| NF-κB 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-κB is associated with pathogenesis and processes of various diseases, such as cancer, autoimmune disease, chronic inflammatory diseases, allergy, and arteriosclerosis, graft rejection. Therefore, many experts suggested recently that NF-(B could be a potential novel anti-inflammatory target. Theoretically, effectively antagonizing NF-κB activity is one of the effective ways to relieve and/or treat those inflammatory diseases.NF-κB could bind to DNA cis-element specifically, Based on this, DNA-binding domain of p65 subunit and Rel homology domain (RHD) of p50 were used as "prayers", via the yeast two-hybrid system from the random 16-peptides cDNA library, Our team have screened 14 polypeptides. Among these polypeptide, 5 interact with DNA-binding domain of p65 subunit and 9 with another subunit's RHD. But, some false positive maybe exist in the yeast two-hybrid technology. So, additional methods must be done to confirm the result. Additionally, through the sequencing of these polypeptides, we found that these polypeptides are hydrophobic ones. So, direct manual synthesis of these polypeptides is very difficult. To get soluble and specific NF-κB interacting polypeptide, 14 polypeptides mentioned above were further confirmed through GST Pull-down assay. It is the base to finally obtain antagonist of NF-κB. The following experiments were performed in this study.1. Construction of prokaryotic expression plasmid of GST-polypeptides fusion protein.Amplified by PCR method, the 14 cDNA of polypeptide were cloned in frame with glutathione S-transferase (GST) tag open reading frame of the pET42a (+) plasmid, respectively. Then identified by PCR and restriction endonuclease analyze. Sequencing result indicated that the sequence of the inserted gene and its open reading frame were completely correct. They were named pET42a/pp1, pET42a/pp2,…and pET42a/pp14.2. Expression of GST-polypeptide fusion proteins in Escharichia coli strain BL-21.The recombinant plasmid were transformed into E.coli BL-21 (DE3) and induced in 0.4mM isopropyl-1-thio-β-D-galactopyranoside(IPTG) at 30℃ for 4 hr.SDS-PAGE revealed GST-polypeptide fusion protein bands located in position of 31kDa, respectively.Their expression level was 8%. On this condition a large scale of GST-polypeptide fusion protein were obtained, and were purified using glutathione-Sepharose immunity affinity.3. Construction of prokaryotic expression vectors of cDNA of NF-κB p50 and p65 RHD .(1) NF-κB (p50 and p65) RHD cDNA were amplified by PCR technology with pRSV- NF-κB1 (p50), pRSV-RelA as templates. The length of the p50 and p65 RHD cDNA was 1104bp and 999bp, respectively.(2) p50 and p65 RHD cDNA was subcloned on pUCm-T plasmid, screened by bule-white spot, Identified by PCR and restriction endonuclease analysis. They were named pUCm-T/p50, pUCm-T/p65, respectively.(3) Digestion pUCm-T/p50 and pUCm-T/p65 by restriction endonuclease, then the cDNA of p50 and p65 RHD were inserted into the prokaryotic expression vectors pET-22b(+) respectively. Screened by Amp, identified by PCR method and restriction endonuclease analysis. The results of DNA sequencing show that the sequences of the inserted gene and their open reading frame were completely correct, then they were named pET-22b/p50 and pET-22 b/p65.4. Expression of p50 and p65 protein in Escharichia coli BL-21 and detection of their DNA binding bioactivity by EMSA.(1) pET-22b/p50,pET22b/p65 recombinant plasmids were transformed into E.coli BL-21 (DE3),and induced in IPTG(0.4mmol/L) at 30℃ for 4 hr. SDS-PAGE revealed tha... |
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