Prokaryotic Expression Of Nono, A Novel DNA Bingding Protein Of The Repressor Element In The Cyclooxygenase-2 Promoter, And Preparation Of Its Polyclonal Antibody

Cyclooxygenase-2 (COX-2) is a key enzyme that catalyzes thesynthesis of prostaglandins. It is not constitutively expressed but can begreatly induced by cytokines, growth factors and mitogens, et al. It hasbeen demonstrated that the abnormal overexpression of COX-2 plays animportant role in inflammation, carcinogenesis, cardiovascular diseasesand the development of diabetes.In the prior studies, we report for the first time a novel repressorelement located between nucleotides -655/-632 of the mouse COX-2promoter. One of the nuclear proteins binding to this element wasidentified as a non-POU-domain-containing, octamer-binding protein(Nono). Nono exhibits multi-functional characteristics in a variety ofnuclear processes, such as RNA processing, transcriptional regulationand DNA unwinding. To confirm the binding of Nono to the repressorelement, the polyclonal antibody against Nono was prepared for theantibody-based supershift assay.The results suggested that the recombinant prokaryotic expressionvector pET-28a(+)-Nono was successfully constructed and the E. coli- based product of His-Nono fusion protein was prepared to immuneBALB/C mouse. High titer of specific polyclonal antibody against Nono,which was detected by ELISA and Western blot, was eventually prepared.The obtained antibodies could be used in supershift assay and furtherfunctional studies.