Construction and identification of recombinant adenovirus vector carrying a N-terminal phosphorylation sites-deleted human I?B? mutant gene / 中国病理生理杂志

ABSTRACT

AIM:To optimize the I?B? mutant(I?B?M)gene derived from human placenta tissue by deleting N-terminal phosphorylation sites of serine 32/36,and to construct and identify its replication-deficient recombinant adenovirus(AdI?B?M).METHODS:The I?B?M gene(203-1 003 bp)was acquired by positional cloning,followed by subcloning it into pShuttle and pGEM-T vectors for further PCR,double digestion,DNA sequencing and homology analysis.Subsequently,the expression unit of pShuttle-I?B?M containing CMV promoter,I?B?M cDNA and poly A signals was inserted into Ad5 vector,after which the resultant recombinant adenovirus AdI?B?M was packaged in 293 cells by cotransfection with lipofectamine.Western blotting analysis and electrophoretic mobility shift assay were utilized to detect the AdI?B?M-mediated expression of I?B?M gene in 293 cells and its suppressive effect on phorbol myristate acetate(PMA)-induced nuclear factor ?B(NF-?B)activation in ECV304 cells,respectively.RESULTS:The relevant nucleotide and amino acid sequence of I?B?M gene was consistent with that of GenBank(accession number M69043).The titer of the prepared AdI?B?M was 4.0?10 12 pfu/L.Moreover,the I?B?M gene was expressed in 293 cells,and potently inhibited the PMA-induced NF-?B activation in ECV304 cells in a dose-dependent manner.CONCLUSION:AdI?B?M is a nonvel vector for both efficient transfer and expression of I?B?M gene as well as specific inhibition of NF-?B activity,providing a promising future for gene therapy of asthma.