Construction Of Eukaryotic Expression Vector Of HCGP-39 And Its Effects On Hepatocyte L02

HCGP-39 is a 40 kDa secreted glycoprotein belonging to the family of mammalian chitinase-like proteins, but without chitinase activity. Elevated HCGP-39 levels are found in serum of patients with diseases characterized by inflammation, fibrosis and tissue remodeling. Several studies have reported that high serum HCGP-39 levels have been associated with a poor prognosis in patients with several solid tumors, but its function in cancer is still not clarified.OBJECTIVE: To construct the eukaryotic expression vector of HCGP-39 and investigate its effects on cell cycle. METHODS: 1.Design sequence-specific primers and use PCR technique to amplify the complete cds of HCGP-39 gene.2.Insert the DNA fragments into pMD18-T vector and carry out sequence analysis.3.Recombine the fragments into eukaryotic expression vector pcDNA3.1(-).4.Transfect cells with recombination vector by lipofectin- mediated DNA transfection and evaluate the effects on cell cycle. RESULTS: DNA sequence analysis proved the complete cds of HCGP-39 gene was obtained by RT-PCR. Restriction endonucleases mapping analysis demonstrated HCGP-39 gene was inserted into pcDNA3.1(-) and eukaryotic expression vector was constructed successfully. FCM shows there is no significant change in cell cycle of hepatocyte L02. CONCLUSIONS: Construction of the eukaryotic expression vector of HCGP-39 is successful. Transfected hepatocyte L02 did not exhibit significant change in cell cycle,but there are some changes in sub-Gl phase in transfected SMMC-7721.