Construction And Identification Of Rnai Lentiviral Expression Vector For Ang2 Gene

ObjectiveTo construct and identify the RNAi lentiviral expression vector for Ang2 gene,and would be useful for the further test the intervention of tumor cells in vitro and the inhibition of nude mouse transplantable tumor.Methods1,Two recombinant plasmids of pSilencer1.0-U6-Ang2-siRNA were constructed by targeting the mRNA of Ang2,and were identified by using enzyme digestion electrophoresis of the restriction endonuclease Hind III and sequencing analysis;2,Connect the recombinant plasmids of pSilencer1.0-U6-Ang2-siRNA and pNL-EGFP vectors which were identified by using enzyme digestion electrophoresis of the restriction endonuclease XbaI.And the Lentiviral transfer plasmids pNL-EGFP-U6-Ang2-siRNA were constructed,then identified by using enzyme digestion electrophoresis of the restriction endonuclease XbaI and sequencing analysis;3,PNL-EGFP-U6-Ang2-siRNA lentiviral transfer plasmid, pVSVG envelope plasmid and pHelper packaging plasmid were cotransfected into 293T cells, resulting in lentivirus. Collect the virus supernatant and determination the viral titer , which wree preparing for the follow-up experiments .Results1,The recombinant plasmids of pSilencer1.0-U6-Ang2-siRNA were constructed successfully, and could not be digested by Hind III. The result of recombinant plasmids sequencing conformed to that of the design;2,The lentiviral transfer plasmids of pNL-EGFP-U6-Ang2-siRNA were constructed successfully and identified by using enzyme digestion electrophoresis of XbaI and sequencing analysis. The results fully in line with expectations;3,EGFP-Ang2-siRNA virus were produced by using the three plasmid lentiviral packaging system. The virus supernatant was collected and viral titers measured for the 9*103/ul.ConclusionsThe RNAi lentiviral expression vectors for Ang2 gene were constructed successfully,and would be useful for the further research of angiogenesis.