重组人粒细胞集落刺激因子Cdna在哺乳动物细胞中高效表达的研究

利用PCR反应、DNA测序、基因重组等技术,构建了两个表达人粒细胞集落刺激因子cDNA的重组质粒pED－GCSF和pEF－GCSF,两质粒分别转染COS7细胞作瞬时表达,转染CHO－dhfr－细胞作稳定表达。结果两质粒在COS7细胞和CHO细胞均获得了表达,pED－GCSF转染COS7细胞48h、72h的表达量分别为5.2×10⁴pg/ml和2.3×10⁵pg/ml,pEF－GCSF转染COS7细胞后48h、72h的表达量分别为2.8×10⁵pg/ml和1.4×10⁵pg/ml。转染CHO－dhfr－细胞,随着加入的氨甲喋呤(MTX)浓度升高,CHO－dhfr+克隆数减少,但平均每个克隆的rhG－CSF表达量升高,在0.5μmol/L MTX下最高表达rhG－CSF细胞株的量是4.46μg/ml/3d。且表达的rhG－CSF注射小鼠腹腔可提高小鼠外周血白细胞的数量。     

人组织型纤溶酶原激活剂(t-PA)组合突变体FrGGI在CHO细胞中的高效表达

为了得到t—PA组合突变体FrGGI在CHO细胞中的高效表达,将表达质粒筛选基因启动子上游的增强子(enhancer)去除．构建了FrGGI真核表达质粒pZLFrGGI。酶切线性化后．采用大剂量DNA电击介导法,转染dhfr基因缺陷型中国仓鼠卵巢细胞系(CHO-dhfr^-)。氨甲喋呤(MTX)筛选转染细胞,混合加压．挑选克隆。在1×10^-7mol／L MTX压力下。获得表达水平达1 500～2500Iu/106细胞·24h的细胞株。此细胞株表达水平稳定,形态良好．倍增时间约为36h．且有进一步提高表达水平的潜能,有望发展为工程细胞株。     

用反式互补表达载体实现全抗体在CHO-dhfr-细胞中的高效表达

目的：通过弱化筛选基因二氢叶酸还原酶基因（dhfr）,构建双顺反子全抗体表达载体,实现全抗体在CHO细胞中的高效表达。方法：将dhfr基因分为分别编码1-105和106～187氨基酸残基的2部分,分别通过linker与亮氨酸拉链GCN4融合,分别通过内部起始位点序列与抗体轻重链基因偶联,构建成2个双顺反子表达载体pIRESLZdhfr—H和pIRESLZdhfrL。用脂质体2000转染CHO-dhfr-细胞,用无次黄嘌呤和胸腺嘧啶脱氧核苷的IMDM筛选培养基筛选阳性克隆。结果：筛选到的阳性克隆表达水平为1．47-3．5μg／（106细胞·d）,经氨甲蝶呤（MTX）梯度加压,在MTX浓度为5×10^-8mol／L时,表达水平达到11．5μg／（10^6细胞·d）。结论：构建了基于亮氨酸拉链二聚化基础的dhfr弱化方式的双顺反子表达载体,实现了全抗体在CHO—dhfr-细胞中的高效表达。     

L615细胞染色体上二氢叶酸还原酶扩增基因的定位

在L615细胞株的细胞中发现有双微体(DM)。同样,这种细胞株的细胞中也有均质染色质区(HSR)的染色体。这条染色体为中度长短,HSR位于这条染色体的中部靠近顶端着丝点处。DM和HSR都是二氢叶酸还原酶(DHFR)基因的顺序,它们是DHFR基因的扩增。由于DHFR基因的扩增,细胞超量产生二氢叶酸还原酶,所以L615细胞对叶酸的同系物药物——氨甲喋呤(MTX)是抗性的。     

