SARS冠状病毒N蛋白的真核细胞定位研究

Cellular localization of SARS - CoV nucleocapsid protein in 293 cells

目的:为进一步探讨SARS-CoV核衣壳蛋白(N蛋白)在病毒复制及细胞通路中的作用,观察了核衣壳蛋白不同区段在细胞内的定位。方法:应用核定位序列分析软件分析N蛋白序列,找出其中的核定位信号序列;构建增强型绿色荧光蛋白(EGFP)与SARS-CoV不同长度核衣壳蛋白区段融合表达的pEGFP-C1重组载体,对293细胞进行瞬时转染,在荧光显微镜下观察核衣壳蛋白不同区段在细胞内的定位。结果:不同的核定位信号分析软件分析的结果不同,Prosite找到的核定位信号位于N蛋白第373-389或374-390aa,而PredietNLS server找到的核定位信号位于N蛋白第36-44aa;全长核衣壳蛋白及第161-422aa区段均定位于细胞质,第1-187aa区段定位于细胞质和细胞核。结论:SARS-CoV核衣壳蛋白的核定位信号可能位于N蛋白第36-44aa。

Objective： To study the role of nucleocapsid protein in virus replication and cell signaling passway ,and to observe the cellular localization of SARS - CoV nucleocapsid protein. Methods： The sequence of SARS - CoV nucleocapsid protein was analyzed by protein analysis programs to identify nuclear localization signals（ NLS）; 293 cells were transfected by the pEGFP- C1 recombinant plasmids which expressed fusion protein of enhanced green fluorescence protein（EGFP） and nucleocapsid protein. At 24, 48, 72 hours post transfection （p. t. ） ,the Green Fluorescent Proteins （GFP） were observed by fluorescence microscope. Results： By using PROSITE, we identified a nuclear localization signal sequence from amino acids 373 to 389 or from amino acids 374 to 390 in the nucleocapsid protein sequence. However, the analysis of PredictNLS server revealed that the nuclear localization signal region was located between amino acids 36 and 44. N1 protein（1 -187aa） was located in both cytoplasm and cell nucleu;N protein（ 1 -422aa） and N2 protein（ 161 -422aa） were located in cytoplasms . Conclusion： The nuclear localization signal is likely to be located between amino acids 36 and 44 in SARS - CoV nucleocapsid protein.