Expression of recombinant human endothelial nitric oxide synthase gene in lung tissue of mouse / 基础医学与临床

ABSTRACT

Objective To find whether heNOS could be expressed in mouse lung after intratracheal administration of recombinant adenovirus vector.Methods heNOS cDNA was obtained by RT-PCR from total RNA which extracted from human HUVEC.The replication-deficient heNOS recombinant adenovirus vector was constructed with a ligation method.High titer of recombinant adenovirus was obtained by chromatographic methods.The expression of heNOS protein was determined by immunohistochemistry staining after intratracheal administration of recombinant adenovirus.Results Sequence confirmed the cloned cDNA containing the whole ORF and the heNOS cDNA was about 3731 bp.It showed 99.93% identity with that of heNOS cDNA in GenBank(163729).The biological activity of heNOS protein was examined in transfected cos7 cell line with heNOS cDNA in eukaryotic expression vector of pcDNA3.0.The replication-deficient recombinant adenovirus vector containing heNOS cDNA was constructed successfully and the purified viral titer was 2.0?1010pfu/mL.After intratracheal administration of the recombinant adenovirus,heNOS expression was found in the majority of bronchial epithelium,alveolar lining cells,endothelial cellsand smooth muscle cells of pulmonary vessels.In control group,little endogenous eNOS immunoreactivity was detected in pulmonary vessels and no eNOS immunoreactivity was shown in bronchial and alveolar epithelial cells.Conclusion The replication-deficient recombinant human endothelial nitric oxide synthase mediated by adenovirus vector constructed could be delivered into the lung tissue of mouse by intratracheal administration of recombinant adenovirus and can be expressed in lung tissue with high-efficiency.