[Construction of recombinant deltaN IkappaBalpha adenovirus and its suppression of NF-kappaB activity in A549 cells].
OBJECTIVE
To explore the regulatory effect of deltaN IkappaBalpha gene on the activity of nuclear factor-kappaB(NF-kappaB).


METHODS
Ser32-and Ser36-deleted IkappaBalpha gene (deltaN IkappaBalpha) was cloned into adenovirus vector, and a replication-defective recombinant deltaN IkappaBalpha adenovirus(Ad-deltaN IkappaBalpha) was generated. A549 cells were divided into three groups: LPS-stimulated groups, Ad-LacZ+LPS group and Ad-deltaN IkappaBalpha+LPS group. Ad-LacZ+LPS group and Ad-deltaN IkappaBalpha+LPS group were infected with Ad-LacZ and Ad-deltaN IkappaBalpha, respectively, two days before LPS stimulation. The NF-kappaB activity of A549 cells was detected by Western blot and electrophoretic mobility shift assay (EMSA). TNF-alpha and IL-6 in the culture supernatant were detecteded by ELISA.


RESULTS
The activity of NF-kappaB and the levels of TNF-alpha and IL-6 from Ad-deltaN IkappaBalpha virus-infected A549 cells were significantly decreased as compared with that of LPS-stimulated group and Ad-LacZ+LPS group.


CONCLUSIONS
The results indicated that deltaN IkappaBalpha may inhibit the activation of NF-kappaB and reduce the release of TNF-alpha and IL-6, suggesting the recombinant deltaN IkappaBalpha adenovirus may be used for anti-inflammatory therapy.
Zhu
Zi-lu
ZL
Department of Biochemistry and Molecular Biology, Nanjing Medical University, Nanjing 210029, China. zhuzilu_2002@yahoo.com.cn
Cheng
Zhi-xiang
ZX
Xiong
Jun
J
Chen
Bing-ying
BY
D000256:Adenoviridae / Q000235:genetics*; D000818:Animals; D045744:Cell Line, Tumor; D004274:DNA, Recombinant / Q000235:genetics*; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D020672:I-kappa B Proteins / Q000235:genetics* / Q000378:metabolism*; D015850:Interleukin-6 / Q000378:metabolism; D008070:Lipopolysaccharides / Q000494:pharmacology; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000037:antagonists & inhibitors* / Q000378:metabolism; D017384:Sequence Deletion; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
D004274:DNA, Recombinant; D020672:I-kappa B Proteins; D015850:Interleukin-6; D008070:Lipopolysaccharides; D016328:NF-kappa B; C000606204:NFKBIA protein, human; D014409:Tumor Necrosis Factor-alpha; D000072000:NF-KappaB Inhibitor alpha