Construction of lentivirus vector containing human LIM mineralization protein-1 (LMP-1) and its expression in rat bone mesenchymal stem cells / 中国骨伤

ABSTRACT

<p><b>OBJECTIVE</b>To construct a recombiant lentivirus vector of human LMP-1 and detect the expression of LMP-1 in infected rat bone mesenchymal stem cells.</p><p><b>METHODS</b>LMP-1 gene from the cDNA library were extracted by Polymerase Chain Reaction (PCR). The LMP-1 genes were connected into lentiviral vectors pGC-FU-EGFP which was linearized by Age I enzyme to produce recombiant lentivirus vector called as pGC-FU-LMP-1-EGFP,then packaged by 293T cells. The virus supernant congtaining LV-LMP-1-EGFP was harvested, concentrated and titrated. The rat BMSCs were transfected with recombiant lentivirus LV-LMP-1-EGFP at the most appropriate MOI. The mRNA and protein expression of LMP-1 were detected by RT-PCR and Western blot.</p><p><b>RESULTS</b>1LV-LMP-1I-EGFP was recombined successfully and the titer reached 2x108TU/ml. 2The efficiency of infection was 93.5% ,which was get after LV-LMP-1-EGFP infected rat BMSCs at the most appropriate MOI=100. The expression of LMP-1 gene was confirmed by RT-PCR and Western blot.</p><p><b>CONCLUSION</b>Lentivirus vector containing human LMP-1 gene is constructed successfully,which can transfected efficiently into rat BMSCs,and the infected rat BMSCs can effectively express LMP-1.</p>