采用Red重组系统构建包装菌株DH-gⅢ

选择感染性噬菌体技术是研究蛋白相互作用的良好手段,获得基因Ⅲ缺陷的辅助噬菌体是构成SIP体系的关键.为制备基因Ⅲ缺失的辅助噬菌体,利用λRed重组系统将完整的噬菌体基因Ⅲ和氯霉素抗性基因整合到大肠埃希菌DH5α的染色体上组氨酸操纵元位点构建包装菌株,经氯霉素抗性筛选并用PCR及组氨酸表型鉴定,获得包装菌株DH-gⅢ;将缺失功能基因Ⅲ的缺陷型噬菌体基因组转化到包装菌株中培养制备辅助噬菌体,以集落形成试验来检测缺陷型噬菌体的感染能力,结果滴度可达到4.3×108,只具备一次感染力,表明包装菌株构建成功,能用来制备辅助噬菌体,有望用于SIP体系.     

选择感染性噬菌体技术及应用

选择感染性噬菌体技术是在传统噬菌体表面展示技术的基础上发展而来的一种新方法,通过将噬菌体感染力的恢复与蛋白间的相互作用偶联起来进行筛选,有效提高了筛选效率,简化了操作过程,降低了本底,为后基因组学的研究提供了便利方法,特别是体内法在理论上适合于库-库筛选,更具有应用价值。     

噬菌体表面呈现技术研究进展

噬菌体表面呈现技术是1985年建立的一种将外源基因表达呈现在噬菌体颗粒表面的方法,可用于建立随机多肽文库、抗体文库等。经特定配基的筛选,可获得与其特异结合的配体分子。通过改构,还可将cDNA产物表达于噬菌体颗粒的尾部构建cDNA文库。SIP技术通过将配体和配基分别与基因Ⅲ蛋白的C末端和N末端融合表达,基因Ⅲ的C-末端参与噬菌体颗粒的组装,配基与配体的结合能够重建基因Ⅲ蛋白的功能,才能形成有感染能力的噬菌体,这样就大大提高了筛选效率。     

用于库-库筛选体系的抗原表达载体的构建

选择感染性噬菌体(SIP)技术是研究蛋白质相互作用的良好手段,体内SIP技术在理论上适于库-库筛选,而双载体共包装聚合噬菌体方式是一条有效途径.为构建适合库-库筛选的抗原表达载体,选用TG10载体作为基本载体进行改造,首先克隆噬菌体M13基因间隔区的序列插入TG10中得到质粒pTMI,克隆基因Ⅲ的N1N2区序列,并在其3′端加上一段G4T柔性多肽,将NIN2插入到pTMI载体中Lac启动子的下游得到pTMIN,化学合成Ptrc启动子序列替换pTMIN中的Lac启动子及部分功能基因序列,构建成抗原表达载体pTTMIN,化学合成c-myc的十肽表位插入到G4S后进行融合表达,采用ELISA和SDS-聚丙烯酰胺凝胶电泳检测抗原的表达,结果显示抗原得到了融合表达.用抗体呈现载体对pTTMIN性能进行鉴定,结果表明抗原表达载体可以与抗体呈现载体高效的共包装.综合以上结果说明抗原表达载体构建成功,可望用于库-库筛选体系.     

噬菌体肽库技术的应用

噬菌体肽库是由大量带有不同肽段的单个噬菌体组成的重组噬菌体库,通过分析筛选到的多肽的结构和序列,可以了解蛋白质分子之间的相互作用。随着生物技术的发展,噬菌体肽库技术在基因治疗、抗原表位定位、确定核酸结合蛋白、基因疫苗研究和药物筛选等方面得到广泛应用并取得了很大进展。     

噬菌体展示文库筛选技术的研究进展

噬菌体展示技术是将编码外源蛋白或多肽的基因片段定向插入到噬菌体的外壳蛋白基因区,使外源蛋白或多肽通过与噬菌体外壳蛋白融合而表达并展示于噬菌体表面,进而筛选表达特异蛋白或多肽的噬菌体,已发展成为生物学后基因组时代一个强有力的实验技术.噬菌体展示文库的筛选是其关键环节.为了提高筛选效率,许多研究者对传统的筛选技术进行了改进,如选择性感染噬菌体、迟延感染性噬菌体、以DNA为基础的筛选方法、亲合力捕获和反复筛选和封闭筛选法等,用于筛选的靶标也越来越具有多样性,使得这一技术有了更加广阔的发展前景.     

噬菌体表面显示技术在生物医学研究中的应用

利用噬菌体表面显示技术,将蛋白质或短肽分子在噬菌体表面表达,可以对抗原表位及蛋白质的相互作用位点进行定位与模拟,并用于蛋白质的体外亲和力优化以及新基因的克隆,在小分子药物的设计和筛选中也具有重要的作用。环肽结构的设计以及筛选策略和方法的改进,逐步推动着噬菌体表面显示技术在免疫学、基因工程药物及基础生物医学研究等研究领域中的成功应用     

噬菌体展示技术及其在寄生虫研究中的应用

噬菌体展示技术是将外源蛋白或多肽的编码基因或DNA序列插入到噬菌体外壳蛋白结构基因的适当位置,使外源基因随外壳蛋白的表达而表达,并随噬菌体的重新组装而展示到噬菌体表面的生物技术.在研究蛋白质识别或蛋白质与核酸相互作用的生物学过程、蛋白质定向改造、研制新型多肽药物、疫苗和抗体等多领域具有重要作用.就噬菌体展示技术基本原理及特点,以及噬菌体展示技术在寄生虫研究中的应用做一简要综述.     

