PCR条件及程序改变对抗体库多样性的影响

用家族特异性免疫球蛋白可变区基因引物两两组合分别对轻、重链进行RT-PCR扩增. 在不同退火温度下得到了全部轻链及大部分重链（13/16）可变区基因. 当先用免疫球蛋白信号肽序列5′-端引物与未扩出基因3′-端引物组合进行一次PCR,再以该产物为模板进行二次PCR,则获得了未扩出三条重链的PCR产物. 这表明通过PCR反应条件的改变及程序调整,可增加所获得可变区基因的种类及数量,从而增加抗体库的多样性.     

特异性单引物PCR

建立特异性单引物PCR,并用于筛选基因克隆重组子。根据α－globin的一段序列设计引物,按照设计好的特性单引物PCR扩增条有α－globin的重组质粒PLNSX－aglobin,具体扩增条件如下：（1）97℃5min变性后,94℃30s,70℃2min30s,30个循环,制备单链模板;（2）94℃1min,12℃5min,16℃3min,18℃3min,20℃3min,22℃3min,25℃1min,30℃3min,37℃3min,72℃6min,低温条件下引物3′端4－5个碱基与所扩单链模板退火配对,扩增得到一系列不同起始位点但同一序列末端长短一的核酸片段;（3）以上述所得片段为模版进行如下条件的扩增：94℃30min,70℃2min,70℃2min,72℃2min,每个循环增加1s）,45个循环。结果,特异性单引物PCR能够从pLNSX－αglobin上扩增出特异带,可有效用于筛选基因克隆重组子。     

利用RACE技术扩增大豆抗病基因同源cDNA 5′末端序列

利用抗病基因保守序列筛选大豆cDNA文库,获得一抗病基因同源cDNA片段,命名为KR3-1。根据KR3-1设计两个基因特异引物（GSP 和 NGSP）,分别与通用引物(UPM)和巢式通用引物（NUP）共同扩增,成功地克隆到了该基因的5′末端序列。该扩增片段长447 bp,与已知序列重叠部分为129 bp。 Abstract:Based on part of a known partial cDNA sequence of a disease resistance gene homolog,KR3-1,obtained by screening a cDNA library from soybean,5′-RACE-PCR was carried out with gene specific primers and universal primers.After the nested PCR reaction,an amplified fragment of 447 bp in length which overlapped the known KR3-1 sequence by 129 bp was obtained subsequently.Thus,a 5′ cDNA end of KR3 was successfully cloned.     

血红密孔菌(Pycnoporussanguineus)漆酶基因的克隆与序列分析

为克隆血红密孔菌 (Pycnoporussanguineus)漆酶基因 ,根据真菌漆酶氨基酸序列保守区设计了 1对简并引物 .以血红密孔菌基因组DNA为模板 ,PCR扩增出长 12 2 7bp的漆酶基因片段 .以此序列为基础 ,通过 5′及 3′RACE技术克隆出漆酶全长cDNA序列 ,序列长为 190 2bp ,其 5′端和 3′端非编码区长分别为 5 1bp和 2 97bp ,开放阅读框长 15 5 4bp ,编码 5 18个氨基酸的蛋白 .该蛋白具有 4个铜离子结合区域 ,预测其相对分子量为 5 6 313 2 ,等电点为 5 5 9,其氨基酸序列与Pycnoporuscinnabarinus漆酶 (lcc3 2 )的同源性最高 ,为 96 % .以该cDNA编码区的两端序列为引物 ,PCR扩增得到漆酶的长度为 2 15 4bp的全长DNA序列 ,序列中包括 10个内含子序列 ,长为 5 2～ 70bp     

一步PCR快速扩增辽宁碱蓬甜菜碱醛脱氢酶cDNA 3'末端序列

根据已获得的辽宁碱蓬甜菜碱醛脱氢酶cDNA的部分序列,设计一条基因特异性引物,与通用引物并用,一步PCR成功地克隆了辽宁碱蓬甜菜碱醛脱氢酶cDNA 3′末端。与常规的3′RACE法相比,一步PCR法具有快速、简便、经济等优点,是一种非常快捷的扩增cDNA 3′末端序列的方法。 Abstract:Based on part of a known cDNA sequence of Suaeda liaotungensis betaine aldehyde dehydrogenase,we successfully cloned the 3′cDNA end of S.lianotungensis betaine aldehyde dehydrogenase using one step PCR with a gene specific primer and universal primer.Compared with the typical 3′ RACE,one step PCR is rapid,simple and inexpensive.It is very rapid to amplify an unknown cDNA 3′end using this method.     

