The Cloning Of Human New Type Antiproteinase Protector Elafin Gene CDNA, Construction Of PEGFP-N1-elafin Eukaryotic Expression Vector And Expression In Eukaryotic Cell

Objective: To clone human new type antiproteinase protector elafin gene cDNA,and construct elafin eukaryotic expression vector,and observe its expression in pulmanory epithelial cell,which will be as the technical base of studying the applied possibility of elafin. Methods: Total RNA was extracted from lung tissues,and elafin cDNA was gotten by means of RT-PCR,then it was cloned into pUCm-T.We confirmed its sequence was consistent with that of elafin provided by Genebank on internet.Secondly, it was subcloned into green fluorescent protein expression vector pEGFP-N1 which transfected E.Coli DH5α,and choosed the positive clone choosed was identified by the cutting of restrict enzyme and electrophoresis.Lastly,employing Northern blotting and ELISA techniques, the transcription and secretion of human elafin in the pulmonary epithelial A549 cell line which was tranfected by pEGFP-N1-elafin were examined. Results: A fragment about 400bp was obtained through RT-PCR and proved to be human elafin gene cDNA according to its sequence,and further filtered the expression vector pEGFP-N1-elafin by the cutting of restrict enzyme and electrophoresis. There was the expression of elafin gene in the cells transfected by pEGFP-N1-elafin and it likely took the mainly expressive form of secretion. Conclusion: The expression vector of elafin gene cDNA have been constructed .There was some evidence that the expression of elafin is mainly by mean of secretion in A549 cell line which showed the potention applications of elafin to treat chronic airway inflammation.