[Cloning and identification of NF-kappaB p50 Rel homology domain and detection of its autonomous reporter gene activity].
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.
Shen
Li-qun
LQ
Research Institute of Surgery, Daping Hospital, Third Military Medical University, Chongqing 400042, China.
Liang
Hua-ping
HP
Lu
Feng-lin
FL
Xu
Xiang
X
Li
Sheng-mao
SM
Hu
Cheng-xiang
CX
Li
Lei
L
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
D016328:NF-kappa B; D052002:NF-kappa B p50 Subunit; D011994:Recombinant Proteins