部分酶解酵母高效电击转化研究

以酵母质粒YCp50为外源DNA,电击转化部分酶解酵母宿主菌AB1380,转化效率稳定在10~6转化子/μg质粒DNA左右,比不酶解酵母或酵母原生质球作受体的电击转化效率高一个数量级以上,也比PEG介导的酵母原生质球转化高3～5倍,而且适合于大片段DNA如水稻YAC分子的转化。达最佳转化时的有关技术参数为:新接菌种通气培养至细胞密度1×10~8～1.5×10~8个/ml;转化时细胞密度控制在1×10~9～1.5×10~9个/ml;每毫升酶解缓冲液加15u溶菌酶(lyticase),30℃下处理酵母5min进行部分酶解;电击时,电场设置在6.25kV/cm、电容25μF,电击后直接铺板。     

HBsAg基因在酵母细胞中的表达

先前从酵母染色体[DNA中得一个作用较强的启动基因片段,使HBsAg基因在其控制下,构建了大肠杆菌一酵母细胞穿梭质粒。这种质粒转化的酵母细胞能生产和装配HBsAg颗粒。经放射免疫分析,超离心沉降研究和电子显微镜观察,酵母产生的HBsAg颗粒与人血清中的HBsAg颗粒十分相似。     

一种简便的适用于酵母双杂交系统的酵母质粒提取方法

目的:建立一种适用于酵母双杂交系统的简便快捷的酵母质粒提取方法。方法:以酿酒酵母为供试材料,用玻璃珠振荡法破除酵母细胞壁,提取酵母总DNA,最后通过电转化大肠杆菌DH10B获得目的质粒。结果:粗提得到的质粒可直接转化DH10B,作为模板用于PCR分析及酵母双杂交后续的序列分析等,大大降低了工作量。结论:该方法简便快捷,经济实用,降低了成本,提高了效率,可以作为一种实验室酵母质粒提取方法。     

人血清白蛋白和粒细胞集落刺激因子融合蛋白的克隆表达

构建重组人血清白蛋白粒细胞集落刺激因子（HSA-hG-CSF）表达载体,用毕赤酵母表达该重组蛋白。PCR扩增出人血清白蛋白基因（HSA）和粒细胞集落刺激因子基因（hG-CSF）,GGGGS作为小肽接头,采用重叠PCR的方法将HSA和hG-CSF拼接起来,与质粒载体pPIC9K连接,转化大肠杆菌感受态细胞DH-5α。抽提质粒,用SalI酶切重组质粒,电转化法导入毕赤酵母SMD1168中,通过表型筛选和诱导表达实验得到蛋白表达工程菌。Western-blotting分析表明融合蛋白具有粒细胞集落刺激因子免疫原性。NFS-60细胞测活实验分析表明体外活性达到约4.0×10^7IU/mg。     

海洋红酵母电转化条件的研究

采用电穿孔的方法对海洋红酵母进行外源DNA的转化.通过调整参数,对影响电转化的主要因素进行探索,初步建立了载体pTEF1/Zeo-rDNA对海洋红酵母的高效电转化方法.结果表明,当采用对数生长中期的菌体制备感受态细胞、电压为900V、质粒浓度为20mg/L和0.2cm电转化杯时,转化率达到最大值,为每微克质粒DNA 52个转化子.经抽样鉴定所得到的转化子均为阳性克隆.首次建立了以海洋红酵母为宿主的高效电转化体系,为外源基因在海洋红酵母中的表达奠定了基础.     

虹鳟生长激素cDNA在酵母中的表达

采用聚合酶链式反应（ Ｐ Ｃ Ｒ） 技术对虹鳟生长激素ｃ Ｄ Ｎ Ａ 进行改造。将改造后的基因克隆到含酵母 Ｐ Ｇ Ｋ 启动子的大肠杆菌酵母穿梭质粒ｐ Ｍ Ａ９１ ,转化酿酒酵母 Ｙ３３ ,构建表达鱼生长激素的酵母工程菌 Ｙ３３（ｐ Ｍ Ａｒ Ｇ Ｈ１６） ,并在酵母中获得表达,表达量约占细胞可溶性蛋白总量的３ ％ 。表达产物作为饲料添加剂投喂罗非鱼,具有明显的促进生长作用     

枯草芽孢杆菌bgls基因在酵母染色体上整合

将枯草杆菌β－1,3—1,4－葡聚糖酶基因(bgls)2．7kbEcoRl片段和酵母染色体Rdna片段克隆到整合型不含酵母自主复制序列(Autonomusly replicating sequence)的大肠杆菌／酵母菌穿梭质粒YIP5上,构建成YIP5-bgls—Rdna的杂种质粒PCZH,转化S．Cerevisiae并得到表达。稳定性测定表明．Y 33(PCZH101)和Y 33(PCZH104)在无选择压力的YEPD培养基中繁殖70代以上,两个转化子90％以上的酵母细胞仍含有质粒并且遗传特征与染色体行为相似。以bgls基因作探针;与酵母染色体DNA的Southern bloot分子杂交证实bgls基因已整合到酵母菌染色体上。     

人成骨蛋白成熟肽基因在毕氏酵母(Pichia Yeast)中的高效表达

采用PCR方法,根据文献报道的人成骨蛋白（osteogenic protein-1,OP-1）成熟肽基因序列,设计并合成一对引物,从构建的含人成骨蛋白基因的质粒中扩增获得大小为420bp的DNA片段,连接到pGEM-T载体进行测序,证明获得人成骨蛋白成熟肽基因片段,并以pPIC9K为表达载体构建重组表达质粒,转化大肠杆菌细胞,经鉴定的阳性重组质粒并线形化,电转化毕氏酵母细胞GS115,于30℃进行甲醇诱导分泌表达,表达产物存在于培养基中,占分泌蛋白的10％.重组表达产物进行Western Blot可以检测到重组表达产物,ELISA检测其具有特异性结合活性.     

glyA基因的克隆及其在毕赤酵母中的表达

根据已知的序列设计引物,以大肠杆菌XL10-Gold总DNA为模板进行梯度PCR,并进行DNA序列测定,其序列与已经报道的glyA基因完全一致。将其克隆到毕赤酵母分泌型表达载体pHBM905C上,获得表达质粒pHBM1001.该质粒转化毕赤酵母GS115所得重组子经PCR验证后成功进行了诱导表达,并初步测定了酶活力。     

扣囊复膜孢酵母β-葡萄糖苷酶基因在毕赤酵母中的克隆与表达

利用PCR技术,从扣囊复膜孢酵母的总DNA中扩增得到β-葡萄糖苷酶（β-Glucosidase）基因（BGL1）,长度为2596 bp,连接到pGEM-T载体上,用限制性内切酶切下目的基因,插入到巴斯德毕赤酵母表达载体pPIC9K中,使之位于α-因子信号肽下游,且与之同框,构建成重组质粒pSHL9K.通过电转化将重组质粒pSHL9K插入到Pichia pastoris GS115菌株染色体中,获得高效表达BGL1基因的毕赤酵母重组工程菌株.重组酶的最适温度为50℃,最适pH为5.4.培养基中β-葡萄糖苷酶活性最高可达47U/mL.     

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.