Kalirin-7 Preparation Of Monoclonal Antibodies And Identification

A large number of Rho guanine nucleotide exchange factors (GEFs) and Rho GTPase activating proteins (GAPs) are used in the CNS to activate specific Rho GTPase family members, thereby inducing various signaling mechanisms that regulate neuronal shape, growth, and plasticity, in part through their effects on the actin cytoskeleton. Kalirin is a large neuronal dual Rho GEF that activates Rac1, RhoA, and RhoG via its two Rho GEF domains. This activation, which is spatially and temporally regulated, allows Kalirin to influence neurite initiation, axonal growth, and dendritic morphogenesis. In addition, this alternatively spliced gene generates developmentally regulated transcripts that yield proteins localized to the postsynaptic density (PSD). Kalirin-7, which interacts with PSD-95, is necessary for dendritic spine formation. In addition, Kalirins have the ability to regulate and influence other aspects of neuronal morphogenesis via proteinprotein interactions with their other domains, including many spectrins, other protein and lipid interaction domains, and a potential kinase. These interactions have implications not only for neuronal morphogenesis but also for vesicle trafficking, secretion, neuronal maintenance, and neurodegenerative disease.Changes in the structure and function of dendritic spines contribute to numerous physiological processes such as synaptic transmission and plasticity, as well as behavior, including learning and memory. Moreover, altered dendritic spine morphogenesis and plasticity is an endophenotype of many neurodevelopmental and neuropsychiatric disorders. Hence, the molecular mechanisms that control spine plasticity and pathology have been under intense investigation over the past few years. A series of recent studies has improved our understanding of spine dynamics by establishing Kalirin-7 as an important regulator of dendritic spine development as well as structural and functional plasticity, providing a model for the molecular control of structural plasticity and implicating Kalirin-7 in synaptic pathology in several disorders including schizophrenia and Alzheimer's disease.In this study, The synthetic peptide comprising the COOH-terminal 19 residues of Kalirin-7 and a cysteine residue was coupled to carrier protein keyhole limpet hemocyanin(KLH) with MBS (m-maleimidobenzoy-N-hydoxysuccinimide ester) as an immunal antigen. The immunal antigen was used to immunize Balb/c mouse. The serumal titer were assessed in the ninth day after the third Immunization. The highest titer is the third mouse: 1:64×104. The B cell that had been taken out from the spleen of the third mouse was fused with SP2/0 cell. By several times clones, we got two hybridomas,3C4 and 4G7, and made relevant antibody. Results of antibody detection:(1) The characterization of selected clones in terms of class and subclass, demonstrating that the antibodies belong to G1 and G2a, subclass withKlight chain. (2) The ascitic titer:3C4 1:4×104 and 4G71:2×104. (3)These were of high-affinity type antibodies with affinity constant 2.25x1011 and 3.64x109. (4) The result of immunohistochemistry detection was fine.