B, 3D, 5A, 6A, 6B, 6D, and 7B. The additive effects of 30 QTLs were positive, contributed by Chuan 35050, the remaining 5 QTLs were negative with the additive effect contributed by Shannong 483. For protein traits, 15 QTLs were obtained and most of them were located on chromosomes 1 D, 3B and 6D, while 20 QTLs for starch traits were detected and most of them were located on chromosomes 3D, 6B and 7B. Only 7 QTLs for protein and starch traits were co-located in three regions on chromosomes 1D, 2A and 2D. These protein and starch trait QTLs showed a distinct distribution pattern in certain regions and chromosomes. Twenty-two QTLs were clustered in 6 regions of 5 chromosomes. Two QTL clusters for protein traits were located on chromosomes 1D and 3B, respectively, three clusters for starch traits on chromosomes 3D, 6B and 7B, and one cluster including protein and starch traits on chromosome 1D.