【作者】 郑海学；

【导师】 谢庆阁；

【作者基本信息】 中国农业科学院 ， 预防兽医学， 2007， 博士

【摘要】 针对非逆转录RNA病毒发展起来的病毒反向遗传学可以实现对RNA病毒基因组结构与功能、复制与表达、病毒致病机制等研究。本研究用T7RNA聚合酶系统和聚合酶Ⅰ系统为基础建立了体内外拯救方法并初步进行应用。一、SVDV HK／70株生物特性测定、生物信息学分析及以T7 RNAP为基础的体外病毒拯救方法的建立和应用为了建立以T7 RNA聚合酶系统为基础的体外拯救病毒方法，选择猪水泡病病毒（SVDV）HK／70株作为细胞质复制的RNA病毒的研究模型。首先，分离和鉴定了该病毒，并测定了该病的一些生物学特性。然后，构建了SVDV全长cDNA并进行序列测定，以此为基础，分析了其相关生物信息学特征。为了鉴定SVDV HK／70株的全长cDNA分子的感染性，以线化的SVDV HK／70株的全长cDNA质粒(pSVOK₁₂)为模板，应用T7 RNA聚合酶系统在体外进行转录，将获得的RNA用脂质体转染法导入IBRS-2细胞，传代培养，可以观察到典型的SVDV致细胞病变效应。使用反向血凝鉴定试验、间接免疫荧光实验、RT-PCR和序列测定进行检测，结果表明，从猪水泡病病毒全长cDNA拯救出了猪水泡病病毒（G-SVDV）；利用常规负染的方法，电镜观察了G-SVDV的形态；测定了G-SVDV的TCID₅₀和LD₅₀，并与亲本毒进行了比较，结果显示G-SVDV与亲本毒的毒力差别不显著。本研究结果证明，我们已经成功构建了猪SVDV HK／70的感染性cDNA克隆，为进一步探索SVDV病毒致病的分子机制及研制新型SVD疫苗奠定了良好的基础。二、以T7 RNAP为基础的体内病毒拯救方法的建立和应用为了建立高效的体内病毒拯救系统，我们利用逆转录病毒转导技术建立了稳定表达T7 RNA聚合酶的细胞系。先克隆出T7 RNAP基因，定向克隆进逆转录病毒载体pBABEpuro，得到阳性重组质粒pT7BABEpuro。共转染包装细胞，获得含有VSV-G膜的假型病毒，含有T7 RNA聚合酶基因。然后把该假型病毒感染靶细胞，把T7 RNAP基因分别整合进BHK-21、IBRS-2和SK6细胞的基因组内。通过抗性筛选，获得了稳定表达具有转录活性T7 RNA聚合酶的细胞系，通过PCR、间接免疫荧光和流式细胞仪（FCM）等技术进行鉴定，结果表明，该T7 RNAP能够被稳定地整合进靶细胞基因组内，细胞系内的T7 RNAP具有较好的转录活性，其活性传代不减弱。最后，利用该细胞系成功拯救出具有感染性的SVDV，并与亲本毒的生物学特性作了比较。该策略使RNA拯救方法简化为一步快速的拯救方法。利用该方法对CSFV C株进行了拯救，进行了拯救病毒的鉴定，并做了兔子致病性试验。三、以真核细胞RNA聚合酶Ⅰ系统为基础的体内拯救系统的建立和应用为了克服那些难以适应甚至没有可适应细胞系的病毒拯救难题，设计构建了完全利用真核细胞聚合酶的RNA病毒体内拯救系统。先克隆出所需的真核RNA聚合酶Ⅰ启动子和终止子序列，建立RNA聚合酶Ⅰ启动转录的重组质粒。然后把SVDV全长cDNA装配进该载体，在IBRS-2细胞内和乳鼠体内成功拯救出了SVDV，第一次证明了聚合酶Ⅰ系统能够高效拯救细胞质复制的正链RNA病毒。并利用该拯救系统对FMDV进行了拯救，首次证明该聚合酶I系统能够转录出至少长8.2 kb的转录本。在此基础上，构建含有外源性生物标记5B 19的SVDV HK／70全长cDNA克隆，利用聚合酶Ⅰ反向遗传拯救系统拯救出含有该标记的病毒。为制备含有基因标记疫苗和建立鉴别诊断方法奠定一定的基础。该设计思路的实现，拓宽了高效病毒拯救的途径，为病毒反向遗传学研究提供了更为高效和广泛应用的病毒拯救技术方法。更多还原

