title: [Cloning and identification of NF-kappaB p50 Rel homology domain and detection of its autonomous reporter gene activity].
abstract: OBJECTIVE
  To clone NF-kappaB p50 Rel homology domain (RHD) gene and construct the "bait" vector in yeast two-hybrid system, and detect the yeast cell toxicity and autonomous reporter gene activity of target gene.
  
  
  METHODS
  Total RNA was extracted from human peripheral blood mononuclear cells and NF-kappaB p50 RHD gene was amplified by RT-PCR, and cloned into pGBKT7. The recombinant plasmid was transformed into yeast AH109. The growth condition of the transformants was observed in the selected medium SD/-Trp. The reporter gene activity of target gene in the yeast cells was verified by filter blotting.
  
  
  RESULTS
  Using restriction enzyme digestion analysis and PCR, the length of inserted gene was confirmed correct. Sequencing result indicated that the sequence of the inserted gene and its open reading frame were completely correct, and then the recombinant plasmid was named pGBKT7-p50. p50 RHD gene had neither autonomous reporter gene activity nor yeast cytotoxicity.
  
  
  CONCLUSIONS
  As a bait plasmid, pGBKT7-p50 could be used in yeast two-hybrid system to screen and capture the polypeptides which interact with p50 RHD.
authors.affiliation: Research Institute of Surgery, Daping Hospital, Third Military Medical University, Chongqing 400042, China.
authors.forename: Li-qun
authors.initials: LQ
authors.lastname: Shen
authors.forename: Hua-ping
authors.initials: HP
authors.lastname: Liang
authors.forename: Feng-lin
authors.initials: FL
authors.lastname: Lu
authors.forename: Xiang
authors.initials: X
authors.lastname: Xu
authors.forename: Sheng-mao
authors.initials: SM
authors.lastname: Li
authors.forename: Cheng-xiang
authors.initials: CX
authors.lastname: Hu
authors.forename: Lei
authors.initials: L
authors.lastname: Li
mesh_terms: D003001:Cloning, Molecular; D017930:Genes, Reporter; D020573:Genes, rel; D006801:Humans; D016328:NF-kappa B / Q000235:genetics*; D052002:NF-kappa B p50 Subunit; D016366:Open Reading Frames / Q000235:genetics; D010957:Plasmids; D011994:Recombinant Proteins / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000235:genetics*; D017422:Sequence Analysis, DNA; D014170:Transformation, Genetic; D020798:Two-Hybrid System Techniques
chemical_list: D016328:NF-kappa B; D052002:NF-kappa B p50 Subunit; D011994:Recombinant Proteins