猪2型圆环病毒ORF2基因在塞姆利基森林病毒RNA复制子衍生的新型真核表达载体中的表达

猪 2型圆环病毒 (porcinecircovirus 2 ,PCV2 )是断乳仔猪多系统衰竭综合征 (postweaningmultisystemicwastingsyndrome,PMWS)的原发性病原。PCV2的ORF2编码病毒唯一的结构蛋白Cap。根据GenBank中公布的PCV2JXL株的序列设计一对引物 ,应用PCR方法从该毒株感染的PK 15细胞中扩增出完整的ORF2基因 ,将此基因克隆于本实验室此前构建的塞姆利基森林病毒 (SemlikiForestvirus,SFV)RNA复制子衍生的新型真核表达载体Psfv1cs中的BamHⅠ位点 ,获得重组质粒pSFV1CS Cap。用pSFV1CS-Cap分别转染BHK-21细胞和293T细胞 ,经间接免疫荧光试验检测表明 ,PCV2 ORF2基因在转染细胞中得到表达。小鼠接种试验表明 ,该重组质粒能诱导小鼠产生特异性抗体.     

短双链RNA在病毒性心肌炎小鼠模型中的抗病毒研究

目的:通过在小鼠病毒性心肌炎动物模型研究短双链小干扰RNA(small interfering RNA,siRNA)对病毒感染和复制的抑制作用,研究RNAi在治疗病毒性疾病的可行性.方法:利用质粒载体将siRNA转染至HeLa细胞和Balb-c小鼠后感染病毒,荧光显微镜现察GFP表达量观察细胞内质粒转染效率和持续时间,通过病毒致细胞病变作用(CPE)保护实验病毒空斑形成实验检测病毒受抑制程度,动物模型中观察动物死亡率和易感组织病理变化评价siRNA的保护作用.结果:在HeLa细胞中针对CVB3 2B区的siRNA能显著抑制柯萨奇病毒B3的感染和复制,抑制率可达90%.动物模型中sigNA质粒可改善动物存活率(30%),并降低易感脏器中病毒含量,减轻病理反应.结论:针对CVB3基因组2B区的siRNA在病毒性心肌炎动物模型中具有保护作用.     

猪圆环病毒2型Cap基因单核苷酸缺失突变体的构建与生物学特性

猪2型圆环病毒(PCV2)是新出现的猪传染病—断乳仔猪多系统衰竭综合征病原.为了分析PCV2基因组核苷酸缺失突变对病毒复制的影响,以PCV2-HZ0201毒株基因组为模板,采用DpnⅠ定点突变技术,构建4个位于ORF2内的单核苷酸缺失突变体感染性克隆△1376PCV2,△1377PCV2,△1378PCV2和△1379PCV2.缺失突变体转染PK-15细胞后用间接免疫荧光检测病毒的拯救效果.结果显示,△1376PCV2感染性克隆能拯救出感染性病毒粒子,△1377PCV2,△1378PCV2和△1379PCV2感染性克隆不能拯救感染性病毒粒子,说明ORF2内1376位核苷酸的缺失不影响PCV2的复制.复制能力测定结果显示,△1376PCV2的复制能力比亲本病毒高10倍左右.促炎因子IL-6的转录本与血清浓度测定结果显示,△1376PCV2感染小鼠后的IL-6转录本与蛋白表达显著高于健康小鼠,且高于亲本病毒.这些数据首次揭示,ORF2内1376位核苷酸缺失的PCV2病毒粒子被提高了复制能力,同时促进了促炎因子IL-6的转录与表达.     

YXXM 基序对J 亚群禽白血病病毒复制的影响

[目的]毒株NX0101是骨髓瘤病变型J亚群禽白血病病毒,其早期感染细胞能诱导PI3 K/Akt信号转导通路的激活,本文针对NX0101毒株是否存在YXXM基序及其作用进行了探讨.[方法]利用TMpred软件对NX0101毒株囊膜蛋白(Env)的氨基酸序列进行生物信息学分析,通过搭桥PCR方法将YXXM基序相应的核苷酸序列突变后,构建突变质粒并转染DF-1细胞,拯救出YXXM突变体毒株NX0101 mt( Y/F,M/A),利用real-time PCR和ELISA方法检测并比较YXXM突变前后毒株在RNA水平和蛋白水平的复制情况.[结果]NX0101毒株Env胞浆区554 -557位氨基酸存在典型的PI3K结合基序YXXM.YXXM基序突变后,病毒RNA转录水平和病毒蛋白合成水平都显著下降.[结论]YXXM基序对NX0101毒株在体外宿主细胞中复制发挥重要的作用.     