【Abstract】 The reverse genetics of RNA virus allows precise study of the mechanisms as antigenicity,virulence, pathogenesis, maturation and replication of the virus at the molecular level. In this study, invitro and in vivo transcription systems were developed.1 Testing of biological properties, analyzing of bioinformatics of SVDV HK/70 strain anddevelopment in in vitro transcription system based on T7 RNA polymerase for the virusTo develop in vitro transcription system based on T7 RNA polymerase （T7 RNAP） for RNA virus,the swine vesicular disease virus HK/70 strain was selected as a model virus of cytoplasmicpositive-strand RNA virus in the present study. Firstly, the virus was isolated and cultured in mouse andIBRS-2 cells. Then, the biological properties such as 50％tissue culture infecting dose (TCID₅₀) andmouse virulence, and so on, were tested. The bioinformatics of the virus genome was also analyzedbased complete genome sequence by some software.At last, the full-length cDNA clone of swine vesicular disease virus HK/70 strain named pSVOK₁₂was constructed in order to study the antigenicity, replication, maturation and pathogenicity of swinevesicular disease virus. In vitro transcription RNA from pSVOK₁₂ transfected IBRS-2 cells and therecovered virus RNA was isolated and sequenced, then indirect hemagglutination test, indirectimmunofluorescence assays, eleectron microscope test, 50％tissue culture infecting dose (TCID₅₀)assays and mouse virulence studies were used to study the antigenicity and virulence of the recoveredvirus. The result showed that the infectious clones has obtained and the virus derived from pSVOK₁₂had the same biological properties as the parental strain HK/70; The full-length infectious cDNA clone,pSVOK₁₂, will be very useful in studies of the antigenicity, virulence, pathogenesis, maturation andreplication of SVDV.2 Development in in vivo transcription system based on T7 RNA polymeraseEstablishment of cell lines stably expressing T7 RNA polymerase by using retroviral gene transfertechnique for rescue of infectious RNA virusReverse genetics based on transfection of in vitro transcribed RNA to target cells has lowefficiency to recover RNA viruses. To develop an efficient and vaccinia virus-free recovery system（reverse genetics）, three stable cell lines （designated as BHKT7, IBRST7 and SK6T7 respectively）constitutively expressing cytoplasmic bacteriophage T7 RNA polymerase （T7 RNAP） were developed.By using retroviral gene transfer technology, the T7 RNAP gene was integrated into the chromosome of these cells （BHK-21, IBRS-2 and SK6） and then these cell lines stably expressing T7 RNAP （BHKT7）were established under selection pressure. The T7 RNAP produced in the BHKT7 cell line was able toefficiently driving in vivo transcription of enhanced green fluorescent protein （EGFP） reporter genecontrolled by the T7 promoter. When the IBRST7/ BHKT7 cell line was directly transfected withlinearized full-length cDNA of swine vesicular disease viruse （SVDV） HK/70, infectious SVDV wassuccessfully recovered. Two-day-old mice inoculated intra-peritoneally with the recovered virus died at72 hours after inoculation. These data showed the T7 RNAP in BHKT7 cells has transcriptional activityand can be used for recovery of infectious RNA virus directly from full-length cDNAs. InfectiousCSFV was also successfully recovered from the SK6T7 cell line, and rabbit virulence studies were usedto study the virulence of the recovered virus.3 Development in the in vivo rescue system based on RNA polymerase I system.For some viruses refuse to grow in laboratory cell cultures, which cannot be recovered in cellcultures, or a limited number of mammalian cell lines are available for culture of virus which cannot betransfected with high efficiencies, which sometimes limits their use in reverse genetics systems forvaccine production and studying basic principles of molecular biology of the virus. To address thislimitation, we established a reverse genetics system that is entirely from a RNA polymeraseⅠ-drivenplasmid required for virus generation. Here, we report the recovery of infectious SVDV entirely fromcDNA, which is a polymerase （pol）Ⅰand polⅡ-driven plasmid constructed which permits intracellulartranscription of the accurate viral RNA （vRNA） and caped mRNA of the viral protein to be derivedfrom the same template resulted in the efficient formation of infectious virus with genetic tags in thegenome from IBRS-2 cell transfected and suckling mice directly inoculated with a RNA polymeraseⅠ/Ⅱ-driven vector. The cDNA-derived viruses behaved identically to wild-type virus in both cell cultureand infected mice. Importantly, the virus can be recovered from the mice directly injected with theplasmid, which also would develop a method to rescue the viruses refuse to grow in laboratory cellcultures.This technology provides an important basis for investigating various fields of virological research.The reverse genetic procedures are simplified to a faster, one step protocol to recover virus andovercome the obstacle to rescue the viruses which have no adaptive cell line. In addition, our findingsdemonstrate that polⅠ/Ⅱ-based vector systems may represent an efficient alternative strategy for therecovery of cytoplasmic positive-strand RNA viruses from cDNA. Then, infectious FMDV was alsosuccessfully recovered from BHK-21 cell line by the in vivo rescue system, it is the first time that theresults shown that the RNA polymeraseⅠcould drive transcripts of about 8.2 kb in length,4 Research on recombinant viruses with marker as vaccines against viral diseases by reversegenetics system To develop a marker vaccination: designing unique antigens to be added to vaccines todifferentiate between natural infection and vaccination. The full-length cDNA clone of SVDVreplication-competent recombinant virus with bio-mark 5B19 was constructed. Then the recombinantvirus was recovered by reverse genetics system.更多还原