The Foundation Of The Diabetes-associated Drug Screening Models Using IκB Kinaseβ And Protein Kinase Cθ As Targets

IKKβ is a kind of important Ser/Thr kinase, which can be activated by some factors. And its downstream signal pathway will consequently lead to P-cell damage and insulin resistance. Accordingly the specific inhibitors of IKKβ may prevent the occurrence of type I DM and cure type II DM. At present the existent specific IKKP inhibitors such as aspirin and sodium salicylate have side effects, which limits their clinical application. Moreover further research mechanisms of insulin resistance, but also play a key role in looking for new drug targets. Besides IKKβ is also associated with chronic inflammatory disease and some types of cancers.PKCθ is another kind of Ser/Thr kinase, which belongs to PKC superfamily. The abnormally activation of PKCθ can also lead to insulin resistance and P-cell damage. So PKCθ can be applied as another drug target of diabetes. And what's more, Since PKCθ ^-/- mice are normal and fertile and their thymus gland are also well-developed. Although PKCθ is expressed in most tissues, maybe its function is abundant. Therefore PKCθ specific inhibitors won't harm normal tissues and can reduce side effect. Moreover PKCθ is a potential drug target of T-cell leukemia. E.coli expression systems haven't complicate post-translational modify function. While mammal cell expression systems have a low-efficient expression amount. So insect baculovirus systems were used. For cloning of IKKβ KD(Kinase domain) /FL(Full Length) and PKCθ, there pairs of specific primers were designed based on the published sequence of these genes. The cDNA was generously provided by two foreignors. The amplified cDNA was subsequently cloned into pFastBacl and its sequence was comfirmed by DNA sequencing analysis. The recombinant plasmid is transformed into DH10Bac^RM competent cells which contain the bacmid with a mini-attTn7 target site and the helper plasmid. The mini-Tn7 element on thepFastBac^TM donor plasmid can transpose to the mini-attfTn7 target site on the bacmid in the presence of transposition proteins provided by the helper plasmid. Colonies containing recombinant bacmids are identified by disruption of the lacZa gene. High molecular weight mini-prep DNA is prepared from selected E. coli clones containing the recombinant bacmid, and this DNA is then used to transfect sf9 insect cells.After that the recombinant virus is released in the medium,which was called as 1st generation virus. The 3rd generation virus is used to infect HighFive insect cells by the concentration of 1:20.In the end the recombinant protein was expressed. Since the protein is 6*His tagged, the 1 mL Ni2+ column is used to purified it. Then the protein is identified it by SDS-PAGE and Western blotting.Enzymology of the IKKp KD/FL and PKC9 is assayed, such as time-dependent line, concentration dependent line, PH dependent line and inhibitory effect of different concentration inhibitor, which proved the fc aidation of the diabetes-associated drug screening models using IkB KinaseP and Protein Kinase CO as targets.