| Biological olfactory systems can recognize and discriminate thousands of distinct odors with very high sensitivity and specificity. Olfaction as the same as vision and audition,is important to the most animals to survive. Most of the organism relys on olfaction to detect food, predators and mates. Since olfacttory receptor (OR) genes were identified and cloned,and the mechanisms of the olfactory perception been elucidated, the interest in olfactory research has been increasing.Olfactory perception in mammals, for example, begins when odorous ligands activate odorant receptor proteins and the interaction of an odorant with a specific OR triggers the activation of an intracellular signaling cascade via a specific G protein, finally generate an action potential, compelted the transdction of chemical information into electrical impulses.then the electrical impulses is transdction to the olfactory bulb, after processing and modification,the impulses input to the cerebral cortex of olfactory signal processing center,decoding the olfactory information, Thus formed on odor perception, implementation of odor identification and discrimination.Olfactory receptor proteins plays an important role in the smelling process,they belong to the family of G-protein-coupled receptor and contains seven-transmembrane domain. And the olfactory receptor gene do not contain an N-terminal import sequence, therefore, the olfactory receptor proteins are not easily localized on the plasma membrane when they are expressed in heterologous cell systems. And many olfactory receptor protein are poor localization on the plasma membrane of heterologous cells, it is attributable to a combination of ER retention due to inefficient folding and poor coupling to ER export machinery, aggregation, and degradation via both proteasomal and autophagic pathways. This also is a hotspots researching in the olfactory receptor expression of olfactory receptors in heterologous cell systems.With the elucidation of their mechanisms of olfactory perception, many types of biomimetic olfactory-based biosensors have been fabricated by combining olfactory functional components with various secondary sensors. the olfactory biosensors are characterized with high sensitivity, rapid response and excellent selectivity. They have great potential commercial prospects and promising applications in such fields as biomedicine, pharmaceutical screening and the quality control of food and water. Meanwhile, olfactory biosensors also provide novel technologies for further studying olfactory transduction, such as the identification of odor-receptor pairs, the characterization of cellular physiology, consequently promote the olfactory studies.Quartz crystal microbalances (QCM) is a kind of quality sensitive sensors, detecting the resonant frequency change Δf and sensor surface quality change Δm of a linear relation, thus as a sensitive detection method is widely used in biological detection. In recent years, the sensors combined with biological specific identification material composition biological sensors, realizing the detection of bacteria, protein, small molecular.Objective:Construction of eucaryotic expression vector of nematodes olfactory receptor gene odr-10, by the method of molecular biology, and expression in the heterologous cell systems Human Embryonic Kidney(HEK-293) cells, provided the sensitive element—the olfactory receptor protein for the biomimetic olfactory-based biosensor.Methods:Inserted the sequence of his labels and rho signal peptide into the N-terminal of odr-10gene, all the gene was cloned into the expression vector pcDNA3.1and the eucaryotic expression vector of odr-10was constructed. The HEK-293cells were infected with the recombinant plasmid by liposome transfection. After48h, the transcription of ODR-10RNA was detected by RT-PCR and the protein expression was detected by western blot and cell immunoflurescence technique was to detect the protrein localization. Functional assays of ODR-10by Ca2+-imaging. Functional immobilization of olfactory receptors ODR-10to the biosensor and detecting of the applications of olfactory receptor-based QCM biosensor.Results:The eucaryotic expression vector of odr-10was constrcted successfully through the restriction analysis and sequencing. The transcription of ODR-10mRNA and protein expression was detected obviously in transfected HEK-293cells. The protein ODR-10localized on the plasma membrane identified by cell immunoflurescence technique. the results of the Ca2+-imaging demonstrate that the olfactory receptor protein, ODR-10, can specifically respond to its natural ligand, diacetyl. The responses of this QCM biosensor to various odorants can specifically responds to the natural ligand of ODR-10, diacetyl, demonstrated that the protein ODR-10can be successfully applied in the olfactory receptor-based QCM biosensors.Conclusion:Successfully expression the olfactory receptor protein of C.elegances, ODR-10in the heterologous cell system Human embryonic kidney(HEK-293) cells.the olfactory receptor ODR-10-based QCM biosensors has the function of recognition odorants,and provides an important basis for developing biomimetic olfactory-based bisosensors... |
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