The Identification Of The Soluble Leptin Receptor In Crucian CARP

Leptin has a variety of physiological functions and was shown to be a pleiotropic hormone, which encoded by ob gene and secreted by adipose tissue into the blood. From the blood stream, Leptin is shuttled across the blood brain barrier coupled to a truncated soluble form of the Leptin receptor and activats certain signal transduction pathways through the binding with the special LPR lied in the hypothalamus to terminate food intake, increase metabolism. Besides its key role in the regulation of food intake and body weight, Leptin is implicated in a series of metabolic processes of the body such as fat synthesis and insulin resistance by combined with LPR loacated in peripheral tissues. Accordingly, it is important to analyze the LPR to understand the physiological role of Leptin. Tartaglia LA first cloned LPR from cDNA expression library in the mouse choroid plexus in1995. In teleosts, the LPR was first identified from marine medaka (Oryzias melastigma) by Wong et al, and then in pufferfish (Takifugu rubripes), Japanese medaka (Oryzias latipes), zebrafish (Danio rerio), Atlantic salmon (Salmo salar), grouper(Epinephelus coioides) and yellow catfish(Pelteobagrus fulvidraco). All of these studies conduct a preliminary study on genomic positioning, tissue distribution and developmental expression regulation. At present, few studies on the expression pattern of LPR in fish tissues have been carried out. We have cloned2LPR isforms from crucian carp in2011. This research will focus on another short LPR isforms in crucian carp and detect it in crucian carp blood.I first extracted total RNA from carp tissue and obtained Leptin gene by RT-PCR. The amplication was cloned into the expression vector pGEX-4T-2, which was induced at37℃with1mM IPTG. The induced expression results showed that fusion protein GST-Leptin formed insoluble inclusion bodies. To increase soluble production, I tried to change induced temperature and IPTG concentration. The final experimental results show that the soluble production of recombinant fusion proteins GST-Leptin is almost not subject to the changes of IPTG concentration, but is very sensitive to temperature variations and the expression level is also greatly reduced with decreasing temperature. Consequently, the insoluble inclusion bodies was solubilized with PBS buffer containing8M urea, and then dialyzed at4℃against a series of different concentrations urea solutions to make GST-Leptin refold. The purification of GST-Leptin through GST Resin is unsuccessful, maybe because the GST-Leptin is not refolded correctly. So I choose expression vector pET32a(+) instead of pGEX-4T-2, the results showed that fusion protein Trx-Leptin is soluble at25℃,0.1mM IPTG and can be used to detection the soluble Leptin receptor(sLR) in carp.According to the203-216and632-645amino acid sequence of extracellular region of carp long Leptin recptor, two polypeptides were synthesised and used to regularly immunize rabbits, and obtained the corresponding polyclonal antibodies. The ELISA detection of antibodies showed that one of them can be used in Western Blot. Anti-LPR203-216and Anti-LPR632-645were used to detect the expression levels of LPR in different tissuses of carp. In this assay Anti-LPR2o3-216does not work well in Westen Blot assay. The detected results by Anti-LPR632-645showed that the expression levels of LPR in liver were significantly higher than gills and muscle, and there were two isforms of LPR in liver.Leptin short recptor cDNA is generated from total RNA of crucian carp gill tissues by RT-PCR. A sLR was detected by Pull-down assay in carp serum, and its molecular weight is about115kD, which employs a different mechanism for the generation of soluble Leptin receptor by intron retention. That is, the coding sequence of LPR containing a retained intron inserted upstream of encoding transmembrane region. This insertion changed the open reading frame and perhaps resulted in a secreted isoform containing only extracellular domains that bind circulating Leptin.There is currently no report about sLR in cypriniformes. My results about sLR can provide a theoretical basis for promoting fish growth, improve the quality of fish and regulation of feeding behavior in fish.