Construction of recombinant adenovirus vector Ad-elafin for nature antibiotic elafin gene and its expression in primary airway epithelium
- VernacularTitle:天然抗菌多肽elafin重组腺病毒载体构建及气道上皮细胞表达
- Author:
Xianzhi DU
;
Xiangdong ZHOU
- Publication Type:Journal Article
- Keywords:
elafin;
adenovirus;
airway epithelium
- From:Journal of Third Military Medical University
2003;0(11):-
- CountryChina
- Language:Chinese
-
Abstract:
Objective To construct the recombinant adenovirus for nature antibiotic elafin protein and observe its expression in primary airway epithelia.Methods The elafin gene was amplified by RT-PCR from lung tissues,then the fragment was inserted into the multiple clone site of pAdTrack-CMV.The linearized shuttle plasmid was homogenously recombined with AdEasy-1 in BJ5183.The candidate clone was further analyzed by restriction endonuclease digestion,PCR,and sequencing.Then the recombinant adenovirus plasmid was digested with PacⅠ and transfected into 293 cells for packaging and amplification.Infection titer and rate were monitored by green fluorescent protein(GFP)expression.Finally the primary airway epithelium was infected with Ad-elafin,and the GFP expression was observed by fluorescence microscope in epithelial cells 24 h after transfection.The elafin protein was detected by ELISA in the supernatant.Results Restriction endonuclease and PCR confirmed that elafin gene was cloned into the adenovirus vector successfully.Twenty-four hours after transfection,the GFP expression was seen by fluorescence microscopy.The concentration of elafin protein was(2.2?0.4)ng/ml in supernatant of transfected group,while that of control group was(1.9?0.3)ng/ml,P
