右旋糖苷蔗糖酶基因的克隆及其序列分析

以筛选的肠膜明串珠菌的基因组为模板,通过聚合酶链式反应(PCR)分别扩增得到右旋糖苷蔗糖酶的基因片段dsr1和dsr2,将基因片段克隆到pUC19载体上并对基因片段组装得到完整基因序列dsrx,通过限制性酶切分析和核苷酸序列分析鉴定,dsrx的序列全长为4,566bp,编码1,522个氨基酸,与GenBank中已注册的U81374核苷酸序列同源性达99％,推导的氨基酸序列与其序列同源性达98．49％。     

幽门螺杆菌Ⅳ型分泌系统cagT基因的克隆表达及其重组蛋白对SGC-7901细胞增殖和分泌细胞因子功能的影响

[目的]初步研究幽门螺杆菌(Helicobacter pylori,H.pylori)NCTC 11637 cagT(HP0532)基因对SGC-7901细胞增殖和白细胞介素-8(IL-8)分泌的影响.[方法]应用PCR技术从H.pylori基因组DNA中扩增cagT编码基因片段,将其定向插入pQE30载体中,双酶切鉴定筛选阳性克隆.测序分析确认后,IPTG诱导表达,表达蛋白以Ni2 -NTA柱进行纯化,并经Western blot鉴定,纯化的蛋白作用于SGC-7901细胞,提取细胞总RNA,RT-PCR扩增IL-8,并用MTT法检测蛋白对细胞增殖的影响.[结果]成功克隆了cagT基因,全长843bp(GenBank 登录号为 EF114758),编码280个氨基酸,与GenBank公布的其他H.pylori菌株基因序列的核苷酸同源性为97%～99%.工程菌诱导后SDS-PAGE显示新生表达蛋白带,相对分子质量约为32kDa,经Ni2 -NTA柱纯化后可获得纯度为98%重组蛋白.经透析复性后的蛋白(终浓度10μg/mL)作用于SGC-7901细胞后,可诱导细胞因子IL-8在mRNA水平上的表达.不同浓度CagT蛋白与SGC-7901细胞作用,细胞增殖的程度随着蛋白浓度的增加逐渐降低,细胞的增殖受到抑制.[结论]CagT蛋白可刺激SGC-7901细胞IL-8的表达,并抑制细胞增殖,为进一步研究其生物学功能奠定了基础.     

奶山羊β-乳球蛋白基因5'、3'调控区的克隆和序列分析

目的:克隆奶山羊β-乳球蛋白(BLG)基因5'、3'调控区,并对其进行序列分析.方法:从徐淮奶山羊组织中提取基因组DNA,采用高保真长模板PCR法克隆得到奶山羊BLG基因4.2 kb的5'调控区和1.8 kb的3'调控区;将纯化后的PCR产物克隆到pGEM-T Easy载体中,经酶切鉴定和序列测定验证克隆的正确性.结果:部分序列分析表明,奶山羊BLG基因5'区与GenBank中登录的山羊和绵羊BLG基因5'区的同源性分别为100%和95%,3'区的同源性分别为99%和93%;克隆得到的奶山羊BLG基因调控区与山羊BLG伪基因显著不同,二者5'区和3'区的同源性分别为83%和88%.结论:克隆得到的奶山羊BLG基因调控区可用于构建乳腺特异性表达载体,用于外源基因在转基因动物乳腺中的高效表达.     

Cpn0308基因真核载体构建及鉴定

目的:构建Cpn0308基因真核表达重组质粒,为肺炎衣原体(Chlamydia pneumoniae,Cpn)核酸疫苗的研制做准备。方法:用PCR技术从Cpn AR39株基因组DNA中扩增Cpn 0308基因,经双酶切、连接等反应,重组入pcDNA3.1/HisA真核表达载体,转化到感受态细胞,再经含氨苄青霉素的LB培养基筛选,酶切、PCR扩增及测序鉴定。结果:从Cpn AR39株基因组DNA中扩增出特异的Cpn 0308基因,约400bp;酶切、重组、转化、筛选鉴定出pcDNA3.1/HisA-Cpn0308重组质粒;序列测定证实与GenBank登录的肺炎衣原体Cpn AR39株Cpn0308基因一致。结论:功地构建了pcDNA3.1/HisA-Cpn0308重组质粒,为肺炎衣原体核酸疫苗的研制奠定了基础。     

右旋糖苷蔗糖酶基因的克隆及其序列分析*

以筛选的肠膜明串珠菌的基因组为模板,通过聚合酶链式反应(PCR)分别扩增得到右旋糖苷蔗糖酶的基因片段dsr1和dsr2,将基因片段克隆到pUC19载体上并对基因片段组装得到完整基因序列dsrx,通过限制性酶切分析和核苷酸序列分析鉴定,dsrx的序列全长为4,566bp,编码1,522个氨基酸,与GenBank中已注册的U81374核苷酸序列同源性达99%,推导的氨基酸序列与其序列同源性达98.49%。     

