Abstract
Objective To construct lentivirus vector expressing human hepatocyte growth factor (HGF) gene containing enhanced green fluorescent protein gene (EGFP).Methods Human HGF gene coding sequences were amplified from pUC-SRα/HGF with flanking attB sites using PCR and cloned into the multiple sites of pDONRTM221 using BP reaction for constructing the entry plasmid pDown-HGF.Then pDown-HGF,pUp-EF1α and pTail-IRES/EGFP were recombined into the target vector pLV.Des3d.P/neo by LR reaction to generate expression vector pLVneo/EF1α-HGF-IRES-EGFP.The expression vector was confirmed by PCR and DNA sequencing.293Fr cells were cotransfected with the recombinant vector and lentiviral packaging system through lipofectamine.The titer of virus was measured by plague assay.Results Both PCR and DNA sequencing analysis revealed that HGF gene was successfully inserted into the target site of pLVneo/EFlα-HGF-IRES-EGFP and the insertion sequence was correct.The green fluorescence was observed in the 293FT cells under fluorescence microscope after transfection and the titer of concentrated virus was 7.9 × 107 TU/ml.Conclusion Lentivirus vector expressing human HGF gene containing EGFP is constructed successfully. Key words: Hepatocyte growth factor; Transfection; Green fluorescent proteins; Lentivirus
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