| NF-KB is a ubiquitous transcription factor, and one of cross-talk points of multiple signal transduction pathway. It plays a key role in basic processes such as regulation of the immune and inflammatory responses, virus replication, cell proliferation and apoptosis. It can regulate transcription and expression of genes such as cytokines, adhesion molecules, chemotaxins, acute phase response proteins and enzymes, which take part in inflammatory responses. Over expression of NF-KB is associated with pathogenesis and processes of various acute and chronic inflammatory diseases. Therefore, many experts suggested recently that NF-KB can be a potential novel anti-inflammatory target. Theoretically, effectively antagonizing NF-KB activity is one of effective ways to relieve and/or treat those inflammatory diseases.To obtain antagonist of NF-KB, the following experiments were performed in this paper.1. Construction of pGBKT7 DNA-BD/p65BD(DNA-BD/p65 binding domain) and identification of its expression in the yeast.(1) By means of primer dimer bridging, the fragment of DNA binding domain of NF-KB p65 subunit was amplified, then inserted into pGBKT7 DNA-BD vector, and pGBKT7 DNA-BD/p65BD was constructed. Sequencing result indicated that the fragment sequence of DNA binding domain of NF-KB p65 subunit and its open reading frame were completely correct.(2) The recombinant plasmid was transformed into yeast AH 109, and the expression of GAL4 DNA-BD/p65BD fusion protein in the yeast cells wasidentified by means of SDS-PAGE and Western-blot. The results indicated that expression of fusion protein showed on the lane of 26kD could be found in the yeast AH109. Gel scans analysis showed that the expressed fusion protein accounted for 5 percent of the total yeast proteins.2. Amplification of GAL4 AD/peptide library, identication of autonomous activation, measurement of cell toxicity and His3 leaky expression in the yeast.(1) Peptide library plasmids were amplified according to the operating manual, extracted by means of alkaline lysis, purificated by means of polyethylene glycol precipitation, and then a lot of pure library plasmids were obtained. Ten clones were selected at random and an automated sequencer analyzed each sequence of their inserted peptides. Sequencing results showed that there was no bias towards one particular random sequence (each of 10 clones contained a different sequence).(2) The recombinant plasmid was transformed into yeast AH 109. The growth condition of the transformant in the selected medium SD/Trp was observed. The results showed that colony size of transformant was the same as that of the controls, p-galactosidase activity of positive clones was tested by using both liquid assay and plate assay. The experimental results suggested that DNA binding domain of NF-KB p65 subunit have neither ability of autonomous reporter gene activation nor yeast cell toxicity. The recombinant plasmid transformant could not grow in the SD/-His medium, and did not have His leaky expression.3. Screening of polypeptides interacted with DNA binding domain of NF-KB p65 subunit.(1) By means of electroperforation, the pGBKT7 DNA-BD/p65BD plasmids and the peptide library plasmids were sequentially transformed into yeast AH 109. Transformants were spreaded on the plates containingSD/-Trp/-Leu/-His medium, 64 positive candidate clones expressing His3 were obtained. These positive candidate clones were cultured in the SD/"Ade/Trp/"Leu/"His medium, and 12 false positive clones were eliminated. -galactosidase activity of remaining positive candidate clones was detected, and 32 clones expressing p-galactosidase were finally obtained.(2) By means of yeast mating, plasmids of positive candidate clones were cotransformed into Y187 respectively with pGBKT7 DNA-BD, pGBKT7 DNA-BD/p65BD and pGBKT7 Lam. p-galactosidase activity of transformants was detected and false positive clones identified. 5 clones were found to possess capability of specially interacting with DN... |
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