银染色测定粘虫核多角体病毒多角体基因序列

LsMNPV DNA用EcoRV酶切进行基因组克降,用AcMNPV的部分多角体基因顺序DNA片段作探针,菌落原位杂交法结合测序筛选到分别含LsMNPV部分多角体基因的重组质粒pLsEV1和pLsPH5。用银染色PCR线性扩增双脱氧法测序,发现LsMNPv的完整基因即位于这两个片段上。LsMNPV多角体基因长741bp,编码区碱基同源性与AcMNPV和MbMNPV分别为80.0％和97.0％,氨基酸同源性分别为89.8和97.5％。氨基酸组成中以谷氨酸(Glu)含量最高,谷氨酰胺和色氨酸含量最低。密码子选用以第三个碱基为嘧啶的密码子频率最高。多角体蛋白N端有一类似信号肽结构的26个氨基酸的疏水区。     

粘虫核型多角体病及其多角体的某些性质

粘虫是禾谷类重要害虫之一。在自然条件下,粘虫能被病毒所感染,导致流行病(Raun,1961;Tanada,1961,1962)。目前,已证实在行军虫(Pseudaletia unipuncta Haworth)中有核型多角体病(Chapman et al,1915;Steinhaus,1951)、质型多角体病(Tanada etal.,1960)、颗粒体病及非包涵体病毒病(Wasser,1952)的存在。Tanada的工作表明:不     

油桐尺蠖核多角体病毒多角体蛋白双向电泳分析

 <正> 油桐尺蠖核多角体病毒(Buzura suppressaria Nuclear Polyhedrosis Virus,BsNPV)是茶树、油桐等的一种主要害虫——油桐尺蠖的病原体,我国首次分离,并已对其进行了广泛的研究。这种病毒的多角体蛋白在单相SDS-PAGE中分子量显示为31 kD的单一多     

茶尺蠖核型多角体病毒多角体蛋白单克隆抗体的制备

为了建立一种基于免疫反应检测茶尺蠖核型多角体病毒的方法,以纯化后的茶尺蠖核型多角体病毒作为抗原,免疫BALB/c小鼠,将小鼠脾脏细胞与小鼠骨髓瘤细胞Sp2/0融合,经间接ELISA筛选及克隆得到了一株稳定分泌单克隆抗体的杂交瘤细胞株,命名为7D3。同时克隆并在大肠杆菌中表达了EoNPV多角体蛋白基因,获得重组多角体蛋白。经Western blotting鉴定,该抗体可与EoNPV的多角体蛋白特异性结合。利用制备EoNPV多角体蛋白的单克隆抗体,建立了间接ELISA测定EoNPV的方法。     

蓖麻蚕核型多角体病毒与苜蓿银纹夜蛾核型多角体病毒多角体蛋白基因同源性的研究

本文报道以苜蓿银纹夜蛾核型多角体病毒多角体蛋白基因mRNA的cDNA的重组质粒PMA-Ⅵ DNA转化E.coli RR_1。采用了多种筛选方法,包括抗菌素抗性筛选,菌落杂交及电泳等方法。快速地筛选出含有PMA-Ⅵ质粒的菌株。并以蓖麻蚕NPV-DNA作探针,通过Southern杂交,表明蓖麻蚕NPV基因组中具有与苜蓿银纹夜蛾NPV多角体蛋白基因同源性的核苷酸序列。     

柞蚕核多角体病毒核多角体基因的定位和克隆

通过Southern转印杂交证明,柞蚕核多角体病毒(Antheraea pernyi nuclear polyhedrosis virus,ApNPV)核多角体基因位于该病毒基因组DNA Bam HⅠ D和E片段上,我们巳将这两个片段分别克隆到pAT153质粒中,并用末端杂交法确定了ApNPV核多角体基因的方向,对含有这一基因的片段进行了限制性内切酶图谱分析,进而对这一基因部分编码区进行了核苷酸序列分析,在用ApNPV这一段序列(222bp)与其他昆虫核多角体病毒AcNPV(AutograPha californica NPV,苜蓿丫纹夜蛾NPV);BmNPV(Bombyx mory NPV,家蚕NPV);OpNPV(Orqyia Pseudotsugata NPV,黄杉毒蛾NPV)核多角体基因相应区段相比较分析中,发现它们之间的同源核苷酸序列比率分别为77.5％、84％和80％。     

