HPV16L1核浆运输的动力学过程  

The dynamic process of of HPV16L1 nucleocytoplasmic transport

作　　者：杨军[1] 王一理[2] 司履生[2] 

机构地区：[1]西安交通大学医学院附属第二医院病理科,西安710004 [2]西安交通大学生命科学与技术学院癌症研究所生物医学信息工程教育部重点实验室,西安710061

出　　处：《微生物学报》2006年第6期917-921,共5页Acta Microbiologica Sinica

基　　金：国家自然科学基金(30300305)~~

摘　　要：利用增强型绿色荧光蛋白(Enhancegreenflurenscentprotein,EGFP)标记不同的截短型HPV16L1蛋白(Humanpapillomavirustype16L1protein,HPV16L1),分析HPV16L1蛋白核定位信号(Nucleuslocationsignal,NLS)的作用。构建重组pFB-EGFP、pFB-EGFP-HPV16L1、pFB-EGFP-HPV16L1△NLS和pFB-EGFP-NLSHPV16L1p转移载体;在DH10Bac宿主菌内经Tn7转座子介导的同源重组后转染Sf-9细胞,获得重组Ac-EGFP、Ac-EGFP-HPV16L1、Ac-EGFP-HPV16L1△NLS和Ac-EGFP-NLSHPV16L1杆状病毒,感染Sf-9昆虫细胞表达相应截短型HPV16L1融合蛋白;利用荧光显微镜和激光共聚焦显微镜观察不同融合蛋白的荧光特性和核浆转运动力学过程。结果发现Ac-EGFP杆状病毒感染的Sf-9细胞内明亮的绿色荧光均匀分布;重组Ac-EGFP-HPV16L1和Ac-EGFP-NLSHPV16L1杆状病毒感染的Sf-9细胞,明亮的绿色荧光主要位于细胞核内;重组Ac-EGFP-HPV16L1△NLS杆状病毒感染的Sf-9细胞,绿色荧光局限于细胞浆内,细胞核内无绿色荧光。说明HPV16L1蛋白羧基端的23个氨基酸(GKRKATPTTSSTSTTAKRKKRKL)具有完全核定位作用,能引导HPV16L1蛋白和EGFP突破核膜屏障进入Sf-9细胞核内。In order to analyse the role of nucleus location signal of Human papiilomavirus type 16 L1 protein （NLSHPV16L1 ）, the various truncated HPV 16 L1 protein were tagged by enhance green flurenscent protein （EGFP）. After the EGFP gene and various truncated HPV16 L1 genes （HPV16 L1, HPV16L1△NLS and NLSHPV16L1 gene segment） were obtained, they were cloned into the baculovirus pFastbac-Hb transfer vector to constructe the recombinanted pFB-EGFP, pFB-EGFP-HPV16L1, pFB-EGFP-HPV16L1△NLS and pFB-EGFP-NLSHPV16L1 transfer vectors. Through Tn7 transposonmediated site-specific in vivo transposition, the foreign gene expression cassette was integrated into a baculovirus shuttle vector （bacmid）. Then the various bacmid DNA were used to transform DHIOBac to generate recombinant Ac-EGFP, Ac-EGFP-HPV16L1, Ac-EGFP-HPV16L1△NLS and Ac-EGFP-NLSHPV16L1 baculoviruses respectively. After Sf-9 cells were transfected with the recombinant baculoviruses respectively, the intraceilular localization of the EGFP tagged fusion proteins containing various truncated of HPV16L1 in Sf-9 cells were visualized by fluorescence microscopy and laser confocal microscopy. The green fluorescence was mainly congregated in the nuclei of Sf-9 cells transfected with the recombinant Ac-EGFP-HPV16L1 and Ac-EGFP-NLSHPV16L1 baculoviruses. The green fluorescence was resorted as a wreath in the cytoplasm of Sf-9 ceils transfected with the recombinant Ac-EGFP-HPV16L1△NKS baculoviruses at all times. The green fluorescence was spreaded through the nuclei and cytoplasm of Sf-9 cells transfected with the recombinant Ac-EGFP baculoviruses. The data suggest that the NKSHPV16L1 （NKS of HPV16L1） should has the full function. It plays an important role in the nuclear import of HPV L1 protein. EGFP could be mediated and transported into the nuclei of Sf-9 cells by the NI-SHPV16L1 also. It is feasible that the NLSHPV16L1 could be used as targeting drug carrier in Targeting drug delivery system（TDDS）.

关 键 词：人乳头瘤病毒16型 核定位信号 核浆运输 增强型绿色荧光蛋白 

分 类 号：R373[医药卫生—病原生物学]