Construction Of Eukaryotic Expression Plasmid PEGFP-N₁-IL10-HGF And Its Expression In T6 Cells

Objective To construct eukaryotic express plasmid pEGFP-N1-IL10-HGF containing human IL10-HGF(LH) recombinant fusion gene and enhanced green fluorescence protein gene ,and to confirm its expression in T6 rat hepatic stellate cells.Methods IL10 cDNA was amplified by RT-PCR from human mononuclear cells, HGF cDNA was amplified by PCR from pUC-SRα/HGF vector, purified fragments were cloned into pGEM-T-Easy vector,recombinant clones were selected by ampicillin and blue-white spot, and then indentfied by digestion analysis and DNA sequencing. LH cDNA was amplified by recombinant PCR from pGEM-T-Easy-IL10 and pGEM-T-Easy-HGF vector, Purified fragments was cloned into pGEM-T-Easy vector,recombinant clones were selected by ampicillin and blue-white spot, and then indentfied by digestion analysis and DNA sequencing. Then the recombinant IL10-HGF ORF was cloned into pEGFP-N1 vector by double-digest direct cloning ,recombinant clones were selected by kanamycin, and then indentfied by digestion analysis and DNA sequencing. The recombinant vector was introduced into T6 cells by lipofectin transfection,The cells were selected by G418,The expression of enhanced green fluorescent protein (EGFP) was observed under fluorescence microscope, the mRNA expression level were indentified by RT-PCR, and the expression level of protein were indentified by western-blot.Results The IL10/HGF/LH gene fragments successfully inserted into the pEGM-T-Easy were confirmed by digestion and DNA senquencing ;the LH gene fragment inserted into pEGFP-N1 vector correctly was indentified confirmed by digestion and DNA senquencing;the expression of enhanced green fluoscence protein was observed in T6 cells transfected with pEGFP-N1-LH and pEGFP-N1 under fluorescence microscope. EGFP protein was localized in whole cell, while LH is expressed mainly in the cell Plasm. The results of RT-PCR and western-blot congfirmed that the recombinant vectors expressed LH mRNA and Protein in T6 cells stably.Conclusion IL10/HGF/LH gene were cloned successfully,The eukaryotic express vector pEGFP-N1-LH containing human LH gene was successfully constructed and expressed in T6 cells stably.