人组织型纤溶酶原激活剂(t-PA)突变体在CHO细胞中的高效表达

构建了二氢叶酸还原酶(dhfr)选择基因启动子上游无增强子(cnhancer)的组织型纤溶酶原激活剂(t-PA)组合突变体FrGGI真核表达质粒pZLFrGGI。将pZLFrGGI酶切线性化,采用大剂量DNA电击介导法,转染dhfr基因缺陷型中国仓鼠卵巢细胞系(CHO-dhfr~-)。用氨甲喋呤(MTX)筛选转染细胞。混合加压后,挑选克隆,在1×10~(-7)mol/L MTX压力下,得到了表达水平达1500～2500IU/10~6细胞·24小时的细胞株。此细胞株表达水平稳定,形态良好,倍增时间为36小时,且有进一步提高表达水平的潜能,有望发展为工程细胞株。     

中国仓鼠卵巢细胞表达新技术

中国仓鼠卵巢细胞（CHO细胞）是基因工程药物生产的最佳表达系统之一,在生物制药中被广泛应用。传统的获得高表达CHO细胞株的方法费时、费力。近年来出现了一些CHO细胞高效表达新技术,它们从克服位置效应,提高基因转录效率、mRNA翻译效率及稳定性、筛选高表达细胞的效率等不同层次调控外源基因在CHO细胞中的高效表达。与MTX加压扩增基因获得高效表达外源基因的方法比较,能够节约时间、减少工作量,不易丢失高表达细胞株。     

proUK-KGDW融合基因在CHO细胞中的高表达

利用常规分子生物学技术,构建了新型高效的proUK-KGDW融合基因的分泌型哺乳动物细胞表达载体。将该载体线性化后转染CHO/dhfr-细胞,经G418筛选获得阳性克隆,然后挑取表达水平较高的克隆进行MTX加压扩增,以提高proUK-KGDW杂合体的表达水平,经2～3轮MTX加压扩增,获得多株表达水平超过10μg/(106细胞·24h)的稳定的高表达细胞株,为proUK-KGDW杂合体的制备及功能研究奠定了基础。     

异源PDGF-A链表达对CHO细胞生长和转化的作用

用DNA磷酸钙盐沉淀方法把含人PDGF(血小板衍生生长因子)A链cDNA的表达质粒pSV_2neo-A转染CHO细胞(中国仓鼠卵巢细胞),然后经G 418(400-800 μg/ml)筛选分离20个转染细胞株。选出其中At_1和Aot7细胞株所进行的实验结果表明,这些细胞的形态和生长行为均发生明显的变化,PDGF-A链mRNA的表达水平比CHO细胞明显增高,胞质有强阳性的PDGF荧光反应,显示有PDGF样蛋白的合成。这些细胞不但生长速率加快,有高密度持续生长的特性,而且能在软琼脂培基上形成大集落和在裸鼠体内接种形成纤维肉瘤,提示外源PDGF-A链基因的表达有使CHO细胞生长失控和发生细胞恶性转化的作用。     

水稻原生质体产生细胞团的冰冻保存和冻后再生植株形成

水稻(Oryza sativa L.)原生质体产生的细胞团加上10-20％的二甲亚枫(DMSO)和10-20％的蔗糖,置于液氮中保存。冻后细胞生存率达到对照的40-50％。存活的细胞在附加2×10~(-5)mol/l 2,4-D 的Linsmier-Skoog(Ls)固体培养基上再生长,然后将形成的愈伤组织块转到附加10~(-6)mol/l NAA,4×10~(-6)mol/l 激动素和10~(-6)mol/l 2 IP 及8％的蔗糖的 LS培养基上分化出芽并形成植株。     

红细胞生成素cDNA在中国仓鼠卵巢细胞中的稳定表达

将(?)红细胞生成素(EPO)cDNA构建的重组表达质粒用电穿孔法引入COS-7细胞,ELISA和红系集落测活结果表明,该重组质粒在哺乳动物细胞中能够表达有生物活性的红细胞生成素。进一步将其转染CHO-dhfr~-细胞,经氨甲喋呤(MTX)加压扩增,混合细胞中各克隆表达水平比较一致,细胞的平均表达水平为2-3μg/10~6 Cells/24hr。细胞冻存后复苏其表达水平与冻存前一致,表明外源基因整合稳定。     

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.