异源多角体蛋白对家蚕核型多角体病毒粒子的包装

利用PCR方法从AcMNPV基因组DNA中分离出多角体蛋白基因 ,将该扩增片段克隆到转移载体pBacPAK8中 ,得到重组转移载体pOAc。将该质粒DNA与线性化的Bm BacPAK6病毒基因组DNA共传染BmN细胞 ,得到了能形成多角体且不产生蓝色空斑的重组病毒hp BmNPV。纯化该重组病毒的多角体颗粒 ,并对多角体蛋白、病毒核酸及多角体病毒颗粒进行分析 ,发现AcMNPV的多角体蛋白能在家蚕细胞中大量表达且能在细胞内识别家蚕核型多角体病毒并组装成多角体颗粒 ;病毒基因组DNA因部分交换 ,其酶切行为发生了相应的变化 ;电镜观察发现经AcMNPV多角体蛋白包装的家蚕核型多角体病毒的多角体颗粒大小为1 2 μm～ 2 9μm ,明显小于野生型家蚕核型多角体病毒的多角体颗粒     

油桐尺蠖核多角体病毒多角体蛋白基因定位与克隆

以[~(32)P]-dATP标记含AcNPV DNA的EcoRI-I片段的重组质粒为探针,在35℃条件下对油桐尺蠖核多角体病毒(BsNPV)多角体蛋白基因进行了定位,将其分别定位在BamH Ⅰ-A,Bgl Ⅰ-A,Bgl Ⅱ-F,EocR Ⅰ-R,Hind Ⅲ-A,Kpn Ⅰ-Ⅰ,Pst Ⅰ-D,Xba Ⅰ-A(或B),Xho Ⅰ-F和G片段上,并以M13mp18为载体,克隆了Kpn Ⅰ-Ⅰ片段。     

大尺蠖核型多角体病毒多角体蛋白基因的结构分析

大尺蠖核型多角体病毒(BsNPV)的多角体蛋白基因(ocu)定位在2．3kb的BamHl—H片段上,用xho1、sma1、HindIII和PstI构建了H片段的物理图谱,并测定了两端636bp序列。在5’端发现了杆状病毒ocu基因共有的典型特征,即：ATG起始区;启动子区(14bp保守序列);TATA box和CATA box区等。单一xhoI位点在ATG上游-15bp处,适于作为构建ocu基因转移载体的插入位点。在这一基因5’端序列中发现了五个反转重复单元CGAGC GCTCG,讨论了这一单元在杆状病毒ocu基因高效表达中的调控功能。     

siRNA介导基因沉默的分子机器、作用模式和应用前景

siRNA(small interfering RNA)介导的基因沉默是细胞内监控寄生的遗传物质、沉默无用的信息模板和调节自然的时空转换的一种分子机制。它与体液免疫和细胞免疫一起形成了动物和人类机体中免疫系统的三大支柱,从不同水平上来对抗体内外有害物质的干扰和侵犯。现已清楚,大约22个核苷酸长的双链RNA能够通过不同途径,以序列特异的方式来高效地沉默含有同源序列的靶RNA分子。这一古老而又迷人的系统现已被公认为是鉴定基因功能、调控基因表达和改变基因表型的简单而有效的方法。可以预计,这一新颖的技术在不远的将来必将形成一条研究功能基因组学、探索信号通路和创造遗传缺陷模型的高速公路,并将开创一条预防和治疗人类疾病的新途径。     

基于基因表达谱的肿瘤分型和特征基因选取

在分析基因表达谱数据特性的基础上,提出了一个将之用于肿瘤分子分型和选型和选取相应亚型特征基因的策略。该策略包括三个步骤：首先采用一个无监督的基因过滤算法以降低用于分型计算的数据的噪声,其次提出了一个概率模型对样本中的分类结构进行建模,最后基于聚类的结果采用相对熵的方法获得对分类贡献大的基因作为特征基因,应用该策略对两个公开发表的数据集进行了再挖掘,结果表明不但获得了其他方法可以得到的信息,而且还提供了更精细、更具有显著生物学意义的信息,具有明显的优越性。     

