麻疹病毒通过受体SLAM对昆虫细胞的感染及其诱发细胞凋亡的机制研究

发布时间：2018-01-10 20:00

本文关键词：麻疹病毒通过受体SLAM对昆虫细胞的感染及其诱发细胞凋亡的机制研究　出处：《武汉大学》2005年博士论文　论文类型：学位论文

【摘要】：已经证明淋巴细胞信号刺激因子SLAM(signaling lymphocytic activation molecule)是麻疹病毒的第二受体。我们在苜宿银线夜蛾核型多角体病毒(AcMNPV)的极晚期启动子Ppolh下游,插入编码增强型绿色荧光蛋白(EGFP)的报道基因,构建了一种来源于杆状病毒的载体——Bacmid-egfp。利用这种带标记的重组杆状病毒感染Sf9细胞,强烈的绿色荧光说明重组杆状病毒的感染。并且,因为不是与外源基因在同一个启动子下,杆状病毒表达系统中EGFP表达对该系统没有影响。我们在Bacmid-egfp中插入Ppol控制的麻疹病毒(measles virus,MV)细胞受体SLAM基因,构建重组杆状病毒rv-egfp-SLAM。在昆虫细胞Sf9中,得到融合有六个组氨酸标签的SLAM,分子量大约为46kD。HPLC证明,经过镍柱纯化的SLAM为比较单一的蛋白。激光共聚焦证明,使用重组杆状病毒表达的SLAM定位在Sf9细胞膜表面。用纯化的蛋白做pull down,发现它可以与麻疹病毒的血凝素H蛋白相互作用,证明杆状病毒表达的SLAM可能具有麻疹病毒受体的功能。进一步的实验证明,当用麻疹病毒感染表达SLAM蛋白的Sf9细胞时,我们可以观察到细胞融合,而且细胞的融合程度与麻疹病毒的感染复数成正相关。在mRNA水平,我们检测到麻疹病毒H基因在Sf9细胞中的转录。免疫细胞组化结果显示了麻疹病毒在SLAM-Sf9细胞中的复制。最后,流式细胞检测说明,麻疹病毒可以诱发表达
[Abstract]:SLAM(signaling lymphocytic activation molecule. It is the second receptor of measles virus. We are downstream of the very late promoter Ppolh of the nucleotypic polyhedrosis virus (AcMNPV). A reporter gene encoding enhanced green fluorescent protein (EGFP) was inserted. A baculovirus-derived vector Bacmid-egfp. was constructed to infect Sf9 cells with this tagged recombinant baculovirus. Strong green fluorescence indicates the infection of the recombinant baculovirus. And, because it is not under the same promoter as the foreign gene. The expression of EGFP in baculovirus expression system had no effect on the system. We inserted Ppol controlled measles virus measurements virus into Bacmid-egfp. The recombinant baculovirus rv-egfp-SLAM was constructed by SLAM gene. In insect Sf9, SLAM with six histidine tags was obtained. The molecular weight was about 46kD. HPLC and the SLAM purified by nickel column was a single protein. The SLAM expressed by recombinant baculovirus was located on the surface of Sf9 cell membrane. The purified protein was used for pull down-down. It was found that it could interact with hemagglutinin H protein of measles virus. It is suggested that the SLAM expressed by baculovirus may have the function of measles virus receptor. Further experiments have shown that cell fusion can be observed when Sf9 cells expressing SLAM protein are infected with measles virus. And the degree of cell fusion is positively correlated with the number of measles virus infections. At the mRNA level. We detected the transcription of measles virus H gene in Sf9 cells. Immunohistochemical results showed the replication of measles virus in SLAM-Sf9 cells. Finally, flow cytometry showed that. Measles virus can induce expression
【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2005
【分类号】：R373

【共引文献】