[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).
Yang
Jun
J
Department of Pathology, Second Hospital of Medical College, Xi'an Jiaotong University, Xi'an 710004, China. yangjundr@yahoo.com.cn
Wang
Yi-li
YL
Si
Lv-sheng
LS
D021581:Active Transport, Cell Nucleus; D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D036022:Capsid Proteins / Q000378:metabolism*; D002467:Cell Nucleus / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000235:genetics; D008969:Molecular Sequence Data; D019913:Nuclear Localization Signals; D009856:Oncogene Proteins, Viral / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D018411:Spodoptera / Q000235:genetics
D036022:Capsid Proteins; D019913:Nuclear Localization Signals; D009856:Oncogene Proteins, Viral; D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; C063317:L1 protein, Human papillomavirus type 16