变色栓菌漆酶基因的克隆与序列分析

本研究以PCR扩增的方法,从变色栓(菌Trametes versicolor)的基因组DNA中扩增出了一预期大小的DNA片断,并将其克隆到了pUCM-T载体上。经筛选、酶切、PCR鉴定,序列分析,证明该片断为变色栓菌漆酶基因的克隆。该基因的开放阅读框由1560个核苷酸组成,编码一个由519个氨基酸组成的多肽。与GeneBank中发表的Laccase基因(AY081188)序列比较发现,编码的氨基酸序列同源性为96%,而核苷酸序列的同源性为92%。     

甘薯叶绿体rbcL基因的克隆与序列分析

根据烟草、水稻和菠菜叶绿体的全基因组序列设计引物,以甘薯的叶绿体基因组DNA为模板,PCR扩增包舍甘暑叶绿体rbcL完整基因(GenBank登录号为AY942199)在内的一段序列.序列分析表明:此片段的全长为1 627 bp,包括1 443bp的编码区序列在内.推测编码480个氨基酸,同时构建了此片段的限制性酶切图谱.相似性比较显示,此基因编码区序列与烟草、菠菜、小麦、水稻、玉米、矮牵牛、紫花苜蓿、拟南芥、莨菪、葡萄以及甜菜的rbcL基因核苷酸的同源性为85%～98%,氨基酸的同源性为92%～95%.     

阴道毛滴虫氢化酶体腺苷酸激酶基因的克隆及序列分析

目的-克隆阴道毛滴虫氢化酶体腺苷酸激酶(AK)基因,并测定其序列,进行序列分析。方法-根据AK基因已知序列设计合成一对引物,应用PCR技术从阴道毛滴虫基因组DNA中扩增出AK基因,并将其克隆入pMD18-T simple载体。阳性克隆的重组质粒经酶切及PCR鉴定后,用双脱氧链末端终止法进行基因序列测定。应用BLAST软件辅助分析所测基因与Genbank中阴道毛滴虫氢化酶体AK序列的同源性。结果-PCR扩增得到特异的阴道毛滴虫氢化酶体腺苷酸激酶基因序列。酶切及PCR鉴定获得了正确的PT-AK重组质粒。测序表明,所克隆的AK基因大小为690bp,编码229个氨基酸。序列分析表明,所测基因与Genbank中阴道毛滴虫氢化酶体AK序列具有高度同源性(99．9％)。结论-克隆了阴道毛滴虫氢化酶体腺苷酸激酶基因,序列测定及同源性分析表明,所测基因与Genbank中阴道毛滴虫氢化酶体AK序列具有高度同源性。     

浙江省麻疹病毒的基因特征分析

目的分析浙江省流行的麻疹病毒(MV)的基因特征,为更好地防控麻疹提供科学参考。方法从GenBank检索并下载浙江省所有麻疹病毒株、中国麻疹疫苗株和WHO推荐参考株的血凝素蛋白(H)和核蛋白(N)的基因组序列,利用MEGA 6.0软件进行比对分析,构建种系进化树,确定浙江省麻疹病毒流行的基因型别,并进行同源性和基因变异分析。结果浙江省麻疹病毒以H1基因型为主(27株),其他基因型为辅(2株B3型)。H1a亚型(21/27)占绝对优势,其次为H1b亚型(6/27)。浙江省所有毒株间H蛋白的氨基酸同源性为96.9%~100.0%,与疫苗株Shanghai-191和Changchun-47的同源性均为95.0%~96.0%。浙江省所有毒株间N蛋白羧基末端的氨基酸同源性为82.7%~100.0%,与疫苗株Shanghai-191的同源性为85.8%~89.5%,与疫苗株Changchun-47的同源性为87.3%~91.0%。结论麻疹病毒H1基因型为浙江省麻疹流行的优势株,与现行参考疫苗株(A基因型)差异较大,因此针对麻疹病毒H1基因型疫苗的研制是今后浙江省麻疹病毒防控的关键。     

新城疫分离毒HN基因的分子特性和片段同源相关性

选取国内1997-2005年分离的新城疫病毒(Newcastle disease virus,NDV)24株,经蚀斑纯化克隆其血凝素-神经氨酸酶(HN)基因,与在GenBank发表的36株国内外不同时期的NDV毒株,进行氨基酸遗传变异分析,并利用SPSS8.0软件对其不同片段的氨基酸进行同源相关比较。结果显示:国内所有NDV分离毒株氨基酸高度同源,同源性为94.4%-99.4%;与LaSota、Clone30疫苗株等的氨基酸同源性为86.9%-89%;与强毒株F48E9的氨基酸同源性为87.9%-89.9%;与国外NDV的氨基酸同源性为87.2%-96.2%。系统发育分析表明:国内NDV分离毒HN遗传距离较近,而与LaSota、Clone30和F48E9遗传距离较远。国内NDV分离毒均缺乏538-540位糖基化位点。不同片段与全长的氨基酸同源性高度相关,且与前80个氨基酸相关最密切。     

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.