筛选环孢霉素A适体的SELEX技术的建立

体外合成一个全长78个核苷酸,中间含35个随机序列的随机单链寡核苷酸序列(ssDNA)文库,运用指数富集的配体系统进化(SELEX)技术,以环孢霉素A(CsA)为靶目标,以磁珠作为筛选介质,利用生物素 链酶抗生物素 辣根过氧化物酶系统,检测每轮ssDNA文库与CsA的亲和力,筛选并鉴定CsA特异性的适体.经过11轮的筛选,ssDNA文库与CsA的亲和力呈上升趋势.将第10轮筛选产物克隆测序并运用相关软件进行一级结构和二级结构分析.随机挑选的19个克隆适体,根据一级结构的同源性可分为5个家族,二级结构预测以茎环(发夹)为主,这可能是适体与CsA作用的部位. CsA特异性的适体将用于酶联法、免疫荧光法等对CsA进行检测.     

SELEX法体外筛选胃癌细胞适配子方法的建立

目的:建立SELEX技术筛选胃癌细胞SGC-7901适配子的方法,并初步鉴定获得的SGC-790 1细胞适配子.方法:体外合成全长88bp中间含52bp随机序列的ssDNA文库 ,通过优化PCR扩 增条件,利用地高辛-抗地高辛抗体-碱性磷酸酶系统测定亲和力,经SELEX反复筛选获得胃 癌SGC-7901细胞的特异性适配子.将最后一轮筛选产物克隆、测序并用相关软件分析适配子 序列的一级结构和二级结构.结果:经12轮SELEX筛选,ssDNA文库与SGC- 7901细胞的亲和力由0.16上升至1.14,表明特异性适配子得到逐步富集.22个克隆子测序, 有4个序列完全一致,二级结构预测茎环可能是适配子与胃癌细胞作用的结构基础.结论:成功建立了SELEX技术体外筛选胃癌细胞SGC-7901高亲和性适配子的方法.     

金黄色葡萄球菌外毒素B特异性适体的筛选及其应用

目的:利用指数富集配基的系统进化(SELEX)技术,筛选能与金黄色葡萄球菌外毒素B(SEB)特异、高亲和力结合的单链DNA(ssDNA)适体,并将该适体应用于患者血清标本的检测。方法:从体外合成的96核苷酸随机ss-DNA文库中,以羧基磁珠作为筛选介质,经逐步PCR扩增、筛选,获得针对SEB的高亲和力、高特异性适体;利用荧光素标记适体测定筛选过程中各轮结合力;利用酶连接适体方法检测适体特异性和结合力。结果:经过13轮筛选,ssDNA文库与SEB的结合百分率从1.1%提高到39.8%,增加了36倍;获得的ssDNA适体(A11)针对SEB的特异性强,与金黄色葡萄球菌表面蛋白A(SPA)结合低,并能初步识别患者血清。结论:利用SELEX技术筛选获得了特异结合SEB的高亲和力的ssDNA适体,为金黄色葡萄球菌的临床诊断与治疗奠定了基础。     

灭活铜绿假单胞菌适体的筛选

利用指数富集配基的系统进化(SELEX)技术,以灭活的铜绿假单胞菌为靶标,从体外合成的96 nt随机ssDNA文库中筛选与铜绿假单胞菌特异结合的适体.在第12轮和第14轮与其它假单胞菌属进行反筛策略,并进行了适体的结合亲和力的测定,再分别利用ClustalX、Mega2和Mfold sever软件分析适体的一级和二级结构.研究结果表明,经过15轮筛选,随机ssDNA文库与铜绿假单胞菌结合的A值从0.022上升到0.448.反筛与未反筛的结合A比值最高比为53倍.经过第15轮反筛后的24个阳性克隆子测序,根据软件分析,其可分成10个家族.每个家族都有其共同的保守序列（除第10个家族）,其中有2条序列几乎完全一致(F23和F47),同源性达到97%,A值分别高达1.598和1.508,Kd为14.55和77.46 nmol/L,间接说明适体与铜绿假单胞菌的结合力明显增高.     

CP4- EPSPS转基因蛋白的适体筛选与亲和性分析

目的:通过筛选获得CP4 - EPSPS转基因蛋白的最优适体,为应用于检测作准备.方法:体外合成长度为78个碱基的随机ssDNA文库,应用指数富集配基的系统进化(SELEX)技术,以CP4 - EPSPS转基因蛋白为靶目标进行15轮筛选,将筛选到的适体群进行克隆、测序,用DNAMAN软件对每个适体的保守序列和二级结构进行分析并应用生物素-抗地高辛碱性磷酸酶显色系统进行亲和性分析.结果:成功获得靶目标的最优适体,即D04号适体,结合率最高(2.2745)为最低的5.9倍.结论:筛选流程合理、可行,实验结果较理想.     

