【作者】 项海涛；

【导师】 柳纪省； 胡永浩；

【作者基本信息】 甘肃农业大学 ， 预防兽医学， 2009， 硕士

【摘要】 目前,对于口蹄疫的防制主要采用屠杀、隔离和免疫接种,其中疾病流行前的免疫接种是特异性保护家畜免遭感染的有效途径,但现有疫苗接种动物后都会存在免疫空白期,即诱导免疫动物产生体液和细胞免疫反应,等待有效抗体产生的一段时间。动物如果在这段时间内遭到病毒的入侵往往会引起该病的流行。为探索解决免疫动物在疫苗接种的免疫空白期内发病这一难题,本研究以Asia I江苏株为模板,引入RNA干扰机制,进行了双功能疫苗的探索研究,并取得下述进展:1.对pBudCE4.1载体进行改造,将其CMV启动子替换为U6启动子,成功改造出适合于双功能疫苗研究的双启动子载体pBudCE4.1-U6。2.利用绿色荧光蛋白的表达与沉默表达对载体pBudCE4.1-U6的双启动子分别进行功能效率鉴定,结果表明对载体的改造是成功的,载体pBudCE4.1-U6的两个启动子均能启动目的基因的表达。3.针对Asia I江苏株的3D基因设计3条小RNA干扰片段,并将之与免疫组合基因P12A+3C克隆入双启动子载体pBudCE4.1-U6,成功构建出了3个双功能疫苗质粒。4.双功能疫苗质粒转染靶细胞BHK-21,有延迟细胞病变的作用,说明双功能载体能够转录出siRNA,对病毒的转录复制有一定的抑制效果。5.间接免疫荧光检测结果表明双功能载体能够同时启动口蹄疫免疫基因在BHK-21细胞中的表达。更多还原

【Abstract】 At present, slaughter, isolation and vaccination are major methods to control FMD, and vaccination before epidemic of FMD is the specific efficient method to protect animals against virus entry. But a challenge to the development of vaccines against microbiology is the exist of immunization black and the latent period, FMD would outbreak when animals challenged with virus during the immunization black as well as animals inoculate the vaccine during the latent period. In order to solve this problem, we introduced RNAi into the bi-functional vaccine research, using Asia I/JS strain as the model to confirm the feasibility of the conception.1. Replaced the CMV promoter of vector pBudCE4.1 by U6 promoter, developed a dual-promoter vector pBudCE4.1-U6.2. Tested the functions of the promoters of vector pBudCE4.1-U6 by expressing EGFP and Interfering EGFP expression. Results demonstrated that both CMV promoter and U6 promoter could play its role.3. We successfully constructed three bi-functional plasmids expressing shRNAs targeting 3D gene of Asia I/JS strain FMDV and expressing immunization protein P12A+3C simultaneously.4. The antiviral potential induced by bi-functional plasmid was evident in the face of challenge of with a homologous FMDV and the inhibition was inconspicuousness.5. Detection of indirect immunofluorescence showed that immunization protein was expressed in BHK-21 cells.更多还原