导　　师： 才学鹏

学科专业： I0602

授予学位： 博士

作　　者： ;

机构地区： 中国农业科学院

摘　　要： 羊痘(capripox,cp)是由绵羊痘病毒(sheeppoxvirus,sppv)和山羊痘病霉(goatpoxvirus,gtpv)引起的绵羊和山羊的一种急性、热性、接触性传染病.病畜以体温升高,全身性丘疹或结节、水疱、内脏病变乃至死亡为特征.口蹄疫(foot and mouth disease,fmd)是由口蹄疫病毒(foot and mouth disease vims,fmdv)引起的一种急性、热性、高度接触性传染病.口蹄疫和羊痘都是危害性非常严重的疾病,均被世界动物卫生组织(oie)列为a类重大传染病.本文利用已在我国广泛使用的山羊痘弱毒疫苗株(av41),利用病毒在细胞内的同源重组,构建表达asial型口蹄疫p1-2a和3c基因的重组山羊痘病毒弱毒株.为了构建表达多个外源基因的重组山羊痘弱毒株,文中还采用随机克隆的方法,筛选出该毒株(av41)的一个复制非必需区. 1.选择胸苷激酶(thymidine kinase,tk)基因区域作为外源基因插入位点构建重组病毒.提取gtpvav41株基因组dna,设计特异性引物扩增tk基因及其两侧翼片段,获得了长度为2078bp的tk基因及两侧的同源臂.其中tk基因的orf为534bp,编码178个氨基酸.核苷酸和氨基酸同源性分析表明,gtpvav41株tk基因与参考毒株的核苷酸和氨基酸的同源性在95.5﹪-100.0﹪之间,说明羊痘病毒tk基因具有高度的保守性. 2.为了使重组病毒能高效表达外源基因,构建一个真核生物基因表达盒p7.5-egfp-p,通过痘苗病毒p7.5启动子来启动绿色荧光蛋白和fmdv asial/ynbs-58株衣壳蛋白前体p1-2a3c的表达.以山羊痘病毒tk基因序列中的kpn i酶切位点为插入点,将整体表达盒插入山羊痘病毒转移载体骨架puc119-tk 之中,构建了共表达fmdv p1-2a3c基因和绿色荧光蛋白基因的重组山羊痘病毒转移载体ptk-p7.5-egfp-p. 3.病毒转移载体ptk-p7.5-egfp-p转染gtpv av41株感染的bhk-21细胞,转染后24h就可见到特异性荧光.经绿色荧光筛选重组病毒,rt-pcr检测 fmdv p1-2a、3c基因,抗原捕获elisa检测 fmdv 抗原,结果表明,成功获得了能稳定、正确表达目的蛋白的重组山羊痘病毒株tk<'->/egfp<'+>/fmdv p1-2a3c<'+>/gtpv.毒价测定结果表明,重组毒在bhk-21细胞上的霉价为10<'5.5>tcid<,50>/0.1ml. 4.提取 gtpv av41 株病毒基因组用 hindⅢ酶切,酶切片段克隆到puc18质粒中,得到6个不同大小的hindⅢ片段puc18-a、puc18-b、puc18-c、puc18-d、puc18-e和puc18-f,分别插入p7.5-p7.5-lacz报告基因构建了pua1,pub1,puc1,pud1,pue1,puf1等6个重组转移载体质粒,转移载体转染gtpvav41株感染的bhk-21细胞.在x-gal存在条件下通过蓝白斑筛选,获得了一株稳定表达lacz基因的山羊痘病毒重组弱毒株,筛选出了gtpvav41株的1个复制非必需基因片段. 本文首次在国内外尝试用山羊痘弱毒疫苗毒株为活载体表达fmdv基因,为反刍动物烈性传染病的防治开创了新的思路.羊痘病毒活载体的构建,将为反刍动物疾病基因工程活载体疫苗的研究奠定基础. Capripox/(CP/), caused by sheeppoxvirus or goatpoxvirus, is an acute feverish and contagious disease in sheep or goats. It characterized by fever, generalized papules or nodules, vesicles, internal lesions, and death. Foot and mouth disease /(FMD/), caused by foot and mouth disease virus, is an acute feverish and highly contagious disease. Both FMD and CP are hazardousextremely serious disease, and be classified as group A disease by Office International des Epizooties/(OIE/)and group first disease in China. In this study, we construct a recombinant attenuated goat pox virus for coexpression FMDV Asia1 P12A3C based on the TK gene and homologous recombination in the cells. Meanwhile, a replication non-essential region was selected by using random cloning method in order to express multigenes in recombinant goatpox virus. /(1/) TK gene was selected as the extraneous gene cloning site. The TK gene and its flanking fragment was amplified with a pair of specific primers. The PCR product was 2078bp in length and the ORF of TK gene was 534bp encoding 178 amino acids. The identity analysis showed that AV41 shared 95.5/%-100.0/% identity rates with the reference strains in levels of nucleotides and amino acids. The results showed that TK gene was highly conservative. /(2/) To express foreign genes in recombinant virus with high efficacy, construction of a eukaryotic gene expression cassette P7.5-EGFP-P was obtained. Then the P7.5 controlled EGFP and the P12A3C gene of FMDV was constructed. Then the segments was ligated into pUC119TK by the kpn I which was chosen as the insertion site for the construction of transfer vector .The transfer vector was named pTK-P7.5-EGFP-P. /(3/) The transfer vector PTK-P7.5-EGFP-P transfected BHK-21 cells which infected GTPV AV 41. Specific fluorescence could be seen at 24h after transfection. Screened by Green fluorescent, RT-PCR and antigen capture ELISA, the results showed that P12A and 3C genes were expressed successfully. The tilter detection showed that the tilter of the recombinant was 10~/(5.5/)TCID50//0.1mL in BHK-21 cells. /(4/) The goatpoxvirus cultured in Primary lamb testicles was purified by sucrose density gradient centrifugation. Genomic DNA was extracted and digested by HindIII .After cloned into PUC18 , six fragments were obtained: Puc18-A、Puc18-B、Puc18-C、Puc18-D、Puc18-E and Puc18-F . Choosing single restriction enzyme site to insert P7.5-P7.5-LacZ ,the resulting plasmids PUA1, PUB1, PUC1, PUD1, PUE1, PUF1 were acquired and transfected BHK-21 cells with CPV subsequently. After plaques selection, passages and purifications at presence of X-gal, one cloned CPV fragments was identified as replication nonessential regions. To my knowledge, this study is the first time to use goatpoxvirus as live vector to express FMDV gene in the world. It advanced a new way of prevention ruminant's diseases. The construction of goatpoxvirus live vector will lay the foundation for the genetically engineered live vector vaccine of ruminant diseases.

关 键 词： 病毒病 口蹄疫 山羊痘 病毒基因 活载体表达

分 类 号： [S855.3]

领　　域： [农业科学] [农业科学] [农业科学]