Construction And Preliminary Observation Of Lentiviral Vector Of Target PI Over-expression

Background: Type 1 diabetes(T1D)is an autoimmune disease by T-cell.Recent studies show that proinsulin(PI)is the primary autoantigen.As we all known,resting antigen-presenting cells(APCs)presenting antigen can induce T-cell tolerance,and APCs are derived from hematopoietic stem cells(HSCs).Besides,due to the basis of our initial success by using non-targeted lentiviral vector(LV),and the fact that major histocompatibility complex class II(MHC II)genes are expressed selectively in APCs and highly in dentritic cells,we will construct a targeted LV which mediates the PI gene expression under the control of mice MHC II,and PI gene would be targetedly expressed in the resting APCs.Subsequently,the constructed targeted vector can be transferred into non-obese diabetes mice(NOD mice)aged 4-5 weeks old(i.e.mice with prediabetes),so as to reconstruct the immune tolerance of NOD mice in the initiation stage of T1 D to achieve the purpose of preventing and treating T1 D.Therefore,in this experiment,lentivirus vectors as a carrier tool to over-express PI were constructed for the treatment of autoimmune diseases such as T1 D.Objective: To construct a lentivirus vector for expressing the targeted PI,where resting APC target the expression of PIMethods: On the basis of non-targeted lentiviral vector(that is p CDH-CMV-MCS-EF1-cop GFP)synthesis by genetic engineering the targeted lentiviral vector,and connect the PI with T4 DNA Ligase gene into the vector.The corresponding venom was obtained after transformation,extraction and packaging,and infected with hematopoietic stem cells isolated by magnetic beads from NOD female mice at 6-8 weeks.Finally,the results were compared with those of untargeted lentiviral vectors.To evaluate the infection efficiency of two lentiviral vectors.Results:1.The lentivirus vector skeleton was modified to successfully construct the targeted viral vector,and the CMV.PI LV with overexpression of PI and H2-Ea.PI LV with characteristic expression of PI were obtained by packaging 293 T cells.2.The isolated HSCs can be infected by CMV.PI LV and H2-Ea.PI LV...