【作者】 李得江；

【导师】 方维焕；

【作者基本信息】 浙江大学 ， 预防兽医学， 2010， 硕士

【摘要】 猪瘟(classical swine fever, CSF)是猪的一种最重要的传染病,其病原是猪瘟病毒(HCV,裁CSFV)。猪瘟毒为RNA病毒,属于黄病毒科瘟病毒属成员。CSF病理特征是微血管壁变性,致使内脏器官多发性出血、梗塞和坏死,以出血和发热为主要特征,呈急性或慢性经过,对养猪业危害极大。新近研究表明,在CSF流行地区,其症状呈现非典型化,甚至在免疫猪群中也有发病；CSFV流行毒株已经从以前的group 1转向group 2。有迹象表明我国目前使用的group 1兔化弱毒C株疫苗对group 2 CSFV流行毒株难以提供有效保护。本研究目的是：(1)初步探明浙江省猪瘟病毒的分子流行病学特征；(2)制备并鉴定猪瘟病毒SM株E2蛋白的单克隆抗体：(3)猪瘟病毒SM株E2蛋白单克隆抗体识别位点的鉴定。1、猪瘟病毒的检测及分子流行病学研究在CSFV-E2基因的上、下游保守区域分别设计两对简并引物用于套式RT-PCR检测。结果表明,该体系具有很好的特异性和灵敏性,检测下限为1400拷贝数的CSFV基因组。根据不同毒株E2基因中Mspl酶切位点排布的不同,建立了鉴别检测疫苗株和野毒株的RFLP方法。应用此方法对2009年浙江地区猪场采集的59份组织样品进行CSFV检测,结果发现9份样品能扩增出CSFV特异性条带,阳性PCR产物经Mspl酶切鉴别鉴定,4份样品中含有疫苗株；4份为野毒感染,其余1份能同时检出疫苗株与野毒株。根据普遍应用的CSFV E2基因5’-端高变区190bp序列进行遗传进化分析,发现2009年实验室检测的流行于浙江省及周边地区的CSFV都属于genotype 2.1b。我国目前使用的疫苗株C株属于subgroup 1.1,在进化树中与本地区的流行毒株进化关系较远。2、猪瘟病毒SM株E2蛋白单克隆抗体的制备与鉴定为了进一步探索E2蛋白N端高变区域内与抗体识别相关的一些关键氨基酸位点差异对不同毒株抗原结构的影响,我们以猪瘟经典强毒株SM株E2为抗原免疫BALB/c小鼠,应用杂交瘤技术结合IFA筛选,获得了1株持续、稳定分泌小鼠抗SM株E2蛋白的单克隆抗体的杂交瘤细胞株2B10,亚型鉴定为IgG2a。IFA显示2B10能与SM毒株反应。结果表明：单抗2B10识别的抗原表位只存在于SM毒株中。3、单克隆抗体2B10识别位点的鉴定制备的单克隆抗体2B10通过Western blot和间接ELISA检测显示,该单抗与重组蛋白E2-AD有较强的反应性,且该单抗能与截短的E2-BC发生反应,表明该单抗识别的线性表位位于E21-125位氨基酸残基内。应用获得的这株单抗进行流行毒株E2抗原多样性分析,结果表明,单抗2B10能只识别group 1中的SM病毒,而不能与group 1中的疫苗株和group 2中的任何流行毒株发生反应。证明了单抗的抗原识别位点只存在与SM毒株中,而通过与流行毒株和疫苗株的截短的E2-BC基因序列比较发现了一些差异位点,对这些位点进行点突变后制备突变蛋白。取单抗与突变蛋白进行了Western blot和间接ELISA检测,结果发现该单抗识别位点为P709位点。上述试验结果为深入开展CSFV病毒变异与抗原多样性的关系研究和新型高效疫苗开发奠定了良好基础。更多还原

【Abstract】 Classical swine fever (CSF) is a highly contagious disease of swine and wild boars, causing significant economical losses in a wide range of the world. The causative agent of this disease is classical swine fever virus (CSFV), a member of the Pestivirus genus within the Flaviviridae family. CSFV is an enveloped RNA virus with the whole genome length being approximately 12.3 kb. The typical pathological change caused by CSFV is degeneration of vascular wall, which may result in hemorrhage, infarction and necrosis of internal organs in addition to fever. CSF is contracted mainly through direct contact. Pigs carring CSFV often act as sources of transmission, and can spread the virus to the environmennts via faeces before any clinical syndroms can be observed. Recent research indicates that the prevalent genotype of CSFV has changed to group 2 from group 1 to which the vaccine C-strain belongs, suggesting that current vaccine could not provide efficient protection against infections by field CSFV.The present study was aimed to:(1) probe the molecular epidemiology of CSFV in Zhejiang, (2) characterize monoclonal antibodies against E2 protein of CSFV strain SM, (3) identify the recognition sites of monoclonal antibodies on the E2 protein. Results thus obtained may provide the foundation for further analysis of antigenic diversity of classical swine fever virus.1. Detection and molecular epidemiology of CSFVTwo pairs of primers were designed for nest RT-PCR based on the conserved regions upstream and downstream of E2 gene. This PCR reaction exhibited good specificity and could detect as low as 1400 copies of CSFV genome. An RFLP approach was developed to differentiate vaccine strain and wild strains based on the distinct distribution of Mspl enzyme restriction sites in their E2 genes. This method was applied to screen CSFV in 59 sapmles from swines in Zhejiang Province. The results revealed that 9 samples were recognized by PCR, with 4 belonging to vaccine strains,4 to wild strains, and the remaining representing the mixture of vaccine strains and wild strains. Phylogenetic analysis of the 190bp variable region of E2 gene indicates that the field viruse isolates recovered in Zhejiang and neibourghing areas belonged to genotype 2.1b, which were divergent from the group 1 isolates including the vaccine C-strain.2. Characterization of E2 monoclonal antibodies from CSFV strain SMTo investigate the effects of some key amino acids involved in the antibody recognition within highly variable region of E2 protein on the antigen structures, we vaccinated BALB/c mice with E2 protein from CSFV classical virulent strain SM, and obtained one hybridoma cells (2B10) that could secrete antibodies against the E protein via hybridoma technique and IFA screening. Subtyping indicated that the monoclonal antibody was IgG2a isotype.2B10 could react with strain SM in IFA assay. These reaults demonstrated that the recognition sites by 2B10 only existed in strain SM.3. Identification of recognition sites of the monoclonal antibody 2B10Monoclonal antibody 2B10 had strong reaction with recombinant protein E2-AD, and also reacts with truncated E2-BC by Western blot and indirect ELISA, indicating that the linear epitopes were located within the 1-125 amino acid resdues of E2. The diversity of E2 protein from prevalent strains was analyzed using two monoclonal antibodies.2B10 could recognize SM strain belonging to group 1, but failed to recognize vaccine strain of the same group and wild strains of group 2. Given that the vaccine strain or wild strains could not react with 2B10, the recognition sites were present only in strain SM. Some amino acids differences were found through comparison of E2-BC sequences between wild strains and the vaccine strain. These amino acids were site-mutated, and mutant peptides were expressed. These peptides were subsequently subjected to reaction with 2B10. The results demonstrated that the recongnition site for 2B10 was P709 in E2 of CSFV strain SM.In summary, this study has provided good foundation for continuing research on the relationship between variations of CSFV E2 gene and antigenic divergence as well as on the development of more effective anti-CSF vaccine.更多还原