经典和高致病性PRRSV疫苗株嵌合病毒的构建及免疫原性分析
发布时间：2018-10-12 16:47
【摘要】：猪繁殖与呼吸综合征(Porcine reproductive and respiratory syndrome,PRRS)是一种由PRRS病毒(PRRS virus,PRRSV)引起的猪病毒性传染病,主要临床特征为妊娠母猪繁殖障碍和各年龄阶段猪呼吸系统疾病。国内于1996年首次分离到PRRSV,2006年由PRRSV变异株引起的高致病性猪繁殖与呼吸综合症(HP-PRRS)给我国养猪业造成了巨大的经济损失,已成为严重危害我国养猪业的传染病之一。目前,疫苗免疫仍是防控PRRS的有效方法,但现有的经典和高致病性PRRSV疫苗在诱导机体免疫反应和交叉保护等方面存在差异,而关于这种差异产生的分子基础尚无报道。因此,本实验选择经典和高致病PRRSV疫苗株为研究对象,以期通过构建嵌合病毒揭示PRRSV疫苗株产生这种免疫学反应差异的原因或基因区域,同时为PRRSV基因组结构与功能的研究提供新方法和思路。本实验在HP-PRRSV HuN4-F112疫苗株的全长cDNA分子感染性克隆pHuN4-F112基础上,利用反向遗传操作技术,将pHuN4-F112基因序列分别替换为经典PRRSV CH-1R疫苗株的相应基因,最终构建CH-1R株的全长cDNA分子感染性克隆,并将CH-1R基因组第454位的C沉默突变为T产生1个SpeI酶切位点作为鉴定拯救病毒的分子标记。将含CH-1R株基因组全长cDNA质粒线性化后体外转录合成病毒RNA,转染BHK-21细胞包装病毒粒子,24 h后将细胞液上清接种MARC-145细胞,拯救出病毒。通过细胞病变(CPE)观察、间接免疫荧光(IFA)和检测分子标记证明病毒拯救成功。进一步比较拯救病毒与亲本病毒的病毒滴度、增殖动力学曲线,发现两者的病毒学及生物学特性没有显著差异,以上结果表明PRRSV CH-1R疫苗株感染性克隆构建成功。本实验以经典PRRSV疫苗株CH-1R的全长感染性克隆pCH-1R为骨架,利用反向遗传操作技术分别将其ORF1a、ORF1b和ORF2-7替换为HP-PRRSV HuN4-F112的相应片段,构建3个全长cDNA嵌合质粒。将构建的3个重组cDNA质粒经体外转录后转染BHK-21细胞,并于MARC-145细胞中传代,拯救出嵌合病毒,分别命名为rCH-1R-T1a、rCH-1R-T1b和rCH-1R-T27。病毒一步生长曲线结果表明,3种嵌合病毒在MARC-145细胞中的生长特性与亲本病毒rCH-1R基本一致;嵌合病毒接种PRRSV阴性仔猪结果显示,嵌合病毒试验组血清中PRRSV N蛋白抗体阳转率分别为3/3(rCH-1R-T1a)、2/3(rCH-1R-T1b)和1/3(rCH-1R-T27),而rCH-1R免疫组血清N蛋白抗体检测始终为阴性。以上结果表明,PRRSV ORF1a和ORF1b区域在病毒诱导N蛋白抗体产生中起关键作用。
[Abstract]:Porcine reproductive and respiratory syndrome (Porcine reproductive and respiratory syndrome,PRRS) is a kind of porcine viral infectious disease caused by PRRS virus (PRRS virus,PRRSV). The main clinical features are reproductive disorders in pregnant sows and respiratory diseases in pigs of all ages. The highly pathogenic porcine reproductive and respiratory syndrome (HP-PRRS) caused by the PRRSV variant strain was first isolated in China in 1996. It has caused huge economic losses to the pig industry in China and has become one of the infectious diseases that seriously harm the pig industry in China. At present, vaccine immunization is still an effective method to prevent and control PRRS, but the existing classical and highly pathogenic PRRSV vaccines are different in inducing immune response and cross-protection, but the molecular basis of this difference has not been reported. Therefore, this study selected classical and highly pathogenic PRRSV vaccine strains as the research object, in order to reveal the cause or gene region of the difference in the immunological response of PRRSV vaccine strain by constructing chimeric virus. At the same time, it provides a new method and train of thought for the study of PRRSV genome structure and function. On the basis of the full-length cDNA molecular infectious clone pHuN4-F112 of HP-PRRSV HuN4-F112 vaccine strain, the pHuN4-F112 gene sequence was replaced with the corresponding gene of classical PRRSV CH-1R vaccine strain by reverse genetic manipulation. Finally, the full-length cDNA molecular infectious clone of CH-1R strain was constructed, and the C-silencing site at position 454th of the CH-1R genome was mutated to T producing a SpeI restriction site as a molecular marker to identify and rescue the virus. The full-length cDNA plasmid containing CH-1R strain was linearized and transcriptional synthesis virus (RNA,) was transfected into the packaging virus particles of BHK-21 cells in vitro. After 24 hours, the supernatant of the cell fluid was inoculated with MARC-145 cells to save the virus. By observing cytopathic (CPE), indirect immunofluorescence (IFA) and detection of molecular markers proved that the virus was successfully saved. Further comparing the virus titer and proliferation kinetics curves of saving virus and parent virus, it was found that there was no significant difference in virology and biological characteristics between them. The above results indicated that the infectious clone of PRRSV CH-1R vaccine strain was successfully constructed. In this experiment, three full-length cDNA chimeric plasmids were constructed by replacing ORF1a,ORF1b and ORF2-7 with the corresponding fragments of HP-PRRSV HuN4-F112 by reverse genetic manipulation with the full-length infectious clone pCH-1R of classical PRRSV vaccine strain CH-1R as the skeleton. Three recombinant cDNA plasmids were transfected into BHK-21 cells after transcriptional in vitro, and then subcultured in MARC-145 cells to save the chimeric virus, named rCH-1R-T1a,rCH-1R-T1b and rCH-1R-T27., respectively. The results of one-step growth curve showed that the growth characteristics of the three chimeric viruses in MARC-145 cells were basically the same as those of parental virus rCH-1R, and the results of inoculation of chimeric viruses with PRRSV negative piglets showed that the growth characteristics of the three chimeric viruses in MARC-145 cells were similar to those of parental viruses. The positive rates of antibody to PRRSV N protein in chimeric virus group were 3 / 3 (rCH-1R-T1a), 2 / 3 (rCH-1R-T1b) and 1 / 3 (rCH-1R-T27), respectively. These results suggest that the PRRSV ORF1a and ORF1b regions play a key role in virus-induced production of N-protein antibodies.
