Construction And Identification Of Angiopoietin Mouse -1 Lentivirus Expression Vector

Objective:To construct a lentiviral vector carrying angiopoietin-1gene and identify it, so can provide experimental basis for further research in gene therapy of BPD by Ang-1.Methods:Ang-1gene carrying attB recombinant sites was amplified by PCR from pcDNA-Ang-1Plasmid. Using the Gateway technology, entry clone pDown-Ang-1was constructed by BP reaction. The entry clone was identified by PCR and gene sequencing.Expression vector PLV.Ex3d.P/puro-CMV>Ang-1>IRES/DsRed-Express2carring DsRed was obtained from the combination of pDown-Ang-1、pUp-CMV、 pDown-Ang-1、pTail-IRES/DsRed-Express2and PLV.Des3d.P/puro by LP reaction. The expression vector was identified by PCR and gene sequencing. PLV.Ex3d.P/puro-CMV>Ang-1> IRES/DsRed-Express2was transfect into the package cell293FT with helper plasmid PLV/helper-SL3、PLV/helper-SL4and PLV/helper-SL5together by lipofectin to produce mature lentivirus. Determine the virus titer and infection efficiency by the expression of fluorescence.Results:According to the proof of PCR and gene sequencing, entry clone pDown-Ang-1and expression vector PLV. Ex3d.P/puro-CMV>Ang-1>IRES/DsRed-Express2was constructed successfully by BP reaction and LR reaction respectively. There were10621bps in the sequence of Ang-1lentiviral vector PLV.Ex3d.P/puro-CMV>Ang-1>IRES/DsRed-Express2, CMV promoter sequences were from1950bp to2538bp, sequences of Ang-1open reading frame were from2569bp to4065bp, sequences of IRES were from4080bp to4677bp and sequences of DsRed-Express2were from4678bp to5355bpo After PLV.Ex3d.P/puro-CMV> Ang-1>IRES/DsRed-Express2was transfect into the package cell293FT with helper plasmid PLV/helper-SL3、PLV/helper-SL4and PLV/helper-SL5together by lipofectin, high degree infectious lentiviral was produced by the package cells and the virus titer was5× 108TU/ml.Conclusion:Using the Gateway technology, we successfully construct Ang-1lentiviral vector PLV.Ex3d.P/puro-CMV>Ang-1>IRES/DsRed-Express2, and package effieient lentivirus particles, so provide experimental basis for further research in gene therapy of BPD by Ang-1.