抗HPV11／E2基因Ribozyme对靶RNA的体外剪切研究

为了寻找HPV11型引起的生殖系统感染的治疗途径和探讨HPV的致病机理,本实验以HPV11病毒质粒为模板,扩增出HPVll型E2区644bp片段,采用pGEM-T-Easy Vector为载体,构建pTV-644克隆载体,经筛选得到克隆株,提取质粒测序鉴定。采用上海生化所陈农安教授编制的锤头状Ribozyme设计软件进行计算机分析,选择Ribozyme对靶基因的最佳剪切位点,及进行基因同源性分析和生物学功能分析,选择出针对HPVllE2靶基因的RZ2777,在最适条件下进行体外剪切反应,发现人工合成和体外转录得到的Ribozyme分子均能在相应位点准确切割靶RNA分子,选择合适的反应条件切割效率达到60％以上,Km和Kcat值分别为0.63μmol/L、0.12μmol/L,RibozymeL两端的5′-cis-ribozyme和3′-cis-ribozyme自我剪切释放并未影响切割活性,但靶RNA侧翼序列影响了Ribozyme的剪切活性。实验研究表明,Ribozyme可能成为治疗HPVll型引起的尖锐湿疣的有效手段,并有望在分子水平上开辟出基因治疗HPVll病毒感染的另一新天地。     

抗HPV11 E2核酶的计算机设计

 借助计算机软件分析 ,设计出能特异性切割HPV11型 6 4 4ntE2mRNA的核酶 (ribozyme) .遵循Symon′s锤头状核酶结构和GUX剪切位点原则 ,靶序列存在 32个这样的剪切位点 .通过计算机软件分析出核酶的最佳剪切位点 ,并对底物及核酶的二级结构进行预测及进行相应基因生物学功能和基因同源性分析 ,筛选出 2个锤头结构核酶 .针对这两位点设计的核酶分别命名为RZ2 777和RZ32 81.计算机分析显示 ,两核酶与底物切点两翼碱基形成锤头状结构 ,切点所在基因序列具有相对松弛的二级结构 ,位于该基因重要生物功能区内 ,是核酶的理想攻击区域 .通过基因库检索 ,在已知人类基因排除了与上述两核酶切点两翼碱基有基因同源性序列的可能性 .将两核酶用于体外剪切实验取得了良好的实验结果 ,认为借助计算机分析可帮助尽快从多个剪切位点选择出最适核酶     

丙型肝炎病毒RNA特异性核酶的切割活性研究

针对丙型肝炎病毒RNA(HCV-RNA)的5′非编码区和部分C区的二级结构,设计并合成了四个不同的锤头型核酶(ribozyme A, ribozyme B, ribozyme C₁, ribozyme C₂).首先应用体外切割实验筛选出作用于HCV-RNA起始密码子上游GTA↓位点的核酶RzA有较好的活性.为初步验证核酶RzA在细胞内的切割活性,经脂质体介导,将RzA-RNA与另一携带该核酶靶基因的质粒表达载体pCl-neo-luciferase(载体中荧光素酶基因受核酶靶基因的调控)共转染HepG₂细胞.通过测定荧光素酶基因的表达证实了核酶在细胞内有较好的切割活性.在此实验基础上,把RzA基因克隆至pCl-neo质粒表达载体中,再次经脂质体介导,将重组的表达载体pCl-neo-RzA与携带该核酶靶基因的质粒表达载体pCl-neo-luciferase共转染HepG₂细胞,获得了更好的切割效果.     

抗HPV16 E7-ribozyme在CV-1细胞中的稳定表达

构建了由RSV—LTR启动子带动并能在细胞内稳定复制的Ribozyme的自身修剪表达质粒pRSV—Rz523、Ribozyme反义对照质粒pRSV—AE7及人增殖细胞核抗原基因(PCNA)启动子带动的HPVl6 E7片段(+554～+686)的真核表达质粒pPCNA—E7。经G418抗性筛选获得了稳定表达Ribozyme的CV-1细胞克隆,其表达水平约为9．Opmol／lO6个细胞,其中活性Ribozyme的量大于50fmol／lO6个细胞,分离得到的Ribozyme可在体外特异切割E7靶RNA片段。通过共转染Ribozyme(或反义对照)和底物表达质粒并筛选出细胞克隆．研究了Ribozyme在细胞中对底物表达水平的影响。初步结果显示．Rihozyme的导人可使细胞内底物E7的RNA表达水平降低了90％(反义对照使E7 RNA表达降低20％)。上述结果提示：在CV-1细胞中表达的Ribozyme不仅在体外,同时在细胞内具有一定的生物学活性,有可能应用于逆转官颈癌细胞的恶性表型。     

pRNA导入型Ribozyme的设计和体外转录制备

目的:枯草杆菌的包装RNA分子pRNA是新型纳米分子载体,将其同锤头型核酶Ribozyme重组可以构建结构稳定、能进入细胞、主动识别结合和剪切基因RNA的pRNA-Ribozyme.由于目前100 nt以上的RNA分子采用化学合成制备较为困难,实验采用基因重组构建并体外转录制备170 nt的pRNA-Ribozyme....     

抗HPV11E2核酸的计算机设计

借助计算机软件分析,设计出能特异性切割HPV11型644nt型644ntE2mNA的核酶。遵循Symons锤头状核酶结构和GUX剪切位点原则,靶序列存在32个剪切位点,通过计算机软件分析核酶的最佳剪切位点,并对底物及核酶的二级结构进行预测及进行相应基因生物学功能和基因同源性分析,筛选出2个锤头结构核酶。针对这两位点设计的核酶分别命名为RZ277和RZ3281。计算机分析显示,两核酶与底物切点两翼碱基形成锤头状结构,切点所在基因序列具有相对松驰的二级结构,位于该基因重要生物功能区内,是核酶的理想攻击区域,通过基因库检索,在已知人类基因中排除了与上述两核酶切点两翼碱基有基因同源性序列的可能性。并非所有的GUX位点（X：C、U、A）或CUX均可作为核酶的最佳剪切切割反应,为下一步将核酶用于细胞内和体内试验打下基础。     

应用核酶体外切割马铃薯卷叶病毒外壳蛋白基因的研究

针对马铃薯卷叶病毒外壳蛋白基因第356～358位点“GUC”．设计、合成了一种“锤头状”核酶。将核酶基因克隆在体外转录载体PSPT19的SP6启动子下游;同时将PLRVCPcDNA亚克隆在体外转录载体pSPT18的SP6启动子下游。利用SP6RNA聚合酶分别体外转录,获得核酶分子和靶RNA序列。在41℃保温进行核酶切割反应,检测到预期大小且被切开的两个RNA短片段。     

抗BmNPV即刻早期蛋白三联ribozyme在家蚕细胞中的表达

为了阻断家蚕核多角体病毒（BmNPV）的基因表达,以BmNPV的即刻早期蛋白基因(IE)为靶序列,设计了三联ribozyme．体外切割反应表明,该ribozyme能特异地切割靶序列的mRNA;细胞实验表明,细胞中表达的ribozyme也能够特异地切割靶序列,从而使受BmNPV感染的Bm-N细胞中的多角体减少约30％．     

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.