【作者】 李任峰；

【导师】 何启盖； 陈焕春；

【作者基本信息】 华中农业大学 ， 预防兽医学， 2005， 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)引起的偶蹄动物共患的急性接触性传染病,人也可以感染。本病有极强的传染性,可形成大范围流行,造成巨大的经济损失和政治影响。预防和控制该病的关键是高效疫苗和准确的诊断方法的应用。目前用于口蹄疫的诊断技术种类很多,而传统ELISA大多用于检测抗体,其抗原需要完整的病毒,生产过程需要昂贵的培养基,成本高,且存在散毒的危险。检测病毒是确诊病毒感染的标准,单抗具有特异性高的特点,因此基于单抗而建立的诊断方法具有重要的应用前景。此外,有研究表明,将口蹄疫具有免疫原性的结构蛋白基因和裂解酶基因(3C基因)连接起来构建的多抗原基因表达载体,表达后与单一的免疫原性基因相比,具有较高的免疫原性。本研究开展了抗口蹄疫病毒单克隆抗体的制备及初步应用研究和口蹄疫病毒免疫原蛋白在大肠杆菌的融合表达。 1 抗O型口蹄疫病毒单克隆抗体的制备和初步应用 以纯化的O型FMDV为免疫原,免疫BALB/C小鼠,将脾细胞与骨髓瘤细胞SP2/O融合,用ELISA筛选获得2株分泌抗O型FMDV单克隆抗体(McAb)的杂交瘤细胞,分别命名为2G12和2G8。染色体数目分析证明为杂交产物,免疫荧光试验证明两株单抗均能特异性地与FMDV反应。选择其中效价较高的1株(2G12)用于下列实验。以自行制备的兔抗FMDV高免血清IgG为捕获抗体,包被酶联免疫吸附试验微量反应板,以单抗2G12为检测抗体,建立了快速检测FMDV抗原的双抗体夹心ELISA。该方法能检出90ng/100μL FMDV病毒,与猪瘟病毒(HCV)、猪呼吸与繁殖障碍综合症耳病病毒(PRRSV)、伪狂犬病毒(PrV)、猪细小病毒(PPV)和乙脑病毒(JEV)均不起反应。本研究为检测口蹄疫病毒抗原提供了灵敏和特异的方法。 2 口蹄疫病毒多抗原基因(VP1-VP3-3C)在大肠杆菌中的融合表达 应用PCR扩增技术获得1564bp的片段,包含口蹄疫病毒VP1全长基因、VP3部分基因和编码3C裂解酶的全长基因,连接到pMD-18T载体上,在获得重组质粒pMD-18T-VP1-VP3-3C后,进行序列分析;将此片段连接到Pgex-KG载体,构建了重组表达质粒pKG-VP1-VP3-3C,将其转化大肠杆菌BL21(DE3)感受态细胞中,经IPTG诱导表达,收集菌体进行SDS-PAGE电泳,结果表明,VP1-VP3-3C基因在大肠杆菌中获得成功表达,其表达产物为分子量83KDa的融合蛋白,并且能与猪更多还原

【Abstract】 Foot and mouth disease virus, FMDV, is the etiology of a highly contagious animal disease which can be pandemic and cause a severe economical loss worldwide. this disease also has an imoprtant political impact on the afftected nations. The infections happened in human were reported. It has been well realized that development of an effective vaccine, alongside with accurate diagnostic methods, plays a key step in the control and prevention of the disease. Currently, many assays for detection of FMDV have been develped, amongst which an inactivated virus-based ELISA is used to detect antibodies against FMDV. As the manipulation of live virus is involved in process of the preparation of viral antigen, it is risky in dissemination of viruses. More importantly, it is a "gold standard" method to detect virus antigen for confirmation of FMDV infection. A monoclonal antibody-based ELISA for detection of FMDV is supposed to meet the requirement of sensitivity and specificty, however, no report can be found at present. In addition, the fusion expression of antigens in E.coli is predicted to improve the immunogenicity of the subunit vaccine against FMDV. This paper describes the monoclonal antibody preparation, development of antigen captured ELISA, and eukaryotical expression of FMDV genes encoding antigens.1 Preparation and application of monoclonal antibody against FMDVBALB/C mice were immunized with purified FMDV, followed by the fusion of spleen cells with myloma cells, SP2/0, and screening by ELISA. As a result, two hybrodoma cell lines, designated as 2G12 and 2G8, which secreted antibodies against FMDV were developed. The derivation of hybridoma from fusion of two cells mentioned above was identified by analysis of the hybridoma chromosomes. The monoclonal antibodies can specifically recognize FMDV in infected cells by immunofluorescence assay. One of the monoclonal antibodies, 2G12, was used in the test. for further investigating the potential of 2G12 in detecting FMDV by immunoassay, IgG was purifed from rabbit anti-FMDV serum and used as capture antibody to coat microtiter plates. The capture ELISA was optimized by checkerboard assay. Ninety nanogram of FMDV can be detected by this method while no cross reactions with hog cholera virus, porcine respiratory and reproduction syndrome virus, pseudorabies virus, porcine parvavirus, Japanese encephalitis virus were observed. The reproducibility of the assay was alsotested. This ELIS A provides a new sensitive and specific tool for detection of FMDV. 2 Fusion expression of recombinant FMDV VP1-VP3-3C protein in E.coliA1564 bp motif, spanning full length of VP1 gene, partial VP3 and full length of 3C protein, was amplied by PCR and subjected to sequence analysis. The amplified product was enzymaticalUy digested and ligated to pGEX-KG vector to produce an expression plasmid, which was transformed to E.coli BL-21 cells. The protein expression was induced by addition of IPTG and the pelleted cells were analysized by SDS-PAGE and Western blot. The results showed that a fusion protein with size of 83 kDa was expressed and could react with swine anti-FMDV serum. This experiment pave a way for further research on diagnosis and subunit vaccine of FMDV.更多还原