| NF-kB is a ubiquitous transcription factor, and 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 effectively induce the transcription and expression of more than 60 genes such as cytokines, adhesion molecules, chemotaxins, acute phase response proteins, and also regulate the expression of many enzymes, which take part in inflammatory responses. Over expression of NF-kB is associated with pathogenesis and processes of various diseases, such as cancer, autoimmune disease, chronic inflammatory diseases, allergy, and arteriosclerosis, graft rejection. Therefore, it is one of the important and prospective studies to detect and screen antagonistic drugs of NF-kB as a therapeutic target.The yeast two-hybrid system is an effectively genetic tool for detecting protein-protein interactions. The system had been used in this study to screen the polypeptides, which interact with NF-κB from the random 16-peptides cDNA library. Then the positive peptides were inserted into the eukaryotic expression vector. It is the base to study on the function of the polypeptides in the future, and finally obtain the antagonist of NF-kB. The following experiments were performed in this study. 1. Construction of yeast expression plasmid of GAL4 DNA-BD/p50 RHD fusion protein.Amplified by PCR method, then inserted into pGBKT7 DNA-BD vector, the gene of Rel homology domain(RHD) of NF-kB p50 was fused with the gene of GAL4 DNA-BD. Sequencing result indicated that the sequence of the inserted gene and its open reading frame were completely correct, then it was named pGBKT7 BD/p50.2. Identification of autonomous activation, measurement of cell toxicity and His3 leaky expression in the yeast.The recombinant plasmid was transformed into yeast AH109. The growth condition of the transformant in the selected medium SD/-Trp was observed. The results showed that colony size of transformants was the same as that of the controls. (-galactosidase activity of positive clones was tested, and the experimental results suggested that the fusion protein has neither ability of autonomous reporter gene activation nor yeast cell toxicity. The recombinant plasmid transformants could not grow in the SD/-His medium, and did not have His leaky expression.3. Screening of polypeptides interacted with NF-kB p50 RHD.(1) By means of electroperforation, the pGBKT7 BD/p50 plasmid and pGAD GH/peptides library plasmids were sequentially transformed into yeast AH109. Transformants were spreaded on the plates containing SD/-Trp/-Leu/-His medium, 108 positive candidate clones expressing His3 were obtained. These positive candidate clones were cultured in the SD/-Trp/-Leu/-His/-Ade medium, and 80 positive candidate clones express Ade, and 50 clones expressing (-galactosidase were finally obtained.(2) To wipe off the pGBKT7 BD/p50 plasmid, the positive candidate clones were cultured within SD/-Leu medium, and shaked at 250 rpm. Without selective pressure of -Trp, the pGBKT7 BD/p50 plasmid would lose during shaking. The results showed that 38 candidate clones had losed the pGBKT7 BD/p50 plasmid.(3) By means of yeast mating, plasmids of positive canditate clones were cotransformed into Y187 respectively with pGBKT7 BD/p50, pGBKT7 DNA-BD and pGBKT7 Lam. (-galactosidase activity of transformants was detected and false positive clones identified. 9 clones were found to possess capability of specially interacting with NF-kB p50 RHD.(4) Plasmids of 8 positive clones were extracted, then they were cotransformed into Y187 with pGBKT7 BD/p50 respectively. (-galactosidase activity of the transformants were detected, and the rescults indicated that 8 candidate plasmids were truely positive. It was unsuccessful to extract plasmid from clone 9, so clone 9 was taken as template to amplify polypeptide cDNA by PCR method.(5) 8 positive plasmids and purified PCR product of clone 9 were sequenced, and the results showed that the gene sequences o... |
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