The Construction Of Survivin ShRNA Lentiviral Vector

Background and Objective:Multiple Myeloma(MM) is a plasma malignancy.It's a high-incident malignant blood tumor which often occurs in the middle-age and elder. Although a variety of chemotherapy regimens and the application of hematopoietic stem cell transplantation, multiple myeloma remains a disease of poor prognosis. In recent years, with the pathogenesis of MM-depth research, the disorder of apoptosis and anti-apoptosis is one of the main pathogenesis in MM. It was comfirmed that PDCD5 protein can promote apoptosis of multiple myeloma cells, downregulate the survivin expression and enhance caspase-3 activity.Survivin is a member of the IAP family, which can inhibit the caspase-3 activity to adjust the apoptotic pathway. Whether PDCD5 enhancing the activity of caspase-3 depends mainly on the downregulation of survivin remains unclear. To study whether survivin is an important target molecule in PDCD5 enhanceing the activity of caspase-3 apoptotic pathway,we observe whether PDCD5 can increase the caspase-3 activity after downregulating the survivin expression. Since Multiple myeloma cell lines disperse structure and they are nondividing, the transfection efficiency of liposomes and retroviral vectors (MLV) is very low.Lentiviral vector can increase the transfection efficiency and achieve stable gene expression. We will construct a lentiviral vector to silence the survivin expression by RNA interference.Methods:According to the literature,we disigned the survivin shRNA effective sequence for down-regulating the expression of survivin protein. Survivin shRNA primers were synthetized by chemical methods.With using gene recombination technology, survivin shRNA was connected to pRNAT-U6.2/lenti vector plasmid.BamHl, Xhol restriction enzyme digesting and sequencing fragment prove recombinant clones to be right. Expend culturing positive cloning,and plasmid was extracted in quantity.Recombinant plasmid and helper plasmid were cotransfected by LipofectamineTM 2000 to HEK293FT cells.Transfected supernatant was collected, high-speed centrifuged, and then virus particles were collected, stored at-70℃.In infected HEK293T cell lines, GFP fluorescence expression can be observed by the fluorescence microscope.Results:The pRNAT-U6.2/Lenti double enzyme digestion testified a fragment being inserted into the lentiviral vector,and DNA sequencing confirmed the inserted fragment was correct.In the packaging cells, after cotransfection,fluorescence was observed by fluorescence microscope.With the supernatant infecting HEK 293T,fluorescence also can be observed.Conclusion:The survivin shRNA lentiviral vector was constructed successfully for providing the basis for the reseach that whether survivin is an important target molecule in PDCD5 enhanceing the activity of caspase-3 apoptotic pathway.