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目的:研究MDV-1 CVI988 VP22蛋白的转导机制及细胞定位机理,分析该蛋白在表达过程中细胞定位的影响因素。方法:构建表达VP22蛋白的重组人腺病毒,将重组病毒感染的AD-293细胞裂解产物加至正常的MDBK细胞上,通过免疫荧光(IFA)及Western blot鉴定VP22的蛋白转导功能。观察感染后不同时间VP22在AD-293细胞上的定位,并与瞬时表达的VP22蛋白定位比较。结果:重组人腺病毒表达的VP22在MDBK细胞上能够进入几乎所有的细胞,说明VP22蛋白具有很强的蛋白转导功能。进一步鉴定VP22的细胞定位发现,在重组病毒感染的AD-293细胞中,VP22首先聚集于细胞核周围,随后以特殊的荧光粒子的形式散在于胞质中,而AD-293细胞中瞬时表达的VP22及MDV感染的CEF中VP22均均匀分布于细胞核。结论:重组人腺病毒表达的VP22蛋白具有很强的蛋白转导功能,不同重组病毒表达的VP22在细胞中的定位模式有所差别。
OBJECTIVE: To study the transduction mechanism and cellular localization mechanism of VP22 protein in MDV-1 CVI988 and to analyze the influencing factors of cellular localization during the expression of MDV-1 CVI988 VP22 protein. METHODS: Recombinant human adenoviruses expressing VP22 protein were constructed. The recombinant virus-infected AD-293 cell lysate was added to normal MDBK cells. The protein transduction function of VP22 was identified by immunofluorescence (IFA) and Western blot. The location of VP22 on AD-293 cells was observed at different times after infection and compared with the transient expression of VP22 protein. Results: Recombinant human adenovirus expressed VP22 can enter almost all cells in MDBK cells, indicating that VP22 protein has a strong protein transduction function. Further identification of the cellular localization of VP22 found that in recombinant virus-infected AD-293 cells, VP22 first aggregated around the nucleus, followed by specific fluorescent particles in the cytoplasm, whereas transient expression of VP22 in AD-293 cells And VP22 in MDV-infected CEF were uniformly distributed in the nucleus. CONCLUSION: VP22 protein expressed by recombinant adenovirus has a strong protein transduction function. The expression patterns of VP22 in different recombinant viruses are different.