不同毒力水貂阿留申病毒在CRFK细胞中增殖及诱导细胞凋亡特性研究

发布时间：2018-06-13 10:24

本文选题：水貂阿留申病毒 + CRFK细胞　；参考：《吉林农业大学》2017年硕士论文

【摘要】：水貂阿留申病(Aleutian disease of mink,AMD),是危害世界养貂业的主要疾病之一,其病原为水貂阿留申病毒(Aleutian mink disease virus,AMDV)感染表现为慢性、持续性、进行性感染,皮兽群毛皮质量下降,种兽群繁殖能力降低,又称为浆细胞增多症。由于该病致病机理特殊,目前仍未研制出有效疫苗用于防控该病。研究表明,AMDV的限制性低水平复制与表达,可以导致病貂的持续性感染和免疫复合物的形成,引发慢性疾病,而细胞凋亡在这一致病过程中发挥重要作用。通常体外连续培养病毒是研究病毒致病机理以及强毒致弱获得弱毒疫苗侯选毒株最常规的方法。本试验对实验室分离鉴定的强毒株进行体外连续传代培养,对不同代次、不同毒力的病毒增殖特性及凋亡特性进行研究,为AMDV致病机理研究以及弱毒疫苗侯选毒株筛选奠定基础。设计特异性引物,通过反应条件的优化,建立了AMDV荧光定量PCR检测方法,该方法具有较好的特异性、敏感性和重复性。为AMDV临床检测及AMDV在CRFK细胞的增殖评价提供技术支持。对实验室分离鉴定的AMDV强毒株AMDV-DL124和AMDV-DL125株第7代(F7)细胞毒在CRFK细胞中进行体外培养,通过培养条件优化,目前已连续传代至45代(F45)。通过病毒含量测定、间接免疫荧光试验和荧光定量PCR试验对AMDV-DL124、AMDV-DL125毒株F7代及F45代细胞毒和标准弱毒株AMDV-G株在CRFK细胞的增殖特性进行研究。结果表明,AMDV-DL124和AMDV-DL125株病毒F7代毒的病毒含量可达108.5 TCID50/mL、明显高于F45代毒的病毒含量106 TCID50/mL和AMDV-G 105.5 TCID50/mL,而F45代毒和和AMDV-G株间,病毒含量无明显差异;间接免疫荧光试验结果表明,AMDV-DL124和AMDV-DL125株在48-72h增殖速度较快,在12-24h增殖速度较慢;荧光定量PCR试验结果表明,随着培养时间的延长AMDV在CRFK细胞的核酸量不断增加,在感染后12h后增加明显,但AMDV-DL124、AMDV-DL125核酸量的峰值可达2.4×106个拷贝,明显高于F45代病毒和AMDV-G株病毒,而后二者之间无明显差异。上述结果表明,AMDV强毒株AMDV-DL124和AMDV-DL125经CRFK细胞连续培养传代后在CRFK细胞的增殖能力降低。应用流式细胞术及TUNEL法检测AMDV-DL124、AMDV-DL125株F7代及F45代病毒和标准弱毒株AMDV-G诱导CRFK细胞凋亡情况。流式细胞术检测AMDV诱导CRFK细胞凋亡率结果表明,AMDV-DL124、AMDV-DL125株F7诱导CRFK细胞的凋亡率在感染后12h达最大值15.86%,明显高于F45代和AMDV-G株诱导CRFK细胞凋亡率2.94%、1.65%,细胞凋亡主要集中在病毒感染后2~12h。TUNEL法测定结果表明病毒在感染后12h出现明显凋亡;上述结果表明,AMDV-DL124和AMDV-DL125株经CRFK细胞连续传代后诱导CRFK细胞凋亡的能力降低。本研究为后续AMDV致病性与细胞凋亡关系的研究奠定基础。
[Abstract]:Aleutian disease of mink AMDV is one of the major diseases of mink industry in the world. The pathogen of Aleutian mink disease virus AMDV (Aleutian mink disease virusus AMDV) is chronic, persistent, progressive infection, and the quality of fur is decreased. The ability of breeding animals to reduce, also known as plasmacytosis. Because of the special mechanism of the disease, no effective vaccine has been developed to prevent and control the disease. Studies have shown that the restricted low level replication and expression of AMDV can lead to persistent infection and the formation of immune complex in minks and lead to chronic disease. Apoptosis plays an important role in the pathogenesis of AMDV. In general, continuous culture of virus in vitro is the most common method to study the pathogenicity of the virus and to obtain attenuated virus vaccine. In this experiment, the virulent strains isolated and identified in laboratory were continuously cultured in vitro, and the proliferation and apoptosis characteristics of different generations and virulence were studied. It will lay a foundation for the study of the pathogenic mechanism of AMDV and the screening of attenuated vaccine strains. The specific primers were designed, and the method of fluorescence quantitative PCR for detection of AMDV was established by optimizing the reaction conditions. The method has good specificity, sensitivity and reproducibility. To provide technical support for clinical detection of AMDV and evaluation of AMDV proliferation in CRFK cells. AMDV virulent strains AMDV-DL124 and AMDV-DL125 were cultured in CRFK cells in vitro. The proliferation characteristics of AMDV-DL124AMDV-DL125 and AMDV-DL125 in CRFK cells were studied by virus content determination, indirect immunofluorescence assay and fluorescence quantitative PCR. The results showed that the virus content of AMDV-DL124 and AMDV-DL125 strain F7 was 108.5 TCID50 / mL, which was significantly higher than that of F45 generation virus (106TCID50 / mL and AMDV-G 105.5 TCID50 / mL), but there was no significant difference between F45 generation virus and AMDV-G strain. The results of indirect immunofluorescence assay showed that AMDV-DL124 and AMDV-DL125 proliferated faster in 48h to 72h and slower in 12-24 hours, the results of fluorescence quantitative PCR showed that the nucleic acid content of AMDV in CRFK cells increased with the extension of culture time. However, the peak of AMDV-DL124AMDV-DL125 nucleic acid content was 2.4 脳 106 copies, which was significantly higher than that of F45 generation virus and AMDV-G strain, but there was no significant difference between them. The results indicated that the proliferation ability of AMDV-DL124 and AMDV-DL125 in CRFK cells was decreased after continuous culture. Flow cytometry and Tunel were used to detect the apoptosis of CRFK cells induced by AMDV-DL124AMDV-DL125 strain F7 passage, F45 generation virus and standard attenuated strain AMDV-G. The results of flow cytometry showed that the apoptosis rate of CRFK cells induced by AMDV-DL124AMDV-DL125 strain F7 reached a maximum of 15.86 at 12 h after infection, which was significantly higher than that of F45 generation and AMDV-G strain 2.941.65.The apoptosis mainly occurred in diseased cells. The results of TUNEL-mediated dUTP Nick end labeling (Tunel) at 2h after infection showed that the virus showed obvious apoptosis at 12h after infection. These results indicated that the apoptosis of CRFK cells induced by AMDV-DL124 and AMDV-DL125 decreased after continuous passage. This study laid a foundation for the further study of the relationship between the pathogenicity of AMDV and apoptosis.
【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】：S852.65

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