Express And Purify Mice Olfactory Receptor 28 In E.coli And Prepare Rabbit Anti-mor28 Antibody

Each olfactory sense neuron (OSN) choose just one olfactory receptor (OR) fromthe more than 1000 possibilities encoded in the genome and transcribes it from just oneallele. This process generates great neuronal diversity and forms the basis for thedevelopment and logic of the olfactory circuit between the nose and brain.The mechanism behind this monoallelic regulation has been the subject of intensespeculation. Recent genetic experiments have brought the outline of the process intosharper relief, identifying a feedback mechanism in which the first olfactory receptorexpressed, generates a signal that stabilizes its choice,thus maintaining singularselection.There are two possible feedback ways: (1) acting as a feedback factor, OR binds toother transcriptional factor (2) acting as a feedback factor, OR binds to regulationregion of other OR genes.To study the two possible feedback ways just mentioned, we express and purifypart amino acid of mice olfactory receptor28(SMOR28) in E.coli and prepare rabbitanti-SMOR28 antibody. The anti-serum will be used to study OR feedback mechanismby immunoprecipitation and other methods.Part sequence of mice olfactory receptor28 gene (smor28) has been amplifiedfrom the mice genome by utilizing PCR. The products have been ligated into thepET28a ( + ) vector plasmid. We obtain SMOR28 expression vectors pMOR28successfully. Expression vectors pMOR28 were induced, SDS-PAGE assay detectexpressed products. We purify SMOR28 by utilizing Ni-NTA agarose. Purified proteinwas dialysed and freezed-dry to inject rabbit preparing rabbit anti-SMOR28 antibody.Western blotting assay shows that the potency of this rabbit anti-SMOR28 antibody isgood.