嵌合猪圆环病毒PCV1-2的构建及其感染性初步鉴定

猪Ⅱ型圆环病毒(PCV2)是当前严重危害养猪业的重要病原之一。目前,世界上还没有有效疫苗用于该病毒的免疫预防。该研究利用PCR方法,将PCV2的ORF2基因替换猪Ⅰ型圆环病毒(PCV1)的ORF2基因,构建了以PCV1基因组为骨架的嵌合病毒(PCV1-2)分子克隆(pSK2PCV1-2)。将该分子克隆转染PK-15细胞并连续盲传5代,用RT-PCR方法可以在转染后盲传的细胞中检测到PCV1的ORF1 mRNA和PCV2的ORF2 mRNA,但检测不到PCV1的ORF2 mRNA和PCV2的ORF1 mRNA。间接免疫荧光检测显示在盲传第5代的细胞中有PCV2 ORF2蛋白的表达,表达蛋白主要分布于细胞核。该研究初步证实构建的PCV1-2分子克隆转染细胞后可以形成具有感染性的嵌合病毒,从而为更深入研究嵌合病毒生物学特性奠定了基础。     

嵌合型猪圆环病毒1-2型感染性DNA克隆的构建及其对小鼠的免疫原性

[目的]本研究旨在构建嵌合型猪圆环病毒1-2型感染性DNA克隆.[方法]利用PCR技术扩增PCV2 ORF2,克隆入缺失PCV1 ORF2的pSK-PCV1△ORF2中,得到pSK-sPCV1-2.该重组质粒中包含嵌合型PCV1-2全基因组,通过不完全酶切的方法,将嵌合型PCV1-2全基因组串联入pSK载体中,得到含串联双拷贝的嵌合型PCV1-2感染性DNA克隆.[结果]经序列测定,获得含串联双拷贝的嵌合型PCV1-2感染性DNA克隆.PCV1-2嵌合病毒接种BALB/c小鼠,用间接ELISA方法对接种后7、14、21、28、35、42 d的小鼠进行血清抗体检测.结果显示从接种后14 d开始,即有部分小鼠产生了针对PCV2 Cap蛋白的特异性抗体;至接种后42 d,几乎全部接种小鼠的血清均呈阳性.[结论]构建了PCV1-2型感染性DNA克隆,该嵌合病毒能够激发机体产生体液免疫应答.     

一株家养野猪源猪圆环病毒2型的分离与鉴定

应用PCR方法从临诊疑似断奶仔猪多系统衰竭综合征(PMWS)家养野猪病例的淋巴结和脾脏中扩增出预期长度的猪圆环病毒2型(PCV2)的DNA片段,在Dulac细胞中进行分离和培养,扩增全基因组序列后进行同源性分析.结果显示,扩增产物与家猪源参考毒株的序列同源性均在98%以上,该病毒与家猪源病毒差异不大.     

猪Ⅱ型圆环病毒ORF1基因克隆及在E.coli中的表达

猪圆环病毒(porcinecircovirus ,PCV)属圆环病毒科(Circoviridae) ,为单股负链DNA病毒,以滚环方式进行复制,是目前已知的最小的动物病毒之一.PCV有2种基因型即PCV 1和PCV 2 ,两者细胞培养均不引起病变.前者广泛存在于猪源肾细胞中,但并不引起感染猪发病,其基因组为1 75 9bp ;后者首先由Allan等[1 ] 从患断奶猪多系统衰弱综合症(postweaningmultisystemicwastingsyndrome ,PMWS)的猪群中分离到,被证明为PMWS的重要病原.PCV 2主要侵害感染猪的免疫系统[2 ] ,从而诱发猪体的免疫抑制.PCV 2常和呼吸与繁殖障碍综合征病毒(PRRSV)…     

猪圆环病毒2型流行株的分离及其感染性克隆的构建

【目的】通过分离一株猪圆环病毒2型(PCV2)流行毒株,并构建其感染性克隆,为研究PCV2基因功能提供操作平台。【方法】通过PCR方法,从疑似患断奶仔猪多系统衰竭综合症(PMWS)的仔猪淋巴结中鉴定为猪圆环病毒(Porcine circovirus,PCV)2型阳性。把阳性病料接种PK-15细胞传代培养,在培养物中扩增出PCV2的全基因序列。对扩增出的全序列进行序列测定,并与GenBank中公布的5株广东PCV2分离株(GD-pz、GD-gj、GD-jm、GD-ss和GD-sz)进行同源性分析。通过EcoRⅠ和SalⅠ将PCV2全基因组序列克隆进pUC18载体中,获得含PCV2 GD-zq株全基因组单拷贝的重组质粒pPCV2-GD-zq,再通过SalⅠ和HindⅢ把另一个全长拷贝克隆进pPCV2-GD-zq质粒中,使PCV2 GD-zq株基因组DNA以头尾相接的双重复方式克隆进pUC18载体中,获得重组质粒pPCV2-2GD-zq。将pPCV2-2GD-zq DNA纯化和定量后转染PK-15细胞,拯救PCV2 GD-zq病毒。【结果】从PMWS感染的猪淋巴结中分离到了一株PCV2,命名为GD-zq株;序列分析结果显示,GD-zq株全基因组为1 767 bp,与GenBank中公布的5株广东PCV2分离株ORF1核苷酸一致性为97.1%-99.7%,编码氨基酸一致性为98.7%-100%;ORF2核苷酸一致性为93.2%-99.6%,编码氨基酸一致性为92.3%-99.1%;全基因一致性为96.0%-99.6%。pPCV2-2GD-zq质粒转染PK-15细胞后,其通过间接免疫荧光实验(IFA)能从转染细胞及其传代细胞中,检测到拯救出的病毒。【结论】分离了一株PCV2广东株GD-zq,成功构建了PCV2 GD-zq株的感染性克隆。     

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.