Construction Of Truncation Mutants Of Pseudorabies Virus VP22 And Study On Their Function

Protein transduction of VP22, a unique biology phenomena with significant theoretical meanings and potential application value, was discovered in recent years. VP22 can penetrate into cell membrane without receptors or carriers, and transportes from the original expressing cells to surrounding nonexpressing cells. Proteins fused with it still maintain their biology activities. These findings not only challenge the conventional gene transfer techniques, but also open up the possibility for improvement of gene therapy and vaccination, and become one of the hotspots in scientific research.To date, it has been demonstrated that the VP22 homologues from herpes simplex virus type 1 (HSV-1), bovine herpesvirus 1 (BHV-1), Marek's disease virus serotype 1 (MDV-1) and Pseudorabies virus (PrV) have the remarkable transport property. BVP22 and PVP22 have a stonger transport property than HVP22. Moreover,PVP22 has several subcellular localizations. The discovery and the deep research of these proteins provide a new chance to better understand the relationship between its function and structure, and the exact mechanism of protein transduction, and be helpful to develop more efficient delivery tools for gene therapy and vaccination.The present study investigated in newly discovered PVP22 with stronger transport property, analysised the function domains of its subcellular localization and protein transduction via a series of terminal truncation mutants, evaluated transport property of every mutants in vivo and in vitro, studied the relationship between protein transduction and immunoenhancement, enriched the protein transduction system, established a base to better understand the property of transdunction and its underlying mechanism. The main projects are:1. 22 mutants of PVP22 were obtained by PCR, seven C-terminal tructation mutants: Pmut1(1-30aa), Pmut2(1-60aa), Pmut3(1-90aa), Pmut4(1-111aa), Pmut5(1-159aa), Pmut6(1-187aa), Pmut7(1-241aa); seven N-terminal truncation mutants: Pmut8(31-247aa), Pmut9(61-247aa), Pmut10(91-247aa), Pmut11(112-247aa), PmutB(149-247aa), Pmut12(160-247aa), Pmut16(188-247aa); eight two-terminal truncation mutants: Pmut13(61-148aa), Pmut14(188-241aa), Pmut15(160-241aa), Pmut17(149-187aa), Pmut18(149-223aa), Pmut19 (149-213aa), Pmut20(112-187aa), Pmut83(149-231aa), all the mutants were fused with enhanced green fluorescent protein (mut4EGFP), resulting in twenty two expression plasmids encoding PVP22 mutants and mut4EGFP fusion proteins. All of them were approved to be expressed by transient transfection in Hela cells.2. By transient transfection and detection of mut4EGFP fluorescence localization in live cell culture, several potential regions of PVP22 for specific subcellular localization were determined as follows: 1-30aa, 60-90aa, 112-149aa and 187-247aa of PVP22 were required for nuclear targeting; 1-30aa and 187-241aa were sufficient to combine microtubles; 111-159aa may involve in the formation of particles in nuclear, and 1-30aa can enhance this formation; there were two new subsellular localizations which were not detected in wild...