欧洲型PRRSV重组腺病毒疫苗生产工艺及动物模型替代方法建立

发布时间：2018-08-15 13:31

【摘要】：二十世纪90年代,我国首次报道了猪繁殖与呼吸综合征(Procine reproductive and respirator syndrome,PRRS),并且确定其病原为美洲型PRRSV,直到现在该病仍然是影响我国养猪业最主要的疾病之一。从爆发以来,我国主要以美洲型高致病性PRRSV及其变异株为主。近些年,PRRS已经打破了地域的限制,在我国北京、香港、内蒙古等地相继分离出欧洲型PRRSV,增加了我国对PRRS预防与治疗难度。目前为止,还没有专门针对我国境内欧洲型PRRS的疫苗。本研究中,对本实验室构建的欧洲型PRRS重组腺病毒疫苗进行鉴定,利用生物反应器微载体培养系统对欧洲型PRRS重组腺病毒候选疫苗进行扩增,并通过动物替代模型试验及血清学替代实验,对重组腺病毒候选疫苗进行效力检测。通过Western blot、PCR方法、间接免疫荧光以及透射电镜观察等方法,验证欧洲型PRRS重组腺病毒rAd-E35-IL-18构建正确。通过生物反应器培养HEK293细胞并扩增欧洲型PRRS重组腺病毒rAd-E35-IL-18,确定最佳培养参数,HEK293细胞在36-60h之间增长速率较快,在36h时更换DMEM培养液,使细胞以较快速率生长,84h后细胞几乎长满微载体表面,之后细胞生长状态呈下降趋势。将12h、24h、36h、48h、60h、72h取样的病毒液进行病毒滴度测定,结果显示,60h时病毒滴度较高。应用重组腺病毒候选疫苗rAd-ORF3-ORF5-IL-18对小鼠和猪进行免疫原性试验、攻毒试验和病毒载量检测,初步验证小鼠可以作为猪的替代模型。在小鼠替代模型基础之上,对小鼠和猪进行重组腺病毒梯度接种,检测免疫组动物的细胞因子和中和抗体水平。对免疫组进行攻毒保护试验,进一步验证小鼠可作为重组腺病候选疫苗的动物替代模型。获取特异性抗体,测定出各梯度相应疫苗免疫应答效价,小鼠抗PRRSV抗体水平大于1:682.7时,可初步作为样品疫苗检验标准。
[Abstract]:Swine reproductive and respiratory syndrome (Procine reproductive and respirator syndrome) was first reported in China in the 1990s, and the pathogen was identified as American PRRSV.The disease is still one of the most important diseases affecting the pig industry in China. Since the outbreak, American type high pathogenic PRRSV and its mutant strains have been the main ones in China. In recent years, PRRS has broken the limitation of region. In Beijing, Hong Kong, Inner Mongolia and other places, European PRRSVS have been isolated one after another, which has increased the difficulty of prevention and treatment of PRRS in China. So far, there is no specific European PRRS vaccine in China. In this study, the recombinant PRRS adenovirus vaccine constructed in our laboratory was identified, and the candidate vaccine of PRRS recombinant adenovirus was amplified by bioreactor microcarrier culture system. The potency of recombinant adenovirus candidate vaccine was tested by animal substitution model test and serological substitution test. The construction of European type PRRS recombinant adenovirus rAd-E35-IL-18 was verified by Western blottir polymerase chain reaction, indirect immunofluorescence and transmission electron microscopy. HEK293 cells were cultured in bioreactor and the recombinant adenovirus rAd-E35-IL-18 of European type PRRS was amplified. The best culture parameter was determined that the growth rate of HEK293 cells was faster in the range of 36 to 60 h, and the culture medium of DMEM was replaced at 36 h. After the cells grew at a faster rate for 84 h, the surface of the microcarriers was almost covered, and then the growth state of the cells decreased. The virus titer was measured in the virus solution sampled for 72 hours after 12h ~ 24h ~ 36h ~ 48h ~ 48h ~ (60 h). The results showed that the titer of the virus was higher at 60h. The recombinant adenovirus candidate vaccine rAd-ORF3-ORF5-IL-18 was used for immunogenicity test, attack test and viral load detection in mice and pigs. It was preliminarily verified that mice could be used as a substitute model for pigs. On the basis of mouse substitute model, recombinant adenovirus gradients were inoculated to mice and pigs to detect cytokines and neutralizing antibodies in immunized animals. The immunized group was tested for the protection of virus attack to further verify that the mice could be used as an alternative animal model for recombinant adenosis vaccine. The specific antibody was obtained and the titer of immune response of each gradient vaccine was determined. When the level of anti PRRSV antibody in mice was greater than 1: 682.7, it could be used as the test standard of the sample vaccine.
【学位授予单位】：吉林农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.4

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