pSUPER介导的RNAi抗乙肝病毒S基因的研究

目的 研究RNAi抗乙肝病毒的作用.方法 设计并合成一段针对HBV S基因的干扰序列及一段无关的序列,克隆进pSUPER载体中,分别构建pSUPEK-HBS、pSUPEK-nonsense载体,然后将pSUPER、pSUPEK-HBS、pSUPER-nonsense转染进HepG2.2.15细胞中,用ELISA方法对细胞上清中HBsAg、HBeAg进行检测.尾静脉注射HBV转基因小鼠,取血清检测.结果 pSUPER-HBS所表达的siRNA成功的抑制了HepG 2.2.15上清及HBV转基因鼠血清中的HBsAg、HBeAg,抑制率分别达到70%、51%以及60%、42%.结论 针对HBV S区的siRNA能明显抑制HBV的复制.     

三链DNA的形成抑制转录因子与乙肝病毒核衣壳启动子的结合

合成的寡核苷酸可缠绕于DNA双螺旋大沟内并以氢键与其相互作用形成三链DNA结构,这类寡核苷酸常简称为TFO(triplex-form-ing oligonucleotide)。通过TFO与靶基因结合形成局部的三链结构来抑制基因转录的技术被称为反基因策略(antigene strategy)。我们选择乙肝病毒(HBV)核衣壳启动子(Cp)内一近似同聚嘌呤·同聚嘧啶序列(1734～1754,ayw亚型,以EcoR Ⅰ切点为 1)为靶序列,以嘌呤     

利用锁核酸化学偶联FokⅠ核酸酶靶向切割HBV基因的体外实验

乙肝病毒(HBV)是有缺口的双链DNA病毒,侵入人体肝细胞后形成共价闭合环状DNA(ccc DNA)持续复制,并在逆转录过程中随机整合入宿主基因组。慢性乙型肝炎感染者平均每个细胞中含有33个ccc DNA拷贝,半衰期长达35~57 d,很难从体内彻底清除。利用锁核酸抑制HBV转录,是乙肝治疗的新策略。此外,利用基因组编辑技术靶向切割HBV基因组,有望从源头治愈乙型肝炎。基于锁核酸与双链DNA形成三股螺旋的能力、抵御核酸酶及聚合酶的稳定性以及对单碱基错配的敏感性,本研究以靶向切割乙型肝炎病毒为例,设计构建锁核酸修饰的寡核苷酸作为DNA结合域,有效增强对靶基因的特异性识别;同时利用FokⅠ核酸酶分子量小、二聚化时才具有酶活等特点,设计构建FokⅠ切割域二聚体重组蛋白作为DNA切割域;进而通过双功能交联剂GMBS,建立了5′端氨基(-NH2)修饰的锁核酸与N端巯基(-SH)修饰的FokⅠ核酸酶定向化学偶联的方法,并在体外验证了新型工具对HBV基因的靶向切割。该方法为此后在体内进行高特异性、无整合风险的抗病毒基因治疗提供了全新的技术思路。     

靶向HBs基因的shRNA表达载体的构建及其抑制HepG2.2.15表达HBVsAg的作用

探索用PGenesil-1(Pg)构建的靶向乙型肝炎病毒表面抗原(HBsAg)基因的shRNA表达载体PGenesil-1-HBs(简称Pgs),对体外培养HepG2.2.15细胞中的HBV基因及其抗原表达的抑制作用.设计、合成靶向HBV S区的3对DNA序列,分别插入PGenesil-1中构建3个siRNA表达载体Pgs1、Pgs2、Pgs3,经限制性内切酶,DNA序列测定等技术鉴定确认.筛选并确定最佳细胞接种量及重组质粒转染量后,分别或按不同组合转染HepG-2.2.15细胞,48 h后采用半定量RT-PCR检测HBVsmRNA转录水平,免疫细胞化学技术检测HBsAg表达水平,MEIA分别检测细胞裂解液和培养上清中HBsAg和HBeAg的表达水平.结果表明,HBV真核表达载体Pgs1、Pgs2、Pgs3均能不同程度地抑制HepG2.2.15细胞中的HBsAg、HBeAg合成和HBs-mRNA转录.成功构建的HBV真核表达载体Pgs1、Pgs2、Pgs3,其中PgS3能显著抑制HBsAg表达(P＜0.01).多种表达载体联合对抗原表达的抑制作用效率不同.     