噬菌体展示库筛选细胞特异分子的进展

介绍噬菌体展示技术的原理和发展,尤其是噬菌体展示技术在筛选细胞特异分子的策略方面的进展。该技术通过20年的发展已成为一种研究抗原一抗体作用、蛋白质相互作用、蛋白一药物相互作用甚至蛋白质一核酸作用的分析手段,但涉及到以完整细胞、器官或组织等复杂的生物活性分子表面为靶标则筛选效果尚不理想。关键是要减少噬菌体展示分子与靶标的非特异性结合,利用更为严格的经过改进的筛选策略。该技术的优势预示着它将广泛被应用于基础理论和研究实践中。     

噬菌体肽库技术

80年代中期,George P.Smith在前人对丝状噬菌体分子生物学研究的基础上首先提出了噬菌体展示技术(Phage Display),其核心就是将蛋白质分子的表型和基因型巧妙地结合于丝状噬菌体这样一个便于对其进行一系列生化和遗传操作的载体上,从而大大简化了蛋白质分子表达库的筛选和鉴定。基于这一优点,噬菌     

Recent advances in the study of Kaposi's sarcoma-associated herpesvirus replication and pathogenesis

It has now been over twenty years since a novel herpesviral genome was identified in Kaposi's sarcoma biopsies. Since then, the cumulative research effort by molecular biologists, virologists, clinicians, and epidemiologists alike has led to the extensive characterization of this tumor virus, Kaposi's sarcoma-associated herpesvirus(KSHV; also known as human herpesvirus 8(HHV-8)), and its associated diseases. Here we review the current knowledge of KSHV biology and pathogenesis, with a particular emphasis on new and exciting advances in the field of epigenetics. We also discuss the development and practicality of various cell culture and animal model systems to study KSHV replication and pathogenesis.     

The structure, reproduction and taxonomy of Vidalia and Osmundaria (Rhodophyta, Rhodomelaceae)

Comprises species occurring mostly in subtidal habitats in tropical, subtropical and warm-temperate areas of the world. An analysis of the type species, V. spiralis (Sonder) Lamouroux ex J. Agardh, a species from Australia, establishes basic characters for distinguishing species in the genus. These characters are (1) branching patterns of thalli, (2) flat blades that may be spiralled on their axis, (3) width of the blade, (4) primary or secondary derivation of sterile and fertile branchlets and (5) position of sterile and fertile branchlets on the thalli. Application of the latter two characters provides an important basic method for separation of species into three major groups. Osmundaria , a genus known only in southern Australia, was studied in relation to Vidalia , and its separation from the Vidalia assemblage is not accepted. Species of Vidalia therefore are transferred to the older genus name, Osmundaria. Two new species, Osmundaria papenfussii and Osmundaria oliveae are described from Natal. Confusion in the usage of the epithet, Vidalia fimbriala Brown ex Turner has been clarified, and Vidalia gregaria Falkenberg, described as an epiphyte on Osmundaria pro/ifera Lamouroux, is revealed to be young branches of the host, Osmundaria prolifera.     

New or rare chromosome counts in Artemisia L. (Asteraceae, Anthemideae) and related genera from Kazakhstan

Fifteen chromosome counts of six Artemisia taxa and one species of each of the genera Brachanthemum, Hippolytia, Kaschgaria, Lepidolopsis and Turaniphytum are reported from Kazakhstan. Three of them are new reports, two are not consistent with previous counts and the remainder are confirmations of very scarce (one to four) earlier records. All the populations studied have the same basic chromosome number, x = 9, with ploidy levels ranging from 2x to 6x. Some correlations between ploidy level, morphological characters and distribution are noted.     

肝癌中HBV和HCV基因和抗原的分布及意义

采用原位分子杂交方法检测HCV RNA及HBV X基因;采用免疫组织化学方法研究HCV核心抗原,非结构区C33c抗原及HBxAg在肝细胞肝癌中的定位及分布.结果表明(1)HCV RNA、HBV X基因在肝细胞肝癌组织检出率分别为40%(55/136)和82%(112/136).HCV RNA定位于癌细胞的胞浆内,阳性细胞呈散在、灶状及弥漫分布三种形式;HBV X基因在肝癌细胞中的分布呈胞浆型、核型及核浆型,阳性细胞也呈上述三种分布形式;(2)HCV C33c抗原、核心抗原在肝细胞肝癌中的阳性率为81%(133/164)及86%(141/164).C33c抗原定位于癌细胞及肝细胞的胞浆内;核心抗原既定位于癌细胞核中,又可定位于胞浆中.C33c抗原阳性细胞以灶状分布为主;而核心抗原阳性细     

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.