CP4-EPSPS转基因蛋白的适体筛选与亲和性分析北大核心CSCD

目的:通过筛选获得CP4-EPSPS转基因蛋白的最优适体,为应用于检测作准备。方法:体外合成长度为78个碱基的随机ssDNA文库,应用指数富集配基的系统进化(SELEX)技术,以CP4-EPSPS转基因蛋白为靶目标进行15轮筛选,将筛选到的适体群进行克隆、测序,用DNAMAN软件对每个适体的保守序列和二级结构进行分析并应用生物素-抗地高辛碱性磷酸酶显色系统进行亲和性分析。结果:成功获得靶目标的最优适体,即D04号适体,结合率最高(2.2745)为最低的5.9倍。结论:筛选流程合理、可行,实验结果较理想。     

RNA适体研究及其在医药上的应用

核酸适体（nucleic acid aptamer）是从人工合成的随机单链核酸库中筛选出的特异性与靶物质高度亲和的核酸分子,包括DNA适体和RNA适体. 体外获得核酸适体的方法称为指数富集配体系统进化技术,即SELEX（systematic evolution of ligands by exponential enrichment）. 在SELEX技术获得的核酸适体中,RNA适体因其结构的多样性而具有靶分子广、亲和力高、特异性强等特点. 同时,相比传统抗体,RNA适体分子量小、易改造修饰、制备方便且无免疫原性. 因此,RNA适体在基础研究、临床诊断、药物研制等方面展现了广阔的应用前景. 本文综述了RNA适体的产生、特点、作用方式、优势与局限性,并详细介绍了其在医药研究领域的应用.     

基于Cell-SELEX的核酸适体在肿瘤中的应用研究进展

核酸适体是运用指数富集的配体系统进化(systematic evolution of ligand and byexponential enrichment,SELEX)技术从人工体外合成的随机寡核苷酸序列库中,经过多轮筛选后得到的单链DNA或RNA,具有识别靶标范围广、亲和力高和稳定性好等优势。Cell-SELEX技术是指将整个细胞作为靶标筛选特异性核酸适体的技术,近年来已经广泛应用于肿瘤细胞特异的核酸适体筛选。本研究综述了Cell-SELEX技术的筛选过程以及通过cell-SELEX技术产生的核酸适体在肿瘤标志物、肿瘤靶向治疗和循环肿瘤细胞的识别与检测等方面的应用研究进展。     

SELEX技术与适体

SELEX技术是从随机寡核苷酸评议库中筛选与靶分子特异结合序列的组合化学技术。随机寡核苷酸的多种空间结构是筛选基础。该技术对靶分子无特殊要求,筛选出的寡核苷酸被称为适体,适体最突出的特征是与靶分子的特异性高亲和力。     

随机单链DNA文库SELEX筛选寡核苷酸适配子方法的建立

指数富集配基的系统进化(SELEX)技术是一种新的组合化学技术.体外构建了一个长度为81 nt、含有35个随机序列的单链DNA(ssDNA)文库,优化了ssDNA文库扩增为双链DNA (dsDNA)文库的PCR反应条件.通过对比不对称PCR和生物素-链亲和素磁珠分离方法制备ssDNA文库的效果,确定了以生物素-链亲和素磁珠分离方法制备ssDNA.由于脱氧核糖核酸的疏水性导致ssDNA文库与硝酸纤维素滤膜的结合背景过高,因此选择以微孔板为介质,分离与靶蛋白结合的适配子.经过9轮循环筛选,随机ssDNA文库与丙型肝炎病毒(HCV)核心蛋白(C蛋白)的结合率从0.5%上升到32.5%.     

A Unique Protease Cleavage Site Predicted in the Spike Protein of the Novel Pneumonia Coronavirus (2019-nCoV) Potentially Related to Viral Transmissibility

正Dear Editor,In December 2019, a novel human coronavirus caused an epidemic of severe pneumonia(Coronavirus Disease 2019,COVID-19) in Wuhan, Hubei, China(Wu et al. 2020; Zhu et al. 2020). So far, this virus has spread to all areas of China and even to other countries. The epidemic has caused 67,102 confirmed infections with 1526 fatal cases     

Curcuminoids as inhibitors of thioredoxin reductase: A receptor based pharmacophore study with distance mapping of the active site

Curcumin is the yellow pigment of turmeric that interacts irreversibly forming an adduct with thioredoxin reductase (TrxR), an enzyme responsible for redox control of cell and defence against oxidative stress. Docking at both the active sites of TrxR was performed to compare the potency of three naturally occurring curcuminoids, namely curcumin, demethoxy curcumin and bis-demethoxy curcumin. Results show that active sites of TrxR occur at the junction of E and F chains. Volume and area of both cavities is predicted. It has been concluded by distance mapping of the most active conformations that Se atom of catalytic residue SeCYS498, is at a distance of 3.56 from C13 of demethoxy curcumin at the E chain active site, whereas C13 carbon atom forms adduct with Se atom of SeCys 498. We report that at least one methoxy group in curcuminoids is necessary for interation with catalytic residues of thioredoxin. Pharmacophore of both active sites of the TrxR receptor for curcumin and demethoxy curcumin molecules has been drawn and proposed for design and synthesis of most probable potent antiproliferative synthetic drugs.     

