Expression of VEGF165 and VEGF121 genes in MC3T3-E1 cell line and biological function of their products / 浙江大学学报·医学版

ABSTRACT

<p><b>OBJECTIVE</b>To construct vectors stably expressing bioactive VEGF165 and VEGF121 in MC3T3-E1 cells.</p><p><b>METHODS</b>VEGF165 and VEGF121 genes were obtained by RT-PCR from HL-60 cells and inserted into eukaryotic expression plasmid pcDNA3.1(+). The constructed plasmids containing right sequence of target genes were transfected into MC3T3-E1 cells by lipofectin. The cells were limitedly diluted twice and selected by G418, the expression of VEGF was tested by ELISA, Western-blot and immunofluorescence essay. The bioactivity of expressed products was proved by MTT method.</p><p><b>RESULT</b>DNA sequencing indicated the sequences of the obtained VEGF165 and VEGF121 were identical to the those reported in Genebank, and the newly-constructed plasmids were VEGF165/pcDNA3.1(+) and VEGF121/pcDNA3.1(+). The results of ELISA, Western-blot and immunofluorescence showed that the transfected cells could express VEGF165 and VEGF121 proteins. And MTT proved the proteins were bioactive.</p><p><b>CONCLUSION</b>The transfected MC3T3-E1 cells could stably express high levels of bioactive VEGF165 and VEGF121.</p>