体外针对X基因的小干扰RNA的抗乙型肝炎病毒效应研究

目的：探讨体外针对乙型肝炎病毒（HBV）X基因的小干扰RNA（siRNA）对HBV复制和抗原表达的抵制作用。方法：利用siRNA表达框架法设计针对HBVX基因的siRNA,转染HepG2．2．15细胞,RT-PCR半定量检测转染前后X基因的表达;ELISA法测定各组24、48、72hHBsAg和HBeAg的含量;荧光定量PCR检测48h时HBVDNA的变化。结果：制备了HBVX基因的siRNA,转染后24、48和72h,HBVX基因mRNA的量分别减少了57％、78％和40％;siRNA能抑制HBsAg和HbeAg的分泌,抑制高峰在48h,抑制率分别为42％和43％;荧光定量PCR证实HBVDNA的复制亦受到抑制。结论：针对HBVX基因的siRNA在体外具有抑制HBV复制和抗原表达的作用。     

反义前S／S基因转移表达抗乙型肝炎病毒的研究

为了观察反义基因转录表达抗乙型肝炎病毒（HBV）的作用,将HBVayw前S／S基因（PreS／S）片段反向插入逆转录病毒载体质粒,构建preS／S反义基因重组体,经转染PA317细胞后,获得重组逆转录病毒颗粒。用重组病毒转导2．2．15细胞后,第3天即可见细胞培养上清HBV表面抗原（HBsAg）和e抗原（HBeAg）表达量减少,到转导后第5天,细胞培养上清中的HBsAg和HBeAg表达量降到最低水平,HBsAg抑制率为71％,HBeAg抑制率为27％,抑制作用至少可持续到转导后第11天。空载及正向插入基因的重组逆转录病毒转导对2,2．15细胞内HBV抗原表达无抑制作用。     

反义前S/S基因转移表达抗乙型肝炎病毒的研究

为了观察反义基因转录表达抗乙型肝炎病毒(HBV)的作用,将HBV ayw前S/S基因(preS/S)片段反向插入逆转录病毒载体质粒,构建preS/S反义基因重组体,经转染PA317细胞后,获得重组逆转录病毒颗粒.用重组病毒转导2.2.15细胞后,第3天即可见细胞培养上清HBV表面抗原(HBsAg)和e抗原(HBeAg)表达量减少,到转导后第5天,细胞培养上清中的HBsAg和HBeAg表达量降到最低水平,HBsAg抑制率为71%,HBeAg抑制率为27%,抑制作用至少可持续到转导后第11天.空载及正向插入基因的重组逆转录病毒转导对2.2.15细胞内HBV抗原表达无抑制作用.     

多种小分子干扰RNA联合抑制乙型肝炎病毒的体外研究

小分子干扰RNA(siRNA)能够在哺乳动物细胞中引起包括病毒基因在内的基因沉默。为了研究多种siRNA联合应用在体外抑制乙型肝炎病毒(HBV)复制中的效果,本研究设计了12种针对不同HBV靶点的siRNA,转染可稳定分泌HBV颗粒的HepG22．2．15细胞,并采用了酶联免疫法检测上清液中HBsAg和HBeAg的含量,实时定量PCR法检测细胞中HBV的DNA含量。结果发现这12种分子均能在不同程度上抑制病毒复制。进一步研究表明它们对HBV的抑制作用在一定程度上存在剂量效应和协同作用,单分子siRNA在80nmol／L处对HBsAg和HBeAg的抑制率分别可达到约80％和60％,而多分子siRNA组合在20nmol／L处对HBsAg就能达到90％的抑制率,但对HBeAg表达的抑制率提高不明显。单分子siRNA在80nmol／L处对HBVDNA复制的抑制率可达到90％以上,而多分子siRNA组合在20nmol／L处对DNA含量就能达到约90％的抑制率。本研究的结果为进一步开发新的联合应用多种siRNA治疗HBV的途径打下了基础。     

反义前C／C基因转移表达抗乙型肝炎病毒作用的研究

为了观察逆转录病毒载体包装细胞系统介导反义基因转移表达的抗乙型肝炎病毒（HBV）作用,将HBVayw前C／C基因（preC／C）片段反向插入逆转录病毒载体质粒。将重组体转染逆转录病毒包装细胞PA317后,获得重组逆转录病毒。用重组逆转录病毒感染2．2．15细胞后发现,感染后第3天,细胞培养上清HBV表面抗原（HBsAg）和e抗原（HBeAg）表达量即明显减少,抑制作用于感染后第5天达到高峰,其中对HBsAg表达的抑制率为27．0％,对HBeAg表达的抑制率为59.5％。反义基因重组逆转录病毒感染2.2．15细胞对HBV抗原表达的抑制作用至少可以持续到转导后的第11天。空载及正义基因重组逆转录病毒感染对2．215细胞HBV抗原表达无明显抑制作用。此外,反义基因重组逆转录病毒感染对2.2.15细胞HBVDNA复制也有抑制作用．无细胞毒性。     

应用RNAi文库筛选HBV复制相关基因

深入研究HBV复制机理,筛选参与HBV复制的基因,可能为开发抗乙肝病毒新药提供新 的靶点．本文拟建立一种筛选HBV复制相关基因的方法: RNAi文库感染HepG2.2.15细胞后,利用免疫磁珠收集HBsAg表达降低的细胞,提取DNA,PCR扩增siRNA编码序列,将PCR产物克隆入T-easy载体,随机挑选克隆测序,发现DDB1基因可能参与HBV复制．本试验建立了一种筛选HBV复制相关基因的方法,为大规模全基因组筛选参与HBV复制的基因奠定了基础．     

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.