| NF-кB (Nuclear Factor- K B) is a transcriptional factor which exists in most cells of human tissue, especially in immune cells, and plays an important regulational role on multiple gene expression, such as some cytokine genes, growth factor s and cellular adherent factor s genes. Recently more data revealed that over activation of NF- кB was involved in the initiation and developing of acute and chronic inflammation, specially in respiratory system. However it is assumed that inhibition of NF- кB pathological activation becomes an effective and essential way to control or release above diseases. In 1999, Lebruska and his colleagues identified a 90 nucleotide (nt) molecule delivering 31mer core domain of a RNA aptamer that was capable of binding specifically to the target molecule NF- K B. Using this approach, expression of this RNA aptamer structure within host cell by a vector may block the activity of NF- K B binding with DNA response elements in gene promoter region and also inhibit the gene transcription which is regulated by NF-кB as a transcriptional factor. In 2001, Cassiday also confirmed this RNA aptamer structure selectively binds to P50/P50 homodimer form of NF- K B in eukaryotic cells using experiment of yeast three hybrid system.The purpose of this research project was to establish techniques for preparation of NF- K B RNA aptamer cDNA recombinant adenovirus and to study the biological activities of NF- кB RNA aptamer cDNA recombinant adenovirus in the inflammation cell model in vitro. The research results could provide a new strategy to cure some diseases due to over activation of NF-кB in the clinic. Up to now, we could not find out any papers, which are concerned with recombinant adenovirus as a vector to express NF-кB RNA aptamer structure in mammalian cells and studying its biological activities as well as our research project, had published.This research describes: (1) cloning of NF-к B RNA aptamer cDNA; (2) construction of recombinant adenovirus vector containing NF- кB RNA aptamer cDNA; (3) production ofrecombinant adenovirus encoding NF- к B RNA aptamer; (4) assessment of biological activities of NF- кB RNA aptamer cDNA recombinant adenovirus in the inflammatory cell model in vitro.Methods:1 .The fragment of NF- K B RNA aptamer cDNA was inserted into pGEM-7Z vector by the procedure of annealing and ligation of six synthetic oligonucleotides, the restrictive endonuclease Bgl II and Xho I cutting sites at the 5' and 3' terminal of the fragment of NF- K B RNA aptamer cDNA were adapted by PCR with a special pair of primers and a DNA templet of pGEM-7Z-aptamer cDNA and then the cDNA fragment obtained from PCR was cloned into pShuttle-CMV vector to create a new shuttle vector pShuttle-CMV-aptamer cDNA.2. Pme I digested and dephosphorylated DNA of pShuttle-CMV-aptamer cDNA was homologically recombinated with DNA of pAdeasy within BJ5183 cells and plasmid DNA of recombinant adenovirus vector, pAdeasy-aptamer cDNA, was extracted from both DNA co-transformed BJ5183 cells.3. After Pac I digestion of plasmid DNA of pAdeasy-aptamer cDNA, 30kb fragment, which included adenovirus genomic DNA fragment (except adenovirus El, E3 gene) and NF- K B RNA aptamer cDNA fragment, was purified from an agarose gel, and then transfected by Lipofectine into adenovirus packaging cell, HEK293. The recombinant adenovirus Ad-aptamer, a replication deficient-recombinant adenovirus, was obtained from the lysates of HEK.293 pellet by repeat froze-melt procedure.4. Comparing treatment of recombinant adenovirus Ad-aptamer with Ad-CMV or without adenovirus on inflammatory U937 cells stimulated by LPS, the biological activities of recombinant adenovirus Ad-aptamer was assessed as following: 1) assay luciferase activities, a report gene product, which gene was regulated by NF- K B in the inflammatory U937 cell model; 2) measure the contents of IL-1 0 and IL-6 by ELISA in the inflammatory U937 culture medium.Results and Discussions:1.The cloning sites and dir...
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