马尔堡病毒GP蛋白C肽的抑制活性及作用机理研究

目的:研究马尔堡病毒(MARV)包膜糖蛋白(GP)C肽的抗该病毒进入活性及初步作用机制。方法:选取MARV的GP2蛋白上CHR区域序列(611～637残基),合成该序列多肽(C肽)及突变体,利用pVAX1-MGP与pNL-4.3-Luc-R-E-质粒包装假病毒体系,测定各肽对马尔堡假病毒融合的抑制活性,利用圆二色谱、凝胶色谱分析C肽及其突变体对MARV的N肽的相互作用。结果:C肽对马尔堡假病毒融合抑制的IC_(50)为32.67μmol/L,C肽突变体的IC_(50)为9.61～33.47μmol/L,抑制活性高于C肽或与其相当;C肽与N肽形成双螺旋结构,在C肽的7元螺旋体的b、f、c、g位引入离子相互作用氨基酸可提高多肽本身的α螺旋结构含量,一些突变可提高复合物的α螺旋结构含量,但C肽与N肽混合后形成的复合物含量不高。结论:MARV的GP蛋白C肽与N肽相互作用较弱,C肽的抗病毒活性较弱,但改造后可提高活性。     

基质金属蛋白酶2抑制剂的筛选及鉴定

以原核表达的具有明胶水解活性的人基质金属蛋白酶 2的催化区 (MCD)为靶标 ,筛选噬菌体随机环七肽库和十二肽库 .找到 6种与MCD特异结合的小肽 ,将 6种小肽基因分别与GST表达质粒重组 ,进行GST融合表达 ,制备融合蛋白 .采用Glutathione Sepharose 4B亲和层析法纯化融合蛋白 ,通过酶抑制实验、体外侵袭实验检测融合蛋白的活性 .结果表明 ,GST C71能够抑制MCD水解 β酪蛋白的活性 ,并且对人纤维肉瘤细胞HT10 80的体外侵袭有明显的抑制作用     

前肽缺失vWF促进细胞分泌蛋白质剪接的L303E／F309S突变体凝血第八因子

该文旨在探索前肽缺失von Willebrand子（vWF-△Pro）对蛋白质剪接的L303E／F309S突变体凝血第八因子（FVIII）分泌的影响。将vWF-△Pro基因与蛋白内含子融合的F聊重链和轻链基因共转HEK293胞。结果显示,转vWF－△Pro细胞的剪接蛋白FVIll分泌量和活性分别为（196±27）ng／mL和（1．39±0-31）IU／mL,明显高于对照细胞的（116±24）ng／mL和（0．91±0．18）IU／mL。表明vWF－△Pro可提高剪接的L303E／F309S突变体FVⅢ蛋白分泌量和活性。     

△^5—3β，7β-二羟基甾醇及其C-7差向异构体波谱特征及胆碱酯酶的抑制活性

本文对△^5-3β,7β-二羟基甾醇（1—3）和△^5-3β,7α-二羟基甾醇（4～6）的一些核磁共振波谱特征进行了比较。活性测试表明化合物1—6对乙酰胆碱酯酶（AChE）无明显的抑制活性,对丁酰胆碱酯酶（BuChE）则有较强的抑制活性,其中24-亚甲基胆甾-5-烯-3β,7α-二醇（6）的IC50值为9．5μM。通过活性数据比较我们发现7α-羟基甾醇对丁酰胆碱酯酶的抑制活性明显比相应的7β-羟基甾醇高。我们通过计算7位羟基和四环平面之间的二面角角度来尝试解释这些活性差别。     

ATP依赖的人Lon蛋白酶的表达、纯化及其活性鉴定

ATP依赖的人Lon蛋白酶是一种同质寡聚、环状的蛋白酶,主要位于细胞线粒体基质中。许多研究表明,Lon蛋白酶对于维护细胞的内环境稳定起着重要作用,并参与线粒体蛋白质量控制和代谢调控。将pPROEX1 His6-Lon重组质粒在Escherichia coli Rosetta 2菌株中诱导表达用Ni2＋柱亲和层析法纯化,获得纯度较高的目的蛋白。经纯化后,Lon蛋白酶的比酶活达到0.17 U/mg。通过多肽底物Rhodamine 110、bis-（CBZ-L-alanyl-L-alanine amide）[（Z-AA）2 Rh110]的降解检测显示,Lon蛋白酶具有肽酶活性,并被ATP所刺激。Casein和线粒体转录因子A降解实验表明,纯化的Lon蛋白酶具有蛋白水解活性,而且蛋白水解活性依赖于ATP。     

乙型肝炎病毒靶向核糖核酸酶及其突变体的原核表达和活性分析

目的：研究原核表达的乙型肝炎病毒（HBV）靶向核糖核酸酶（RNase）及其突变体（点突变失去RNase活性）的活性。方法：将构建的靶向核糖核酸酶及其突变体基因克隆入原核表达载体pET32a（＋）,转化大肠杆菌BL21（DE3）,以IPTG诱导融合蛋白（HBV核心蛋白与人嗜酸性粒细胞来源的神经毒素的融合蛋白）的表达;表达产物经包涵体纯化、SDS-PAGE和Western印迹鉴定,将纯化的蛋白用透析方法复性;以酵母tRNA为作用底物,应用复性的蛋白进行RNase活性分析。结果：纯化和复性了HBV靶向核糖核酸酶及其突变体;复性的HBV靶向核糖核酸酶可以降解酵母tRNA且具有剂量依赖性,而复性后的突变的靶向核糖核酸酶体不具有RNase活性。结论：原核表达的HBV靶向核糖核酸酶具有较强的RNase活性,为探索HBV靶向核糖核酸酶抑制乙肝病毒复制的机理奠定了基础。     

