<p><b>BACKGROUND</b>
Nuclear factor kappaB (
NF-kappaB) overactivation, requiring
phosphorylation and degradation of its inhibitor
IkappaBalpha, is the basis for chronicity of
airway inflammation in
asthma. Based on our previous
plasmid pShuttle-
IkappaBalpha,
carrying an
IkappaBalpha gene from
human placenta, we optimized a novel
IkappaBalpha mutant (IkappaBalphaM)
gene, constructed and characterized its replication-deficient recombinant
adenovirus (AdIkappaBalphaM), and tested whether AdIkappaBalphaM-mediated overexpression of IkappaBalphaM could inhibit the
NF-kappaB activation in
endothelial cells.</p><p><b>
METHODS</b>IkappaBalphaM
gene (203 - 1003 bp) encoding 267
amino acids, acquired by site-directed deleting N-terminal
phosphorylation sites of
serine 32/36, was subcloned into the pShuttle and pGEM-T vectors for further
polymerase chain reaction (
PCR), restriction
digestion,
deoxyribonucleic acid (
DNA) sequencing and homology analyses. Subsequent to inserting the expression unit of pShuttle-IkappaBalphaM, containing
cytomegalovirus (CMV) promoter, IkappaBalphaM
complementary DNA (
cDNA) and polyadenylic
acid (PolyA) signals, into the type 5
adenovirus (Ad5) vector, the resultant AdIkappaBalphaM was packaged in
human embryonic
kidney (HEK) 293
cells by cotransfection with lipofectamine.
Western blot analysis and
electrophoretic mobility shift assay were utilized to detect the AdIkappaBalphaM-mediated overexpression of IkappaBalphaM in
HEK293 cells and its suppressive effect on phorbol 12-
myristate 13-
acetate (PMA)-induced
NF-kappaB activation in
human umbilical vein endothelial (ECV304)
cells, respectively.</p><p><b>RESULTS</b>The relevant
nucleotides and deduced
amino acids of 801 bp IkappaBalphaM
gene were consistent with those of
IkappaBalpha gene (
GenBank accession number M69043). The titer of the prepared AdIkappaBalphaM was 4.0 x 10 (12) plaque-forming units (pfu)/L. Moreover, the IkappaBalphaM
gene was overexpressed in
HEK293 cells, and potently inhibited the PMA-induced
NF-kappaB activation in ECV304
cells dose-dependently.</p><p><b>CONCLUSIONS</b>AdIkappaBalphaM is a novel vector for both efficient transfer and specific overexpression of IkappaBalphaM
gene, as well as potent inhibition of
NF-kappaB activity, providing a promising strategy for
gene therapy of
asthma.</p>