Found A New Ikk¦Â Binding Protein

With the complete human genome sequence now in hand, we face the enormous challenge of interpreting it and learning how to use these information to understand the functions of sequences, especially genes which we did not know exactly. The methods of studying the function of new genes include bio-chip, mass spectrum, yeast two-hybrid, gene knock-out, RNAi and et al. Each of them has its own virtues and shortcomings. In this article, we used yeast two-hybrid, gene transfection, reporter gene and flow cytometry technologies to perform our research.In the early studies, we analysed 476 genes' sequences, and picked out 13 genes in the end. And we inserted the 13 genes respectively into yeast expression vectors and mammalian expression vectors.Then we used pACT2 cDNA library which have 1500 defined important genes involving oncogene, cell cycle gene, apoptosis gene, disease relative gene, etc. to screen the protein potentially interacting with the 13 genes we selected before by means of cotransformation in yeast strain Y190. As a result, we found 2 couples interaction proteins. In order to verify the proteins interaction, we performed several experiments in vitro and in vivo, including Pull-down, coimmunoprecipitation and co-location in cells. In the end, we confirmed that 11#( Hs.l88881)protein could interact with IKKβ (IkB Kinase β ) that was screened in previous work.In the experiments of detecting the activity of luciferase in NF-kB report vector, we found 11#( Hs.188881)gene can decrease the activity of NF-kB and the inactivation effect of the 11C-terminal on NF-kB is stronger than the 11N-terminal's.We also expressed the recombinant protein GST-11C in prokaryocyte, and got antiserum by immunizing rabbit with GST-11C. The purified antiserum can combine with the full-length of 11#( Hs. 188881), this has been proved by western-blot. The purified antiserum also can be used in the further research, such as the immunohistochemistry, and so on.In conclusion, follow the new strategies for study new genes' functions, we...