Founding A Method That Can Rapidly Detect NF-κB Activity

Nuclear factor-KB(NF-kB)is a transcription factor that regulates many genes, and participates in cellular proliferation, apoptosis, invasion, differentiation and so on. Plenty of researches show that it plays an important role in tumor development, progression and therapy, so researchers believe that NF-kB is a new target for tumor therapy.To obtain human NF-kB P50 cDNA, total RNA was extracted from cell U251 and the segment of NF-kB P50(1308bp,coding 436aa) was amplified from total RNA by RT-PCR technique. The amplified cDNA was cloned into the prokaryotic expression vector pGEX-4T-l and sequenced. The recombinant plasmid pGEX-4T-P50 was then transformed into E.coli BL21(DE3). The fusion protein GST-P50 was obtained by inducing with 0.2mM IPTG at 30℃ for 6 hr. It was approximate 70 kD and accounted about 30% of total bacterial proteins. GST-P50 was purified by GST affinity column and was injected into rabbit to produce polyclonal antibody. ELISA analysis showed that the titer for antibody is 5x105, Western blot analysis showed that the antibody had good specificity for P50 protein.We founded a new method can rapidly detect cellular NF-kB activity. In our experiments, when covered with 100μl GST-P50 (lug/ml), dilutability of antibody is 1/1600, antibody reacted with nucleoprotein for 1 hr at 37℃, sensitivity of detecting NF-kB activity was best and can reach ng level.The new method was examined by LPS-detect-model. U251 cellswere stimulated with 100ng/ml LPS for 0.5,l,2,4,6hr, or were stimulated with 10,20,40,60,100 ng/ml LPS for 1 hr in DMEM culture medium, then detected cellular NF-kB activity by our method. Our results about influence of cellular NF-kB activity by LPS is the same with reported. So completely approved that our method can rapidly, exactly and availably detect NF-kB activity. This study provided a basis for further clarify the function of NF-kB and screen drugs inhibiting NF-kB activity.