HPV16L1核浆运输的动力学过程

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。

The dynamic process of of HPV16L1 nucleocytoplasmic transport

In order to analyse the role of nucleus location signal of Human papillomavirus 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 delta NLS and NLSHPV16L 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 delta NLS and pFB-EGFP-NLSHPV16L1 transfer vectors. Through Tn7 transposon-mediated 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 DH10Bac to generate recombinant Ac-EGFP, Ac-EGFP-HPV16L1, Ac-EGFP-HPV16L1 delta NLS and Ac-EGFP-NLSHPV16L1 baculoviruses respectively. After Sf-9 cells were transfected with the recombinant baculoviruses respectively, the intracellular 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-NLSHPV16L baculoviruses. The green fluorescence was resorted as a wreath in the cytoplasm of Sf-9 cells transfected with the recombinant Ac-EGFP-HPV16L1 delta NLS 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 NLSHPV16L1 (NLS 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 NLSHPVI6L1 also. It is feasible that the NLSHPVI6LI could be used as targeting drug carrier in Targeting drug delivery system(TDDS).