Objective      To construct an eukaryotic expression vector of human S100A9 full length gene pEGFP-N3-S100A9 and to observe its effect on mRNA and protein expression of S100A9 and the proliferation of glioma cell line U251.       Methods      The full length gene of S100A9 (345 bp) was obtained by RT-PCR

and was inserted into pEGFP-N3 vector by gene recombination technique. After verified by restrictive en

zyme digestion and PCR

the recombinant plasmids were transferred into U251 cells by liposome. Total RNA and protein were extracted in 24 h after transfection to detect the S100A9 expression by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting

respectively. The transfected U251 cells were continuously traced by fluorescent microscopy. The cell proliferation was analyzed by MTT assay

and the distribution of cell cycle was assessed by flow cytometry.       Results      The recombinant eukaryotic expression vector pEGFP-N3-S100A9 was successfully constructed

and the gene sequencing result suggested the S100A9 sequence was identical to that published in GenBank (NM-002965). Green fluorescence could be seen by fluorescence microscopy in 8 h after transfection and a peak appeared within 24-48 h. S100A9 mRNA was expressed and a 14×10

3

fusion protein was detected 24 h after transfection. The overexpression of S100A9 enhanced the cell proliferation significantly (P0.01)

and the number of cells decreased at G

1

phase and increased at S phase (P0.05).       Conclusion      The eukaryotic expression vector pEGFP-N3-S100A9 is constructed and expressed successfully in U251 cells

and it can effectively increase the expression of S100A9 and enhance the proliferation of U251 cells.