高效定点突变肠道病毒71 VP1基因的研究

[目的]肠道病毒71(Enterovirus 71,EV71)是引发手足口病(Hand-foot-mouth Disease,HFMD)的主要病原体之一,为了筛选EV71抗原表位,开发新型EV71疫苗,利用优化后的PCR点突变技术定点突变EV71 VP1基因。[方法]设计含有突变位点的互补引物,模板使用量为10 ng,进行单引物PCR,将PCR产物混合后复性,转化至大肠杆菌BL21,筛选阳性克隆并测序。[结果]测序结果表明VP1基因突变率为40%～45%。[结论]该方法对传统的PCR点突变技术进行改进,降低模板使用量,使用单引物PCR,省略DpnⅠ酶解过程,可达到40%～45%的定点突变率,从而降低实验成本、简化操作流程。     

结合单酶切位点的融合PCR技术对SCN1A基因的定点诱变

目的:利用结合单酶切位点的融合PCR技术对癫痫相关基因SCN1A进行定点突变。方法:首先设计两对引物PF1/PR1和PF2/PR2,PF1和PR2均位于突变位点最近的单酶切位点处,而突变位点设计在第一对反向引物(PR1)和第二对正向引物上(PF2)。通过重叠延伸法两次PCR扩增:第一次用PF1/PR1和PF2/PR2分开扩增,以扩增产物作模板,PF1/PR2作引物进行融合PCR,得到的扩增产物即含有所需要的突变位点,最后将扩增片段克隆入pMD18-T载体,经测序筛选阳性克隆。结果:DNA测序表明SCN1A基因所编码的第946位密码子由精氨酸(Arg)突变为组氨酸(His),再通过酶切和连接反应将重组质粒上的突变片段替换SCN1A表达质粒上的对应片段,成功构建了SCN1A突变载体。结论:与现在常用的长距离PCR定点诱导突变相比较,结合单酶切位点的融合PCR定点突变技术具备扩增距离短的优点,大大降低了自发突变的概率,适合于大肠杆菌中易自发突变的较大载体的定点诱变。     

单引物PCR法引入定点突变 *

目的:利用单个突变引物,在含人呼吸道合胞病毒F蛋白基因编码序列的pcDNA3.1(+)-F质粒中,通过单次环形PCR在特定序列位置引入定点突变。 方法: 以双链环状的pcDNA3.1(+)-F质粒DNA为模板,设计分别含有三种目的突变N70Q, I431N, Q270T的三条单引物,分别进行单次PCR。用甲基化DNA特异的限制性内切酶Dpn I处理PCR产物后转化大肠杆菌DH5α,进行克隆筛选,酶切鉴定和测序分析。 结果: 酶切鉴定结果和测序结果均符合预期,利用单引物PCR法成功在含人呼吸道合胞病毒F蛋白基因编码序列的pcDNA3.1(+)-F质粒DNA 中引入了单碱基突变、两个间隔碱基突变及相邻三碱基突变三种目的突变。 结论: 单引物PCR法解决了常规定点突变方法中多个PCR反应,程序繁琐及突变效率低等问题,是一种简便、快速、有效的基因工程定点突变新方法。     

利用DREAM设计和同源重组进行一步定点突变

目的：建立基于DREAM设计和同源重组的简便、快速定点突变方法。方法：设计两条包含突变的反向PCR（inverse PCR）引物,使其5'端互补从而产生同源重组,同时使用DREAM设计方案在上述引物中引入限制性内切酶位点以便突变子筛选。用能扩增长片段的高保真耐热 DNA聚合酶扩增全长的质粒DNA,直接转化大肠杆菌。转化到细菌中的全长质粒DNA PCR产物可利用其末端同源序列发生同源重组而环化。利用引入的酶切位点方便地进行突变子的筛选。结果：我们用该方法成功地对长度大于7 kb的质粒进行了定点突变。结论：本定点突变无需任何突变试剂盒和特殊的试剂,只需一步反应即可完成;利用DREAM设计使克隆筛选简便可靠,高保真耐热DNA聚合酶可保证多数突变子克隆不发生意外突变,而该酶扩增长片段的能力使该方法适合于大多数质粒不经亚克隆直接突变。     