Comparative morphology of the carpel in the Liliaceae: Hewardieae, Petrosavieae, and Tricyrteae

The young pistils in the melanthioid tribes, Hewardieae, Petrosavieae and Tricyrteae, are uniformly tricarpellate and syncarpous. They lack raphide idioblasts. All are multiovulate, with bitegmic ovules. The Petrosavieae are marked by the presence of septal glands and incomplete syncarpy. Tepals and stamens adhere to the ovary in the Hewardieae and the Petrosavieae but not in the Tricyrteae. Two vascular bundles occur in the stamens of the Hewartlieae and Tricyrtis latifolia. Ventral bundles in the upper part of the ovary of the Hewardieae are continuous with compound septal bundles and placental bundles in the lower part. Putative ventral bundles occur in the alternate position in the Tricyrteae and putative placental bundles in the opposite. position in the Petrosavieae. The dichtomously branched stigma in each carpel of the Tricyrteae is supplied by a bifurcated dorsal bundle.     

Selection with two alleles and complete dominance

For a plant selection model with frequency-independent viabilities, fertilities and selfing rates, it is shown that apart from global fixation, for certain parameter combinations a protected polymorphism and facultative fixation (either allele may become fixed according to initial frequencies) may both occur. Facultative fixation requires different selling rates for the dominant and recessive type. Protection of the polymorphism requires resource allocation for male and female function. In this connection the problem of purely genetically caused population extinction is discussed.
For general frequency dependence and regular segregation, the chances for establishment of a completely recessive gene are compared to those of a completely dominant gene. It is proven that the process of establishment of the recessive gene, despite a fitness advantage, may be considerably endangered by drift effects if random mating prevails. The recessive gene may reach the same effectivity in establishment as a dominant gene, only if the recessive homozygote mates exclusively with its own type during the period of establishment.     

Composition and diversity of bird communities in Swedish farmland–forest mosaic landscapes

CapsuleThe amount of forest (at local and landscape scales) and occurrence of residual habitats at the local scale are shown to be the major factors influencing bird community composition in farmland–forest landscapes in central Sweden.

Aims To investigate the importance of local habitat and landscape structure for breeding birds in farmland–forest landscapes in central Sweden.

Methods Breeding birds were censused at 292 points. A detailed habitat mapping was made within 300 m of the points. Within a 300–600 m radius only two major habitats (forests and arable fields) were identified.

Results Cluster analyses of bird communities identified three site types that also differed in habitat composition: (i) partially forested sites in forested landscapes; (ii) heterogeneous sites with residual habitats in mosaic landscapes; and (iii) field-dominated farmland sites in open landscapes. A total of 19 of 25 farmland bird species (restricted to farmland or using both farmland and forest) had the highest abundance in farmland sites with mosaics of forest and farmland, while only six farmland species had the highest abundance in field-dominated sites. The bird community changed from being dominated by farmland species to being dominated by forest species (common in forest landscapes without farmland) at small proportions (10–20%) of forest at the local scale. A major difference in habitat composition between heterogeneous and field-dominated sites was the occurrence of different residual habitats (e.g. shrubby areas and seminatural grasslands). These habitats seemed to influence bird community composition more than land-use, despite covering <10% of the area. Seminatural grasslands were important for bird community composition and species-richness, but grazing seemed to be less important. Among different land-use types, cereal crops were the least preferred fields. Set-asides with tall vegetation and short rotation coppices were positively associated with species-richness of farmland birds.

Conclusion In general, the composition of the landscape was important for bird community composition, although amount and distribution of forests, occurrence of residual habitats and land-use of fields at the local scale had the strongest influence on bird community composition. The possible implications of these patterns for managing farmland–forest landscapes are discussed.     

Chemical constituents from the fruits of Rhus typhina L. and their chemotaxonomic significance

This work describes the isolation and characterization of twenty-nine compounds from the fruits of Rhus typhina L., including eleven flavonoids (1–11), eleven phenols (12–22), two pentacyclic triterpenes (23–24), two organic acids (25–26), one lumichrome (27), one courmarin (28) and one pyrimidine (29) on the basis of their spectroscopic data. Compounds apigenin (1), daidzein (4), orobol (5), 3′, 5, 5′, 7-tetrahydroxyflavanone (6), naringenin (7), butein (8), (-)-catechin (9), quercetin-3-O-α-L-(3″-O-galloyl)-rhamnoside (11), 2-hydroxybenzoic acid (13), 4-hydroxybenzaldehyde (14), vanillin (15), methyl 3,4-dihydroxybenzoate (16), 3,5-dihydroxybenzamide (18), tyrosol (19), caffeic acid (20), 3-(2,4,6-trihydroxyphenyl)-1-(4-hydroxyphenyl)-propan-1-one (21), phlorizin (22), friedelin (23), oleanolic acid (24), 4,4-dimethyl-heptanedioic acid (25), anthranilic acid (26), lumichrome (27), scoparone (28) and uracil (29) have not been recorded before in this plant. This is the first report on the occurrence of compounds 4–7, 9, 11, 13–14, 16, 18–21, 25–29 from the genus Rhus. Moreover, the chemotaxonomic significance of these isolated compounds was also summarized.