【作者】 孙德刚；

【导师】 黄保续；

【作者基本信息】 东北农业大学 ， 临床兽医学， 2007， 硕士

【摘要】 伪狂犬病病毒是疱疹病毒科、α-疱疹病毒亚科的成员，可以引起猪、牛、羊及野生动物的伪狂犬病(又称Aujeszky氏病)，猪是该病毒的天然宿主、也是主要感染源。伪狂犬病给养猪业造成了巨大经济损失，其危害仅次于猪瘟和口蹄疫。作为主要的诊断抗原，伪狂犬病病毒糖蛋白E(glycoprotein,gE)在伪狂犬根除计划中发挥重要的作用。为获得大量的、成本较低的诊断抗原，本研究利用大肠杆菌表达系统对伪狂犬病病毒gE主要抗原区域进行了表达，为gE-ELISA试剂盒的研制提供一定的理论依据和借鉴。本研究主要内容：根据GenBank中伪狂犬病毒gE基因序列，设计了一对引物，PCR扩增长度为558bp的gE基因去信号肽后的主要抗原区域(157-714bp)，将其克隆进测序载体PGEM-Teasy中，成功构建质粒PGEMTeasy-gE。用BamhI和HindⅢ将gE基因主要抗原区域从PGEM-Teasy中切出，然后与同样酶处理的原核表达载体pet32a连接，命名为pet-gE。在IPTG的诱导后，经SDS-PAGE电泳发现43ku处出现特异性蛋白条带。研究了目的蛋白表达量与各个培养条件的关系，优化后的最佳表达条件为IPTG浓度为1mM，诱导时间为5小时时的蛋白表达量最大。经过western-blot分析，表达产物具有很好的抗原性。该研究成功表达出具有抗原性的目的蛋白，为血清学鉴别诊断方法gE-ELISA的建立打下很好的基础，同时gE-ELISA也能对野毒和疫苗毒进行鉴别。更多还原

【Abstract】 Pseudorabies virus(prv),is a member of Alphaherpesvirinae,herpesviridae,which can causeAujeszky’s disease in swine,bovine,sheep and wild animals.PRV infection could cause its naturaland main storing host-swine(also its main infectious origination)a huge loses,the most hugeeconomical lose in all swine infectious diseases except swine plague and aftosa. Glyprotein E ofPseudorabies Virus is known to be an important diagnostic antigen in pseudorabies eradicationcampaign.In order to obtain the gE antigen in large quantity and at a low cost, a gene fragment encodingthe main antigtie domain (157-714bp) of PRV gE without signal peptide was expressed in E coliexpression system. This study may supply theoretical evidence for establishment of gE-ELISA.Using a pair of primers designed according to the revelant nucleotide sequence fromGenBank, the specific genes encoding the main antigetic domains of PRV gE were amplified. ThePCR products of 557bp were cloned into the sequencing vectors PGEM-Teasy.The plasmidPGEMTeasy-gE was constructed successfully. Cut out the main antigetic of PRV gE fromPGEMTeasy-gE with BamhⅠand HindⅢ, and linked with prokaryotic expression vector pet32awhich has been cut by the two enzyme beforehand. The plasmid construced was named PET-gE.After induced by IPTG,a specific strip of 43 Ku appeard in SDS-PAGE.According to the study on the relationship bwteen expression quantity of protein and revelantcultivating conditions, the optimal expression condition were: 5 hours inducing range, the 1mMconcentration of IPTG.On analysis by Western-blot, the expression product has good antigenicity.The target protein with antigenicity was expressed successfully in the study. It laid thefoundation for the establishment of the diagnostic method of serology gE-ELISA,which can tellvaccine virus from virulence virus.更多还原