单引物PCR法引入定点突变

目的:利用单个突变引物,在含人呼吸道合胞病毒F蛋白基因编码序列的pc DNA3.1(+)-F质粒中,通过单次环形PCR在特定序列位置引入定点突变。方法:以双链环状的pc DNA3.1(+)-F质粒DNA为模板,设计分别含有三种目的突变N70Q,I431N,Q270T的三条单引物,分别进行单次PCR。用甲基化DNA特异的限制性内切酶Dpn I处理PCR产物后转化大肠杆菌DH5α,进行克隆筛选,酶切鉴定和测序分析。结果:酶切鉴定结果和测序结果均符合预期,利用单引物PCR法成功在含人呼吸道合胞病毒F蛋白基因编码序列的pc DNA3.1(+)-F质粒DNA中引入了单碱基突变、两个间隔碱基突变及相邻三碱基突变三种目的突变。结论:单引物PCR法解决了常规定点突变方法中多个PCR反应,程序繁琐及突变效率低等问题,是一种简便、快速、有效的基因工程定点突变新方法。     

基于重叠延伸PCR法的定点突变技术

目的:建立一种高效而经济的定点突变方法。方法:采用重叠延伸PCR定点突变技术,引物设计时引入目的突变,以前两次PCR产物为模板,进行第三次PCR,即可获得突变后的目的DNA片段。将此片段连入pMDTM18-T载体后测序验证突变结果。结果:DNA测序表明,待突变位点已由ATTGG突变为ATTTT。结论:成功实现了目的位点的定点突变,重叠延伸PCR法是一种高效且经济的定点突变方法。     

一种高效而经济获得长片段基因突变体的方法

重组PCR方法的发展为体外突变技术提供了更快捷、准确的方法.通过改进引物设计和PCR保真性等方法,应用重叠延伸PCR的原理成功地实现了对BA基因的定点突变,并构建了其含有m1,m2,m3和m4的突变体.实验证明通过这种改进方法可以有效地进行长片段基因的突变,并且这种方法与其他方法相比具有简便、快捷、经济、高效的特点.     

定点突变三种方法的比较研究

目的：通过使用优化后的定点突变三种方法分别对一个新基因重组载体进行定点突变研究,比较这几种定点突变方法的优缺点。方法：重叠延伸PCR法使用Stratagene在线定点突变引物设计程序从而使引物设计简化而可靠;MutaBEST定点突变试剂盒采用胶纯化试剂盒代替说明书推荐的酚-氯仿抽提质粒的方法以提高回收率;使用PrimeSTAR高保真DNA聚合酶和超级感受态试剂盒代替Quikchange定点突变试剂盒的相应组分可使产物不受影响同时降低试验费用。结果：三种方法都能够获得单碱基突变重组载体。结论：QuikChange突变策略通过改进是一种高效、简洁、经济和可靠的定点突变方法。     

改进重叠延伸PCR技术构建定点双突变

目的:目的DNA片段中快速构建位点不同的定点双突变体。方法: 借鉴DNA shuffling技术中DNA小片段延伸扩增获得全长DNA片段的工作原理,与常规基因定点突变技术相结合,改进重叠延伸PCR技术构建定点双突变。结果:对嗜酸热脂肪杆菌(Alicyclobacillus acidocaldarius)Tc-12-31的甘露聚糖酶基因AamanA中两个可能的活性位点E151和E231进行双点突变,先后经过无引物和有引物两步PCR,扩增获得全长DNA,测序结果表明得到预期的定点双突变体;酶活性检测和薄层层析结果表明双点突变体丧失了酶的活性。结论: 改良的重叠PCR技术,能经济、简便、高效地获得双点定点突变体,在酶的催化机理的阐述、蛋白质结构改造等分子生物学领域中具有较高的应用价值。     

以P型职PRSV-VPg为模板多位点突变获得W型PRSV-VPg的研究

以P型PRSV-VPg(Papaya Ringspot Virus,VPg gene)基因为模板利用高保真酶通过两对引物(突变4个位点),PCR扩增获得一条276 bp和一条141 bp的两个小片段.以此为基础,利用长引物延伸和套叠PCR的方法,定点突变剩余5个位点,最后拼接获得W型PRSV-VPg基因.结果表明通过上述方案可以成功改造P型PRSV-VPg基因,获得与W型朋PRSV-VPg同源的目的基因,成功率为100%.该技术可以绕开传统的通过寻找毒源进行RT-PCR方法和人工合成方法获得病毒基因,其技术操作简单,且节约时间和成本,并在人工合成基因、多位点基因定点突变等方面具有很好的借鉴作用和应用价值.     

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.