不同种源韦塔桉RAPD反应条件优化研究

对13个韦塔桉种源的RAPD反应条件,即PCR反应体系、PCR扩增条件等因素进行了研究。结果表明,最佳PCR体系为:DNA模板为10.0μL、双蒸水4.0μL、10×缓冲液2.0μL、25mMMgCl21.5μL、10mMdNTPs0.35μL、0.1μM引物2.0μ1、5UTaqDNA聚合酶0.15μL。最佳PCR扩增条件为:92℃预变性2min,1个循环;92℃变性1min,36℃退火1min,72℃延伸2min,共35个循环;72℃延伸5min,1个循环。     

梅花类黄酮3′-羟化酶基因片段基于基因组DNA的简并PCR法克隆(英文)

用本研究设计的“预先去杂—SDS法”从梅花嫩叶提取到高质量的基因组DNA。根据11条已公开发表的并提交到GenBank的类黄酮3′-羟化酶基因cDNA的假定氨基酸序列的保守区设计2个正向简并引物和3个反向简并引物组成6对引物,仅有1对引物能以PCR法同时从梅花‘南京红须’、‘南京红’和‘粉皮宫粉’的基因组DNA扩增到一个469 bp的核苷酸片段,这3个片段在总体上有99 .72 %的一致性,与11条类黄酮3′-羟化酶基因cDNA的相应区域有65 .57 %的一致性。同时,“GGEK”并非类黄酮3′-羟化酶的特征性模体。这是首次从木本植物的基因组DNA克隆到类黄酮3′-羟化酶基因片段。本研究结果可为梅花类黄酮3′-羟化酶基因全长的克隆奠定基础。     

聚合酶链反应鉴定马肉方法的建立及应用

依据马卫星DNA序列设计了1对引物,建立了PCR鉴定生、熟马肉的方法。引物Ⅰ 5′-GTTGTGCCTTTCAGCTCTAGG-3′,合成浓度2.64μg/μl;引物Ⅱ 5′-ACAGCATCTTG-CATCCAGC-3′,合成浓度2.97μg/μl,设计扩增长度为186bp。PCR扩增体系为50μl,循环参数为94℃ 40s、49.5℃ 50s、72℃ 60s,35个循环后72℃保温延伸7min,产物经3％琼脂糖凝胶电泳鉴定。应用本方法可以从生鲜马肉、煮熟马肉、高压马肉的全基因组DNA中扩增出186 bp的目的DNA条带,而对牛、羊、猪、驼、鹿等15种动物的DNA扩增则呈阴性反应。其     

鸡二价金属转运蛋白1 cDNA的克隆与序列分析

鸡二价金属转运蛋白1（divalent metal transporter 1, DMT1）在动物胃肠道锰吸收过程中起重要作用.根据哺乳动物Dmt1同源蛋白氨基酸序列的保守性设计引物,应用3′RACE(rapid amplification of cDNA ends)技术,扩增并克隆获得鸡小肠Dmt1 cDNA 3′端1 289bp和1 092bp的2种片段,发现其3′端翻译区和非翻译区存在差异. 根据鸡Dmt1 cDNA 3′端片段的测序结果设计引物,扩增获得1个与3′端片段部分重叠的鸡Dmt1 cDNA 5′端907 bp片段,并对其进行了克隆测序. 根据鸡小肠Dmt1 3′RACE片段和5′RACE片段序列信息进行拼接,从而获得鸡小肠Dmt1 cDNA全序列信息.结果表明,鸡小肠Dmt1 cDNA有2种形式,1种全长为1 972个核苷酸,其中5′非翻译区为104个核苷酸,编码区1 695个核苷酸,3′非翻译区为173个核苷酸,编码1个含564个氨基酸残基的蛋白质;另1种形式为1 775个核苷酸,其中5′非翻译区为104个核苷酸,编码区1 593个核苷酸,3′非翻译区为78个核苷酸,编码1个含530个氨基酸残基的蛋白质.据鸡Dmt1 cDNA推测出的2种形式蛋白质的氨基酸序列与人、大鼠和小鼠的Dmt1蛋白具有高度同源性,它们的同源性分别为82%、82%、80%,和 84%、84%、83%. 对推测氨基酸序列进行疏水性和跨膜区分析表明,Dmt1蛋白为1种跨膜整合蛋白,具有膜转运蛋白糖基化位点和底物结合位点的保守序列.     

广西甜茶ISSR-PCR反应条件的建立与优化

为了探讨广西甜茶ISSR实验中的多种因素对实验结果的影响,采用单因素法对PCR反应体系中的5个潜在因素(Mg2+, dNTP,引物, Taq酶和模板DNA)在5水平上进行优化实验,并进一步优化了反应程序中的退火温度和循环次数。结果表明：25滋L的最佳反应体系为,1×PCR Buffer、MgCl22.0 mmol/L、dNTP 0.2 mmol/L、引物0.8滋mol/L、Taq DNA聚合酶0.75 U、模板DNA 80 ng;最佳反应程序为：在94℃下进行3 min预变性;随后循环扩增35次(包括94℃变性1 min,54℃退火1 min,72℃延伸1 min);最后在72℃下延伸10 min。本研究建立的广西甜茶的最佳ISSR-PCR反应条件为进一步进行遗传多样性研究奠定了基础。     

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.