【作者】 李刚；

【导师】 王君伟；

【作者基本信息】 东北农业大学 ， 预防兽医学， 2006， 硕士

【摘要】 口蹄疫（Foot-and-Mouth Disease，FMD）是由口蹄疫病毒（Foot-and-Mouth Disease Virus，FMDV）引起的牛、羊、猪等偶蹄动物感染的一种烈性传染病，该病的发生和流行严重危害畜牧业的健康持续发展，造成巨大的经济损失，因此世界各国相当重视对该病的研究和防治。口蹄疫病毒衣壳由VP1、VP2、VP3、VP4 4种结构蛋白各60个拷贝组成，其中VP1蛋白又称1D蛋白，其基因位于基因组的2977-3615位，编码213个氨基酸，VP1暴露在病毒颗粒的表面，是诱导产生中和抗体的主要成分。 本试验以口蹄疫病毒的细胞培养毒为材料，通过RT-PCR方法获得了此株病毒结构基因的核苷酸序列，同法获得了口蹄疫疫苗株结构基因的核苷酸序列。对此株口蹄疫病毒与参考毒株的VP1、VP2、VP3、VP4的各核苷酸序列进行了比较，发现主要差异在可以诱导产生中和抗体的VP1上，根据VP1基因序列绘制系统进化树（国际上通用VP1基因绘制系统进化树），发现此株病毒属于O型FMDV的SEA拓扑型。将此株口蹄疫病毒与口蹄疫疫苗株进行比较发现，VP1完全相同，差异在VP2、VP3上。 以已经构建好的pMD18-T-VP1质粒为模板，PCR扩增得到FMDV VP1目的基因，将目的基因克隆到pMD18-T载体中，转化宿主菌TG1后得到重组质粒pMD18-T-VP1，测序证明此VP1基因序列准确无误。对重组质粒pMD18-T-VP1和原核表达载体pET-30a（+）分别以相同的限制性内切酶SacⅠ和HindⅢ消化，经连接酶连接，转化宿主菌TG1，筛选出阳性克隆pET-30a（+）-VP1。随后将此阳性质粒pET-30a（+）-VP1转化宿主菌BL21（DE3）pLysS中，经IPTG诱导VP1基因的表达，收集不同时间的菌液进行SDS-PAGE、Western blotting分析和dot-ELISA分析，结果表明VP1基因在大肠杆菌中获得了高效表达，表达的目的蛋白的分子量约为34ku，但表达产物未检测到抗原性。 对重组质粒pMD18-T-VP1和真核表达载体pBlueBacHis2A分别以相同的限制性内切酶SacⅠ和HindⅢ消化后，经连接酶连接，转化宿主菌TG1，筛选出阳性克隆pBlueBacHis2A-VP1。将pBlueBacHis2A-VP1质粒与Bac-N-Blue^TM DNA共转染Sf9昆虫细胞，噬斑筛选重组病毒，将纯化的重组病毒感染Sf9细胞，表达了32.6ku目的蛋白条带，dot-ELISA分析表明表达产物具有抗原性。 以真核表达的融合蛋白为包被抗原建立了间接ELISA方法，用来检测动物体内FMD的抗体水平。更多还原

【Abstract】 FMD is a virulent infectious disease caused by FMDV that infects artiodactyl such as cattle, sheep, pig, et al. Outbreak and prevalence of the disease endanger the good development of graziery seriously and cause great financial loss, so all the world highlight study and contol of the disease. Capsid of FMDV is composed of VP1, VP2, VP3, VP4 of 60 copies, of which VP1 protein is called 1D protein, too. Its gene is in 2977-3615 of genome and code for 213 amino acids. VP1 is located on the surface of virus and is the main component that induces neutralizing antibody.Material of the study was FMDV from cell culture, nucleotide sequence of structural gene of FMDV and FMD vaccine strain were acquired by RT-PCR. Compared with FMDV reference strains, it was found that there was difference in VP1 gene that induces neutralizing antibody, and Phylogenetic tree was drawn according to VP1 gene sequence（VP1 gene sequence is the criterion of drawing Phylogenetic tree in the world）. Compared with FMD vaccine, it was found that there was difference in VP2、 VP3, not VP1.VP1 target gene was amplified by PCR with plasmid pMD18-T-VP1 as template, then it was cloned to vector pMD18-T and transform TG1 strain of E.coli, so recombinant plasmid pMD18-T-VP1 was acquired, sequencing assured that the sequence of VP1 was correct. Recombinant plasmid pMD18-T-VP1 and prokaryotic expression vector pET-30a（+） were cut by the same restriction endonucleases SacI and HindIII and ligated together, transformed TG1 strain of E.coli with ligation mixture, then the positive clone pET-30a（+）-VP1 was identified. Transforming BL21（DE3）pLysS with positive plasmid pET-30a（+）-VP1, inducing expression of VP1 gene with IPTG, collecting bacteria at different times, then doing SDS-PAGE, Western blotting and dot-ELISA analysis, the result showed that the gene of VP1 had been expressed well in E.coli and molecular weight of the target protein was 34ku, but the product did not have antigenicity.Recombinant plasmid pMD18-T-VP1 and eukaryotic expression vector pBlueBacHis2A were cut by the same restriction endonucleases SacI and HindIII and ligated together, transformed TG1 strain of E.coli with ligation mixture, then the positive clone pBlueBacHis2A-VP1 was identified. Cotransfecting insect cell Sf9 with plasmid pBlueBacHis2A-VP1 and Bac-N-Blue^TM DNA, screening recombinant virus by plaque, Sf9 cells were inoculated by pure recombinant virus, then 32.6 ku target protein was expressed, dot-ELISA analysis showed that the product had antigenicity.Indirect ELISA that can detected the valence of antibody was developed with the fusion protein expressed by eukaryotic expression system as antigen.更多还原