Construction And Idenithcation Of The PTEM Expression Plasmid GGF-PTEM

Objective To extract the wild-type PTEN gene from lympholeukocyte, cloned into theeukaryotic expression vector pEGFPN1transfected into osteosarcoma MG63cell lineand expression of PTEN protein. This eukaryotic expression vector, can provideexperimental basis to further explore the value of the PTEN gene in the occurrence andmetastasis of osteosarcoma.Methods①pEGFPN1/PTEN Construction and identification of eukaryoticexpression vector: the total RNA of the human lymphocyte was extracted underthecondition without RNA enzyme conta mination. The complete encoded sequence ofPTEN gene was amplified by reverse transcription-polymerase chain reactionfromhuman lymphocyte Amplified the PTEN gene fragment by reverse transcriptionpolymerase chain reaction (RT-PCR), the recovery of purified PCR products after theend of the electrophoresis analysis,The amplified fragment was inserted into eukaryoticexpressionvector pEGFPN1.PCR screening and restriction enzyme digestion andsequencing to identify the recombinant vector,sequenced to analyze the homology withthe GeneBank database using the BLAST software.②MG63cells transfected with therecombinant human PTEN protein expression and detection: Take purificationsterilization recombinant plasmid pEGFPN1/PTEN, transfected into MG63cells with the transfection reagent Lipofecta mineTM2000. Transfection efficiency was observedby an inverted fluorescence microscope to detect the expression of GFP greenfluorescent protein.48h after transfection, cells were collected by RT-PCR and Westernblot analysis of PTEN mRNA and protein expression in MG63cells.Results①reverse transcription polymerase chain reaction (RT-PCR), successfullyamplified a1209bp open reading frame of PTEN cDNA fragment. Successfully clonedinto the eukaryotic expression vector pEGFPN1; will build of carrier pEGFPN1/PTENdo of EcoRI, BamH I restriction enzyme digestion and PCR, respectively, to obtain asize with its corresponding PCR product of the inserted fragments; by sequencing andBlast analysis The recombinant plasmids were successfully constructed.②The liposometransfected into MG63cells24hours after the visible expression of green fluorescentprotein, transfected cells were collected after48hours, RT-PCR and Western blotconfirm recombinant PTEN eukaryotic expression vector to express PTEN mRNA andMG63cells in the PTEN size consistent protein electrophoresis strip, Western resultsshow54kD seen at positive article.Conclusion The eukaryotic expression vector of PTEN gene has been successfullyconstructed,which may provide a basis for further researches.