Objective: To investigate the biological effects of PTEN gene on the malignant glioma cell line SHG-44. Firstly, A recombinant eukaryotic expression plasmid containing PTEN gene fused with EGFP (enhanced green fluorescence protein) gene was constructed. Secondly, the expression of the recombinant vector in human glioma cells was detected.
Methods: (1) The human PTEN gene was amplified by RT-PCR and inserted into pEGFP-N1 that was selected by T-A subclone, and the recombinant expression vector was obtained. After the recombinant plasmids were transfected into glioma SHG-44 cells by cation polymex, expression of fusion protein was tested. (2) The stable transfected cells were selected by G418 and amplified. Light microscopy and growth curve were used to measure the effects of PTEN expression on cell morphology and proliferation. Expression of GFAP (glial fibillary acidic protein) was detected immunohistochemically.
Results: (1) The positive recombinant was sequenced and demonstrated to have the same sequence as that of PTEN gene in GenBank. It was proved that the eukaryotic expression vector pEGFP-PTEN have been constructed successfully and expressed in SHG-44 cells. (2) The SHG-44 cell growth was changed obviously. The expression of GFAP was increased.
Conclusion: The construction of PTEN eukaryotic expression vector containing green fluorescence protein gene will lay the basis for carrying out further studies on the function of PTEN gene.