Cannabinoid â…  Receptor Monoclonal Antibody Preparation And Identification

Since its discovery and cloning, cannabinoid type1receptor has benn shown to play an important role in a variety of physiological functions by meadiating the effects of endogenous cannabinoid system. CB1receptors (CB1R) exist mainly in the brain tissues. They are expressed in the central nervous system (CNS), neurons, interneurons, astrocytes and peripheral nerve, also participate in the regulation of Mammalian physiological activities by neuromodulator and hormone regulation. CB1R also play an important role in various physiological effects of Endogenous cannabinoid syste (ECS). Dysfunction in ECS can result in serious abnormalities. CB1R antagonists can modulate ECS activation state so as to restore the balance, to reduce the harmful effects. The use of CB1R antagonists block CB1R can reduce food intake and fat accumulation, while also can oxidize free fatty acids, reduce the adverse factors secreted by adipose cells, improve the level of adiponectin, be beneficial to promoting the metabolism of fat and carbohydrate metabolism; Moreover CB1R antagonists can reduce the risk of cardiovascular disease by reducing weight, decreasing the waistline and improving various metabolic risk factors; The CB1R expressed in the central and peripheral nervous system have specific role in easing and preventing pains of the central nervous system and peripheral tissues. It has been shown that CB1R antagonists play an very important role in pain signaling through regulation of neurotransmitter release; In terms of addiction, activating CB1R can regulate many neurotransmitter release, such as dopamine, y-amino butyric acid and glutamic acid, which are related to relapse process of drug craving. The ventral tegmental area (VTA) which secretes dopamine and its main projection area of nucleus acumens play an important role in addictive drugs’ effect of stimulation. Related experiments show that injecting morphine in rats’ VTA or injecting amphetamine in nucleus acumens can restore its demands to heroin or cocaine. There also are many CB1R in the system of GAB A intermediate neuron axonal endings, exciting these receptors can restrain the release of GABA. while GABA can exert inhibitory effects to DA projection neurons. That’s to say. exciting CB1R in the level of VTA can promote the release of DA. CB1R antagonists can’t only inhibit the addictive behavior, reduce the dependence on the addictive substance also can prevent addiction relapse. It’s not very clear that the mechanism of the effects of cannabinoids on drug demand regulator, the present research thought that the brain areas in which CB1R highly expressed have important role in recurrent behavior and conditioned reflex, which contain of prefrontal cortex, amygdala, nucleus acumens, striatum and hippocampus. What’s more, the recent studies on brain imaging show, the suggestive induced craving during drug addiction also has a similar loop. Therefore, cannabinoid receptor agonists can promote its craving to heroin or cocaine by enhancing the release of intermediate neurotransmitter DA mostly. Therefore, further studies of CB1R have obvious significance, while report about the preparation of CB1R monoclonal antibody and its use is few at home, more are polyclonal antibodies against the CB1R, so it’s very necessary to prepare monoclonal antibodies against the CB1R successfully.In the present experiment, three amino acid sequence of CB1R were selected by using a variety of software prediction. Then, three corresponding peptides were synthetized and used to carry out chain and key Kong Qilan protein (KLH) crosslink immunization, at last immunized Balb/c female mice, after the second immunization took mouse serum at the ninth day and measured separately the tail blood potency and inhibition. According to the measured results we selected mouse spleen cells which have high potency and good response inhibition as the fusion cells donor to fuse with myeloma cell SP2/0. Screening, cloning promptly after fusion, five stable cell lines producing anti-CB1R MAbs were obtained. We analyzed their isotypes, titer, and affinity and found that those MAbs belong to the IgG1and IgG2b subclasses with κ light chain, respectively. The purified ascites were used in immunohistochemistry and Western blot ting and found that the preparation of the antibody have high titer and affinity. The antibody can also be used for immunohistochemistry and Western blot.