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目的:探讨马传染性贫血病毒(Equineinfectiousa-nemiavirus,EIAV)驴白细胞减毒疫苗(DLV)免疫马后,外周血单个核细胞(PBMC)中IFN-γmRNA转录水平与免疫保护应答的关系,揭示DLV的免疫保护机制。方法:应用分子克隆及实时定量RT-PCR技术,建立马PBMC中IFN-γmRNA转录水平的定量检测方法,在不同时间点定期观察4组(疫苗免疫组、阴性对照组、强毒株阳性对照组及EIAV自然感染组)12匹马PBMC中IFN-γmRNA的转录水平及分布特征,同时监测体温变化等指标。疫苗株免疫动物8个月后,用EIAV强毒株攻击,观察攻击前后IFN-γmRNA转录水平的变化。结果:疫苗免疫马在免疫期内,PBMC中IFN-γmRNA转录的量显著高于阴性对照组及自然感染组(P<0.01)。免疫后用EIAV强毒株攻击,IFN-γ转录的量继续升高,4匹免疫马均获得完全保护;强毒株阳性对照组IFN-γmRNA转录的量随疾病的进展波动,发热期明显下降。结论:本研究首次证明,EIAV减毒疫苗可诱导马PBMC中IFN-γ基因高效转录,其转录水平与DLV的免疫保护密切相关。此结果在分子水平为阐明DLV的免疫保护机制提供了新的实验依据。
OBJECTIVE: To investigate the relationship between the level of IFN-γmRNA transcription and the immune protection response in peripheral blood mononuclear cells (PBMCs) after immunization with equineinfectiousa-nemiavirus (EIAV) donkey leukocyte attenuated vaccine (DLV) Immune protection mechanism. Methods: Quantitative detection of IFN-γ mRNA level in PBMCs was established by molecular cloning and real-time quantitative RT-PCR. Four groups (immunized group, negative control group and virulent positive control group were observed regularly at different time points. And EIAV natural infection group) in 12 horses PBMC IFN-γmRNA transcriptional level and distribution characteristics, while monitoring body temperature changes and other indicators. Eight months after vaccination, the mice were challenged with virulent strains of EIAV and the changes of IFN-γ mRNA levels before and after challenge were observed. Results: The amount of IFN-γmRNA transcripts in PBMC of vaccinated horses during immunization was significantly higher than that of negative control group and natural infection group (P <0.01). After immunization with EIAV virulent strain, the amount of IFN-γ transcribed continued to increase, and all four immunized horses were completely protected. The amount of IFN-γmRNA transcripts in positive control group fluctuated with disease progression and the fever period decreased significantly . Conclusions: This study demonstrated for the first time that EIAV attenuated vaccines can induce efficient transcription of IFN-γ gene in PBMCs of horses and the transcriptional level is closely related to the immune protection of DLV. This result provides new experimental evidence for elucidating the immune protection mechanism of DLV at the molecular level.