鞭毛蛋白FliC突变体／HPV18L2N融合蛋白的表达与纯化

目的：人乳头瘤病毒（HPV）的持续性感染导致女性宫颈癌的发生。HPV的次要衣壳蛋白L2可以诱发交叉中和多种型别HPV的中和抗体,但是单独免疫L2诱发的抗体滴度较低。鼠伤寒沙门氏茵鞭毛蛋白FliC是一种有效的佐剂。删除FliC超变区域的突变体可与外源抗原融合表达并且显著增强外源抗原特异性抗体的产生。本研究旨在构建鞭毛蛋白FliC超变区删除突变体与HPV18L2N（aa．13—154）的融合基因,通过大肠杆菌原核表达系统表达F1ic突变体与HPV18L2N的融合蛋白并纯化,为研究鞭毛蛋白的佐剂活性及新型HPV18L2疫苗奠定基础。方法：以鼠伤寒沙门氏菌鞭毛蛋白编码基因fliC为模板,通过重叠PCR法构建删除fliCD3区域（fliCAD3）、D3＋CD2a区域（fliCAD3CD2a）、D3＋D2区域（fliCAD2D3）的突变体,同时将HPV18L2N基因插入置换突变体的超变区删除区域。含有重组基因的表达载体在大肠杆菌中诱导表达,经SDS—PAGE及Westernblot鉴定分析。表达的融合蛋白经Ni—Sepharose亲和层祈纯化及Q-Sepharose离子交换层析去除内毒素。纯化后的融合蛋白经Native—PAGE鉴定分析,通过鲎试剂凝胶法测量蛋白溶液中的内毒素含量。结果：构建了pET22b．fliCAD3／18L2N、pET22b—nic△D3cD2a／18L2N、pET22b—fliCAD2D3／18L2N重组载体。重组载体在大肠杆菌以包涵体形式高效表达,且主要以单体形式存在。结论：通过原核表达及层析法纯化,成功获得了无热源、高纯度的鞭毛蛋白FliC突变体与HPV18L2N的融合蛋白,为增强HPVL2免疫原性提供了一种新的途径,为进一步研制HPV18L2疫苗奠定了基础。     

人胰高血糖素样肽-1突变体基因的克隆及表达

目的：克隆人胰高血糖素样肽-1突变体（^2Gly-hGLP-1)基因,高效表达GST-^2Gly-hGLP-1融合蛋白.方法:在获得重组hGLP-1基因工程菌基础上,利用定点突变技术改造其第2位丙氨酸为甘氨酸,经酶切克隆于pGEM-7z( )载体中,构建pGEM-4T-3/^2Gly-hGLP-1融合表达规模.SDS-PAGE和凝胶扫描分析,融合蛋白以可溶形式存在,其表达量占菌体总蛋白的29.7%。表达产物经亲和层析纯化后纯度在95%以上,免疫印迹证实,该融合蛋白可被异性hGLP-1（7-37）抗体所识别。结论：为产业化规模制备hGLP-1突变体提供技术线路。     

β-内酰胺酶抑制多肽的串联表达及抑制活性研究

细菌对β-内酰胺类抗生素及β-内酰胺酶抑制剂产生耐受性使新型β-内酰胺酶抑制剂成为抗生素领域的研究热点。我们曾通过酵母双杂交系统筛选到了一个β-内酰胺酶的结合多肽SIPIS04-01,体外实验表明其具有抑制氨苄西林的活性,为提高其表达量,本文尝试基因串联表达方式-随机定向串联法成功构建了密码子改造之后的小肽SIPIS04-01编码基因SIPIS04-01N的双拷贝质粒pYG563,使表达产物中融合蛋白占细菌蛋白总量的比例较单拷贝表达提高了48.4%。纯化所得小肽SIPIS04-01N对TEM-1类型的β-内酰胺酶具有体外抑制活性,并测定了抑制常数Ki。     

植物螯合肽合酶基因AtPCS2的表达调控

植物螯合肽合酶（pcs）受重金属离子激活,并以还原型谷胱甘肽为底物合成植物螯合肽（PCs）,在植物和真菌的重金属解毒机制中起重要作用．拟南芥基因组中有两个编码PCS的基因AtPCS1和AtPCS2,但AtPCS1单基因功能缺失即可导致相应的突变体cad1—3对镉高度敏感,其体内也检测不到PCs;而体外表达分析表明,AtPCS2具有完全的PCs合酶活性,预示植物体内可能存在AtPCS2的负向调控机制．基于该推测,构建了CaMV35S启动子驱动的AtPCS2基因编码区与c—Myc抗原标签融合的过表达载体．结果表叽在cadl-3的MV35S／AtPCS2：cMyc的异位表达株系中,AtPCS2的mRNA和蛋白都保持较高的表达量．不仅如此,AtPCS2具有植物螯合肽合成能力,并完全互补了cad1-3突变体的镉敏感性状．AtPCS2和EYFP的融合蛋白在细胞质有明显表达,在细胞核也检测到一定信号．以上结果表明,AtPCS2在植物体内可能主要受转录水平调控,而且可能具有调节PCs合成以外的其他生化功能．     

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.