摘要
Translation reading frame of human plasminogen activator inhibitor 1 (PAI 1) cDNA was amplified from total RNA extracted from glomerular mesangial cells by using reverse transcription polymerase chain reaction (RT PCR) and inserted into plasmid pUC19 in a sense orientation. Sequencing results revealed that PAI 1 cDNA had new initial codon ATG, but no signal peptide sequences, and translation reading frame was in agreement with PAI 1 cDNA derived from human endothelial cells. PAI 1 cDNA determined by sequencing was inserted into prokaryotic expression plasmid pBV220, and recombinant plasmid pBV220 PAI 1 which had high level expression in Escherichia coli was obtained. Recombinant PAI 1 protein attained to approximately 45% of total bacterial proteins. Western blotting showed that there was a specific band in the region of 43.0 ku. Latent recombinant PAI 1 with 97% purity was obtained from inclusion bodies after denaturation, renaturation and purification by FPLC. Recombinant PAI 1 activated by treatment with 4.0 mol/L guanidinium hydrochloride had significant inhibition activity to u PA and shared bioactivity in common with natural PAI 1.
Translation reading frame of human plasminogen activator inhibitor 1 (PAI 1) cDNA was amplified from total RNA extracted from glomerular mesangial cells by using reverse transcription polymerase chain reaction (RT PCR) and inserted into plasmid pUC19 in a sense orientation. Sequencing results revealed that PAI 1 cDNA had new initial codon ATG, but no signal peptide sequences, and translation reading frame was in agreement with PAI 1 cDNA derived from human endothelial cells. PAI 1 cDNA determined by sequencing was inserted into prokaryotic expression plasmid pBV220, and recombinant plasmid pBV220 PAI 1 which had high level expression in Escherichia coli was obtained. Recombinant PAI 1 protein attained to approximately 45% of total bacterial proteins. Western blotting showed that there was a specific band in the region of 43.0 ku. Latent recombinant PAI 1 with 97% purity was obtained from inclusion bodies after denaturation, renaturation and purification by FPLC. Recombinant PAI 1 activated by treatment with 4.0 mol/L guanidinium hydrochloride had significant inhibition activity to u PA and shared bioactivity in common with natural PAI 1.
基金
ProjectsupportedinpartbytheNationalNaturalScienceFoundationofChina (GrantNo .394 2 10 0 5 )andChineseMilitaryScienceFoundation .
