Construction And Expression Of Lentiviral Vector Carrying PEX Gene

ObjectiveThe noneatalytic carboxyl-terminal hemopexin domain(PEX) of matrix metalloproteinase, as an endogenous angiogenesis inhibitor, plays a domaint role in inhibiting neoplasms invasion and hematogenous metastasis. The present study aims to construct the lentiviral vector containing MMP-2-PEX gene and observe its expression and secretion in 293FT cells , to provid effective experimental foundation for further studying the effect of PEX gene in neoplasms treatment.MethodsTotal cellular RNA was extracted from the HT1080 cells using TRIzol reagent, MMP-2-PEX gene was reverse transcribed and amplified by PCR ;Lentiviral vector containing MMP-9 signal peptide and MMP-2-PEX gene was constructed by recombinant DNA technology;Recombinant lentivirus was packaged in 293FT cells with four plasmids of lentiviral system by lipofectamine 2000;The presence of mRNA transcripts of exogenous PEX was observed by RT- PCR;the translational level expression and secretion of PEX gene in 293FT cells supernatants were detected by Western-blot analysis;Chicken chorioallantoic membrane (CAM) assay was taken to evaluate activity of PEX protein. ResultsThe recombinant lentiviral expression vector carrying PEX gene and signal peptide was constructed and verified by restricted endonuclease digestion and sequencing;The presence of mRNA transcripts of exogenous PEX was enhanced;The PEX expression and secretion could be detected in 293FT cells by Western blot;Exogenous PEX protein possessd biologic activity of neovascularization inhibition.ConclusionsLentiviral expression vector carry PEX gene was constructed successfully, system of gene transfer and expression constructed provid effective experimental foundation for further studying the effect of PEX gene in neoplasms invasion and hematogenous metastasis.