Construction Of MTOR ShRNA Recombinanted Lentiviral Vector And Virus Transfect T Lymphocyte

mTOR is a highly conserved protein, Molecular weight of289kDa. one of thefamily members of serine/threonine protein kinase PIKK, through the pluralityof signal paths to achieve the regulation of cell growth and proliferation. PI3K/AKT/mTOR and LKB1/AMPK/TSC/mTOR through the role of the downstreamsignaling molecule p70S6K the activity and phosphorylation to affect thetranslation of the mRNA within the cell are two more important signal of all,they controlling cell growth and proliferation[1]. Delgoffe[2] found that theknockout of of mTOR gene frap1CD4+T cells can’t be differentiated into Th1、Th2or Th17effector cells in mice, and the cells are found to adjust different intothe T help cells through select activation of mTORC1and mTORC2. Rashomologenriched in brain as the active molecules of mTORC1directly involvedin T cells differentiation regulation, and the T cells can’t be differentiated intoTh1and Th17when expression of Rheb is too low. Addition, excludedrapamycin associated protein1(frap1) gene T cells may be largely differentiatedinto the CD4+CD25+FOXP3(+) Tregs. In the study, using RNAi technologyto build mTOR shRNA lentiviral vector,as a foundation that the inhibition of Tcells mTOR gene by RNAi.Objective: To construct a mTOR shRNA lentiviral vector and transfect Tcells and provide the fundamental research of restraining T cells mTOR genes byRNAi. Methods: designing and synthetizing the shRNA segment for mTOR geneof mice,link it with enzyme cleavage carrier of pLKD.UBC.GFP.U6andtransfect into DH5α together,extraction posit-ive cloning plasmid andsequencing, which was used to infect293T cells.The titers of virus were testedaccording to the expressing levels of GFP. Extract the virus and transfectactivation of T lymphocytes cultured in vitro, and detect the rate of transfection.Results: DNA sequencing and PCR results demonstrated that the mTOR recombinanted lentiviral vector was constructed and transfect T lymphocytessuccessfully.Conclusion: The mouse mTOR recombinanted lentiviral vector wassuccessfully constructed and packaged.and the transfection efficiency isrelatively well,and provide the fundamental research of restraining T cells mTORgenes by RNAi.