Protective Effects Of Human Elafin Gene Recombinant Plasmid Transfection To Airway Epithelium Integrity

OBJECTIVE: To construct recombinant plasmid of human nature new antiproteinase protector elastase-specific inhibitor(elafin) gene and explore the molecule mechanism of protective effects of human elafin gene recombinant plasmid transfection and expression to airway epithelium integrity,these provided theory and experiment background for employing recombinant elafin in clinical therapy of chronic airway inflammation.METHODS: Firstly,elafin PCR product was gotten by means of PCR,then it was cloned into green fluorescent protein(GFP) expression vector pEGFP-C1 which transfected E.Coli DH5α,and the positive clone choosed was identified by the cutting of restrict enzyme and electrophoresis and its sequence was confirmed to be consistent with that of elafin provided by genebank on internet.Secondly, transfecting it into A549 cells ,then co-incubation with polymorphonuclear granulocyte(PMN) and stimulating these cells with lipopolysaccharide(LPS).After 24hour,western blot,MTT technique, substrate detect technique and enzyme-linked immunoadsordent assay(ELISA) were performed respectively to detect expression of zonula occludens-1(ZO-1) in cells,adhesive capability of cells with collagen,the vital force unit of neutrophil elastase(NE) and elafin level in colture supernatant.RESULTS: A fragment 459bp was obtained through PCR,further filtered the expressive vector pEGFP-C1-elafin by the use of restrict cutting enzyme and...