Construction And Identification Of Recombinant Lentivirus Vetor PWPXLd-Raf

SignificancePancreatic cancer is one of the common and malignant tumors of digestivesystem, which is ranked fourth in western countries. In recent20years, the incidencerate of pancreatic cancer has been increased rapidly in China. Meanwhile, themortality rate is closely correlated with the incidence rate, which is relatively low in5years after diagnosis. Further studies are required to elucidate the key molecule cluesfor diagnosis and treatment in pancreatic cancer. Accordingly, to improve the survivalof patients with pancreatic cancer, there is urgent need to develop effective treatmentfor this disease. The key of improving therapeutic is to expand the understanding ofmolecular mechanism of pancreatic cancers, which will facilitate research to developnovel molecular-targeted agents and new treatment for the disease.Ras gene was first discovered in the descendants of the Sarcoma virus mouse.After that, people also found the similar gene in the human cells: H-ras、K-ras andN-ras. Abnormal Ras gene can be caused by gene mutation, gene insertion,over-express, and so on, which will lead to tumorgenesis. Mutation of K-ras is foundin more than90%of pancreatic cancer, which suggest that Ras gene is involved in thepancreatic tumorgenesis. However, the role of Ras downstream signaling pathway isstill unknown.Ras-Raf-Erk signal transduction pathway is one of the downstream signalingpathways of Ras gene which mediates signaling from receptor tyrosine kinases.Activated Ras is tightly bound to GDP(Guanosine Diphosphate),which is exchangedby GTP(Guanosine Triphosphate) upon binding of extracellular stimuli. The signal initiated by mutation activated Ras gene is involved in tumorigenesis includingRas-Raf-Erk signal transduction pathway. Raf is mitogen-activated protein kinasekinase,it can phosphorylation and activated downstream mitogen-activated proteinkinase,also excitation turosine or threonine residue.There are3kinds of subtype inRaf, B-Raf is the most strong kinase in this pathway,there are more than30pointmutation site about B-Raf in the human tumor cell,so it maybe the best potentialitytherapeutic target.ObjectIn this study, we made a lentivirus constructor which can over-express the B-Rafkinase to infect pancreatic primary acinar cells, then examine the expression level ofB-Raf downstream signal p-Erk in the acinar cells to certify the constructor work. Thenext step can inject these cells to the pancreas of nude mice in order to mimicpancreatic cancer animal model. This will elucidate the role of the Ras-Raf-Erk signaltransduction pathway in the tumorigenesis of pancreatic cancer.Method1、 To clone a lenti-virus constructor pWPXLd-Raf then transformation it tocompetent cell Stbl3.2、 Maxi purification the recombination plasmid.3、 Packaging a lenti-virus and concentrate the supernatant.4、 To prepare and culture the293T cells and primary pancreatic acinar cells.5、 Infection lenti-virus to293T cells and pancreatic acinar cells.6、 To check the level of p-Erk expression from both kinds of cells by WesternBlotting analysis.ResultEnzyme digestion and electrophoresis were used to confirm the target gene isinserted into the lenti-virus constructor successfully. Then the293T cells and acinarcells were infected by concentrated lentivirus, Western Blotting analysis wasperformed B-Raf down stream signal p-Erk were both high level expression.pWPXLd-Raf constructor work. Finally,pancreas of nude mice will be injected withthese acinar cells for the next step. Conclusion1、Construction recombinant lentivirus vetor pWPXLd-Raf and culture primarypancreatic cells are successfully.2、Lentivirus can infection293T cells and primary pancreatic cells.3、Recombinant lentivirus pWPXLd-Raf constructor can over-express B-Raf proteinkinase.