【学位授予单位】：吉林农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S855.3
本文编号：2266868
[Abstract]:Porcine reproductive and respiratory syndrome (Porcine reproductive and respiratory syndrome,PRRS) is a kind of porcine viral infectious disease caused by PRRS virus (PRRS virus,PRRSV). The main clinical features are reproductive disorders in pregnant sows and respiratory diseases in pigs of all ages. The highly pathogenic porcine reproductive and respiratory syndrome (HP-PRRS) caused by the PRRSV variant strain was first isolated in China in 1996. It has caused huge economic losses to the pig industry in China and has become one of the infectious diseases that seriously harm the pig industry in China. At present, vaccine immunization is still an effective method to prevent and control PRRS, but the existing classical and highly pathogenic PRRSV vaccines are different in inducing immune response and cross-protection, but the molecular basis of this difference has not been reported. Therefore, this study selected classical and highly pathogenic PRRSV vaccine strains as the research object, in order to reveal the cause or gene region of the difference in the immunological response of PRRSV vaccine strain by constructing chimeric virus. At the same time, it provides a new method and train of thought for the study of PRRSV genome structure and function. On the basis of the full-length cDNA molecular infectious clone pHuN4-F112 of HP-PRRSV HuN4-F112 vaccine strain, the pHuN4-F112 gene sequence was replaced with the corresponding gene of classical PRRSV CH-1R vaccine strain by reverse genetic manipulation. Finally, the full-length cDNA molecular infectious clone of CH-1R strain was constructed, and the C-silencing site at position 454th of the CH-1R genome was mutated to T producing a SpeI restriction site as a molecular marker to identify and rescue the virus. The full-length cDNA plasmid containing CH-1R strain was linearized and transcriptional synthesis virus (RNA,) was transfected into the packaging virus particles of BHK-21 cells in vitro. After 24 hours, the supernatant of the cell fluid was inoculated with MARC-145 cells to save the virus. By observing cytopathic (CPE), indirect immunofluorescence (IFA) and detection of molecular markers proved that the virus was successfully saved. Further comparing the virus titer and proliferation kinetics curves of saving virus and parent virus, it was found that there was no significant difference in virology and biological characteristics between them. The above results indicated that the infectious clone of PRRSV CH-1R vaccine strain was successfully constructed. In this experiment, three full-length cDNA chimeric plasmids were constructed by replacing ORF1a,ORF1b and ORF2-7 with the corresponding fragments of HP-PRRSV HuN4-F112 by reverse genetic manipulation with the full-length infectious clone pCH-1R of classical PRRSV vaccine strain CH-1R as the skeleton. Three recombinant cDNA plasmids were transfected into BHK-21 cells after transcriptional in vitro, and then subcultured in MARC-145 cells to save the chimeric virus, named rCH-1R-T1a,rCH-1R-T1b and rCH-1R-T27., respectively. The results of one-step growth curve showed that the growth characteristics of the three chimeric viruses in MARC-145 cells were basically the same as those of parental virus rCH-1R, and the results of inoculation of chimeric viruses with PRRSV negative piglets showed that the growth characteristics of the three chimeric viruses in MARC-145 cells were similar to those of parental viruses. The positive rates of antibody to PRRSV N protein in chimeric virus group were 3 / 3 (rCH-1R-T1a), 2 / 3 (rCH-1R-T1b) and 1 / 3 (rCH-1R-T27), respectively. These results suggest that the PRRSV ORF1a and ORF1b regions play a key role in virus-induced production of N-protein antibodies.
【学位授予单位】：吉林农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S855.3
【参考文献】
相关期刊论文 前1条
1 郭宝清,陈章水,刘文兴,崔益洙;从疑似PRRS流产胎儿分离PRRSV的研究[J];中国畜禽传染病;1996年02期
，本文编号：2266868
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