GENE FLOW IN CHAMAECRISTA FASCICULATA (LEGUMINOSAE) II. GENE ESTABLISHMENT

The role of gene establishment in gene flow was investigated in a population of the annual legume Chamaecrista fasciculata by determining the effect of interparent distance on progeny fitness throughout the entire life history. A decelerating gain in progeny fitness with increasing interparent distance was observed. Selfed progeny suffered a 2-fold fitness disadvantage compared to progeny derived from mating events between individuals in the same neighborhood. Progeny derived from within neighborhood crosses had lower fitness than progeny from crosses between neighborhoods. Coupling the effect of interparent distance on gene establishment with information on gene dispersal resulted in a considerable increase in estimated gene flow. However, gene flow was still limited, as the average neighborhood area corresponded to a circle with radius of approximately 3.0 m. Yearly fluctuations in population size and variation in reproductive output lowered the estimate of N_e below the census estimate to approximately 100 individuals. The role of a seed bank in increasing the estimate of N_e was found to be insignificant. It is likely that genetic drift plays a major role in determining the distribution of genetic variation in this population.     

GENE FLOW IN CHAMAECRISTA FASCICULATA (LEGUMINOSAE) I. GENE DISPERSAL

Both pollen and seed dispersal components of gene flow were examined in the annual plant Chamaecrista fasciculata (Leguminosae) and quantified in terms of Wright's neighborhood area. Pollen dispersal was estimated by measuring pollinator flight movement throughout the flowering season and the contribution of pollen carryover to pollen dispersal was determined by comparing pollinator flight movement with dispersal of electrophoretic markers in an experimental transect. Phenological effects on the probability of fruit set were measured to determine whether pollinations should be weighted differentially across the flowering season. The outcrossing rate, a major determinant of the role of pollen dispersal in gene flow, was estimated from electrophoretic analysis of progeny arrays and by measuring the proportion of nongeitonogamous pollinator flight movements. Seed dispersal was measured in a prairie habitat and in experimental plots without surrounding vegetation. Seed dispersal was small in comparison to pollen dispersal in both environments. Fruit set was low at the beginning and end of the flowering season, periods when flower density is low and pollinator flight distances are large. Although the outcrossing rate was high (t = 80%) and pollen carryover substantial, pollen dispersal was limited. Averaged over 4 years, neighborhood area, based on both seed and pollen dispersal, was 17.6 m², and corresponds to a circle of radius 2.4 m. The observed limited gene dispersal suggests the population of C. fasciculata is genetically subdivided into small breeding units of related individuals.     

GENE FLOW IN LICHENS

Experimental studies of the evolutionary biology of lichen fungi have been hampered by massive difficulties of in vitro culture and artificial crosses are still not possible. Gene flow in these organisms is demonstrated here for the first time by the analysis of secondary products in the progeny of individuals from natural populations of mixed chemotypes of the Cladonia chlorophaea complex. All of the chemotypes in this study have been interpreted as distinct sibling species. In the Appalachian Mountains of North Carolina, however, the grayi and merochlorophaea chemotypes are found to belong to a single interbreeding populations that is reproductively isolated from the cryptochlorophaea chemotype. In the Coastal Plain, the cryptochlorophaea chemotype hybridizes with the local endemic perlomera chemotype. This study has major impact for species concept in lichens because consideration of neither morphologial tendencies nor biogenetic relationships of the secondary products could have predicted its result.     

精子介导鱼类基因转移和聚合酶链反应检测技术

金鱼精子与美洲大绵ｗｅｉ的抗冻蛋白基因一起保温３０分钟后,再与卵子受精,共获得１４５尾成鱼和若干胚胎。从胚胎和成鱼中提取ＤＮＡ经聚合酶链反应（ＰＣＲ法）扩增和Ｓｏｕｔｈｅｒｎ ｂｌｏｔ分子杂交表明,外源的抗冻蛋白基因进入了部分受体鱼的染色体组内。测定了４５尾一年龄实验鱼中,有１２尾显示出明确的杂交带,阳性率为２６％。     

HIV—1CN嵌合基因与IL—2基因共表达产物诱导产生CTL的实验研究

经脂质体介导共表达中国流行株HIV-1gag-gp120基因与IL-2基因的核酸疫苗质粒pGPIL-2转染BHK-21细胞,以间接免疫荧光法鉴定其表达.取pGPIL-2免疫鼠脾细胞,检测pGPIL-2诱导的细胞毒性T细胞杀伤活性.杀伤实验结果证明,经基因免疫获得的CTL效应细胞,可杀伤HIV-1\{CN\}嵌合基因转染的靶细胞.提示pGPIL-2可有效诱导CTL的产生,该研究结果为进一步设计中国流行株HIV-1核酸疫苗提供了重要实验依据.