【作者】 赫冬梅；

【导师】 钱凯先； 沈桂芳；

【作者基本信息】 浙江大学 ， 细胞生物学， 2005， 博士

【摘要】 口蹄疫和猪瘟都是当今世界上严重危害家畜的烈性传染病,发病率高,流行快,危害严重。目前,疫苗接种是预防口蹄疫和猪瘟病毒发病的主要手段。传统的灭活疫苗和弱毒疫苗在防治和消除这两种疾病的大规模流行中发挥了重大作用。但随着传代次数的增加和对不同细胞的适应性增殖,疫苗毒株可能发生抗原漂移,加之数十年来病毒在大规模免疫接种压力下出现抗原变异,现有的传统疫苗已不能适应新形势的需要。而且由于其潜在的危险性,使其应用受到很大限制。随着分子生物学和分子免疫学的深入发展,迫切需要研制新型的基因工程疫苗。 转基因植物作为生产疫苗的生物反应器,具有表达量高,成本低,容易生产等优点。另外,植物表达的抗原蛋白不需要纯化,可以作为口服疫苗直接食用。如:番茄,大豆,牧草,烟草等。VPl和E2分别是口蹄疫病毒和猪瘟病毒的核心抗原决定簇,能够诱导机体产生保护性抗体反应。研究表明,VPl和E2已经在多种表达系统内得到了表达。如:烟草和苜蓿表达的VPl抗原蛋白喂饲小鼠后,能够诱导特异性抗体免疫反应。为此,本文中将口蹄疫病毒抗原基因VPl和黏膜免疫佐剂霍乱毒素B亚基(CTB)融合基因在马铃薯内重组和表达;同时,利用植物叶绿体基因组高效表达的特点,把猪瘟病毒抗原基因E2重组并转化进烟草和衣藻叶绿体基因组中,使其高效表达。为利用植物生物反应器研制口蹄疫和猪瘟基因工程疫苗提供理论和实践依据。 论文的第二部分研究了将口蹄疫病毒抗原基因VPl和霍乱毒素B亚基融合基因通过农杆菌转染的方法转化到马铃薯基因组中,进行重组和表达。通过抗性筛选,得到了具有卡那霉素抗性的转化植株。对马铃薯基因组DNA的PCR扩增、SotJthernblot、Western blot和ELISA分析表明,外源基因已经整合进马铃薯基因组,并得到了表达。 论文的第三部分主要研究了烟草叶绿体表达载体PTRE2的构建,并通过基因枪法转化烟草叶绿体基因组,通过壮观霉素抗性筛选,获得了一株转化植株苗。经过两轮抗生素筛选后,分别用CSFV的E2基因及烟草叶绿体同源片段的引物对抗性植株的叶绿体DNA进行PCR扩增和酶切Southern blot分析,结果表明目的基因已更多还原

【Abstract】 Both classical swine fever (CSF) and Foot- and- mouth disease (FMD) are highly contagious disease of livestock, which are classified by the Office International des Epizooties (OIE) as a list A disease. Classical swine fever virus (CSFV) and Foot-and-mouth disease virus (FMDV) are the causative agent of classical swine fever (CSF) and Foot- and- mouth disease (FMD) respectively. At present, Vaccine is effective method to prevent the disease. Conventional vaccines are based on the utilization of inactivated and attenuated virus which have considerable risk although they have proved to be effective for the prevention and controlling of the disease. The most widely used conventional vaccination of classical swine fever is The Chinese strain which was safe and effective but interferes with serodiagnosis. So it is necessary to develop safe and effective alternative vaccines.The structural protein VP1 of foot-and-mouth disease virus, which has been shown to contain critical epitopes responsible for the induction of neutralizing antibodies, has been expressed in different expression systems and plants. Production of complete or partial VP1 in a diversity of expression systems has been performed in the search for an effective and inexpensive alternative which would be highly immunogenesis. In this study, we developed a expression vector pBI121CTBVP1 by fusion of the foot and mouth disease virus (FMDV) VP1 gene and the cholera toxin B subunit (CTB) gene with a flexible linker tetrapeptide (GPGP) at the c-terminal of CTB, and then cloned into intermediate vector pBI121-patatin containing specific-tuber patatin promoter of potato. Furthermore, we fused the ER targeting signal (SEKDEL) encoding sequence at the c-terminal of VP1 in order to improve expression of fused gene and distribution. The transgenic potatos were obtained by Agrobacterium-mediated transformation and selection on kanamycin resistance agent. The transgenic plantlets were identified by PCR, Southern-blot and the production of fused protein was confirmed and quantified by Western-blot and ELISA assays. The results demonstrated that the fused genes were expressed stablely under control of specific-tuber patatin promoter up to 0.1%-0.13% though it was fant in leaves, and thetransgenic potato could be as oral vaccine candidate defense of foot-and-mouth disease.The E2 protein of CSFV contains major antigenic determinants that is conserved and involved in neutralization by antibodies. The expression of E2 protein in insect and in E. coli has been reported. Animal immunization and the challenge against CSFV experiments have been conducted using the recombinant E2 protein. Here, we studied the expression of CSFV E2 gene of shimen strain in tobacco and C. reinhardti chloroplast by biolistic bombardment transformation. The transformants were identified by PCR, Southern blotting, Western blotting after selection on resistant medium. ELISA quantification assay showed that the expressed E2 protein accumulated up to 1.0-2% and 2% of the total soluble protein in tobacco and C. reinhardti chloroplast respectively. In order to confirm the neutralizing activity of the protected antibodies induced by E2 antigen, neutralization assay were carried out on mices. The results of the study showed that the protein E2 expressed in tobacco and C. reinhardti chloroplast could elicit animal bodies to produce antibodies. This indicated that the expressed E2 proteins have a certain of immunogenicity. Our finding provides a new way to develop plant-drived vaccine against CSFV.更多还原