HN蛋白携带TC标签的重组新城疫病毒的构建及鉴定

发布时间：2018-07-23 21:01

【摘要】：新城疫病毒(NDV)是在世界范围内引发家禽发病和死亡的主要病因。HN作为重要的囊膜蛋白,在NDV吸附入侵过程中发挥重要作用。本研究利用反向遗传技术,在HN编码区的N端插入由6个氨基酸组成的TC标签(tetracysteine),成功转染细胞拯救获得TC标记的重组病毒La Sota-HN-NTC,经鉴定La Sota-HN-NTC能够在鸡胚中有效复制,生长曲线与亲本病毒La Sota相似。Western-blot结果显示,La Sota-HN-NTC中HN蛋白的表达量与La Sota相比无明显变化。La Sota-HN-NTC的毒力稍低于La Sota,并且TC标签能够在重组病毒中稳定存在。La Sota-HN-NTC感染的细胞中能够观察到明显的绿色荧光,表明TC标记的HN蛋白能够正常表达并被双砷染料特异性染色。NDV吸附试验表明,La Sota-HN-NTC吸附细胞受体的能力没有因为TC标签发生改变。激光共聚焦监测到了La Sota-HN-NTC感染细胞中HN蛋白的动态分布。本研究为监测HN蛋白在NDV感染过程中的动态分布,阐明HN蛋白在NDV感染中的作用机制提供了工具和基础。
[Abstract]:Newcastle disease virus (NDV) is the main cause of poultry morbidity and mortality in the world. HN is an important envelope protein and plays an important role in the process of NDV adsorption and invasion. In this study, reverse genetic technique was used to insert a 6-amino acid (tetracysteine), tag (tetracysteine), into the N-terminal of HN coding region to successfully transfect the TC-labeled recombinant virus La Sota-HN-NTC. The recombinant virus La Sota-HN-NTC was successfully transfected into the HN coding region. The recombinant virus La Sota-HN-NTC was identified to be able to replicate effectively in chicken embryos. The growth curve was similar to that of parent virus La Sota. Western-blot analysis showed that the expression of HN protein in La Sota-HN-NTC had no significant change compared with La Sota. La Sota-HN-NTC was slightly less virulent than La Sota, and TC label could stably exist the infection of .La Sota-HN-NTC in recombinant virus. Visible green fluorescence can be observed in the cells, The results showed that TC-labeled HN protein could express normally and was stained by double arsenic dye. NDV adsorption test showed that the ability of La Sota-HN-NTC to adsorb cell receptor was not changed because of TC label. The dynamic distribution of HN protein in La Sota-HN-NTC infected cells was detected by confocal laser. This study provides a tool and basis for monitoring the dynamic distribution of HN protein in the process of NDV infection and clarifying the mechanism of HN protein in NDV infection.
【作者单位】：东北农业大学动物科学技术学院;中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室禽呼吸道病研究团队;
【基金】：黑龙江省应用技术研究与开发计划项目(PC13S02) 国家自然科学基金项目(31502088) 黑龙江省自然科学基金项目(QC2015038)
【分类号】：S852.65

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