Study On The Interference Of Tk-RNAi Bacteria To The Stable Expression Of HIV Key Proteins In SW480 Cells

RNAi technology has great application potential in the research of gene function and the treatment of diseases,and it is a hot topic to apply RNAi technology to anti-HIV-1 therapy.With further research,RNAi technology in the anti-HIV virus experimental research has made some gratifying achievements.However,there are still a lot of problems.One of the main problems is how to ensure the efficiency of RNAi interference fragments into cells.For most of the lower organisms,the success rate of ds RNA or si RNA is more higher,but the success rate is not high for higher organisms such as mammal and human cell lines.And at present,most of the research on the RNAi of HIV virus gene is carried out in cultured cells rather than the cells in vivo,so the clinical application of RNAi in the treatment of AIDS is still facing with many challenges.Tk-RNAi bacteria can help RNAi to be applied to the clinical treatment of AIDS.Transkingdom RNA interference is a novel approach for si RNA delivery,which uses non-bacteria to produce and deliver therapeutic short hairpin RNA(sh RNA)into target cells to induce RNAi.this paper is to apply transkingdom RNA interference to disturb the expression of HIV gene,and to provide a theoretical basis for the development of a new type of drug resistant to HIV infection which use bacteria as RNA interference vector.We constructed two fusion expression plasmids,p EB-nef-myc and p EGFP-C1-gag,and then transfected them into SW480 cell with liposome.The expression of nef protein and gag protein was detected by Western blot.SW480 cell lines stably expressing nef and gag were successfully established after G418 screening.Then we constructed two tk-RNAi plasmids,TRIP-sh RNA-nef and TRIP-sh RNA-gag,which were respectively transformed into BL21(DE3)by electroporation,and obtained the tk RNAi-nef bacteria and the tk RNAi-gag bacteria.After acridine orange staining,we observed that tk RNAi-nef bacteria and tk RNAi-gag bacteria still could intrude into the SW480 cells by confocal microscope.Finally,Western blot results showed that the Tk-RNAi bacteria could downregulate the expression of HIV-1 nef and gag in SW480 cell lines,effectively and specificly.