Kalirin-7单克隆抗体的制备与鉴定

发布时间：2018-10-14 13:34

【摘要】：Rho鸟嘌呤核苷酸交换因子(guanine nucleotide exchange factors, GEFs)和Rho GTPase激活蛋白(GTPase activating proteins, GAPS)在中枢神经系统(Central nervous system, CNS)被用于激活特殊Rho GTPase家族成员,因而引发多种调节神经的形态、生长和可塑性的信号机制,在一些位置通过它们影响肌动蛋白细胞骨架。Kalirin是一个大的神经双Rho GEF,它通过其两个Rho GEF区激活Rac1 RhoA和RhoG。这个激活受时间和空间上的调控,并允许Kalirin影响神经突(neurite)的起始、轴突生长和树突状形态形成。这个选择性剪接基因产物进一步转录,其产生的蛋白定位在突触后致密区(postsynaptic density, PSD)。树突棘的形成需要与PSD-95相互作用的Kalirin-7。而且,Kalirin能调节和影响神经形态形成的其他方面,这通过蛋白质-蛋白质间不同的区域相互作用完成,包括很多血影蛋白(spectrin)区、其他蛋白及脂类相互作用区,还可以和一个潜在的激酶相互作用。这些相互作用提示,其不仅与神经形态发生有关,也与囊泡运输、分泌、神经维护和神经变性的疾病有关。树突棘结构和功能上的改变促进多种生理过程,例如突触传递和突触可塑性,进而表现与行为上,其中包括学习与记忆。另外,树突棘形态发生和可塑性的改变是很多神经发育紊乱和神经病紊乱的一种内在表现型。因此,在近些年中关于调节树突棘可塑性的分子机制,及相关的病理分子机制一直是一个研究热点。近来一系列的研究明确了Kalirin-7是树突棘发育过程中重要的调节因子,也是树突棘结构可塑性和功能可塑性中重要的调节因子,这些使我们对树突棘的动态变化有了进一步的理解,同时提供了一个关于树突棘结构可塑性的分子调节机制模型,该模型说明了Kalirin-7在一些机能紊乱性疾病(包括精神分裂症和阿尔茨海默症)的突触病理中的作用。我们合成了Kalirin-7 C末端的19个氨基酸的序列和一个半胱氨酸残基的由20个氨基酸组成的短肽。这条20个氨基酸的短肽是半抗原,用BMS(m-maleimidobenzoy-N-hydoxysuccinimide ester)将这条短肽与KLH(钥孔嘁蓝蛋白)交联起来,制备成抗原,免疫雌性Balb/c小鼠。第三次免疫后的第9天测血清效价,3号小鼠效价最高,为1：6.4x104。取3号小鼠脾脏中的B淋巴细胞与SP2/0细胞进行细胞融合。经过细胞克隆化后,最终获得分泌抗体的杂交瘤细胞两株,3C4和4G7,并制备出相应的抗体。抗体鉴定结果如下：(1)对筛选出的两株抗Kalirin-7的抗体的单克隆细胞的细胞培养液进行鉴定,测定3C4亚类为IgG1,4G7亚类为IgG2a,它们亚型均为Kappa;(2)用间接ELISA检测的方法测得抗Kalirin-7的抗体3C4小鼠腹水效价为1：4×104,4G7小鼠腹水效价为1：2×104；(3)用间接ELISA检测的方法测得抗Kalirin-7的单克隆抗体3C4亲和力常数为2.25x 10~(11),4G7亲和力常数为3.64×109；(4)免疫组化鉴定结果良好。
[Abstract]:Rho guanine nucleotide exchange factor (guanine nucleotide exchange factors, GEFs) and Rho GTPase activator (GTPase activating proteins, GAPS) (GTPase activating proteins, GAPS) are used to activate special Rho GTPase family members in the central nervous system (Central nervous system, CNS). The signaling mechanism of growth and plasticity through which actin cytoskeleton is affected at some point. Kalirin is a large neural double Rho GEF, that activates Rac1 RhoA and RhoG. through its two Rho GEF regions This activation is regulated by time and space and allows Kalirin to affect the initiation, axon growth and dendritic formation of (neurite). This selective splicing gene product is further transcribed and the resulting protein is located in the postsynaptic dense region of (postsynaptic density, PSD). The formation of dendritic spine requires Kalirin-7. to interact with PSD-95 Moreover, Kalirin regulates and affects other aspects of neuromorphogenesis, through protein-protein interactions in different regions, including many of the (spectrin) regions of the hemosiderin, and other protein and lipid interactions. It can also interact with a potential kinase. These interactions suggest that they are related not only to neuromorphogenesis, but also to vesicular transport, secretion, nerve maintenance and neurodegenerative diseases. Structural and functional changes in dendritic spine promote a variety of physiological processes, such as synaptic transmission and synaptic plasticity, which in turn contribute to performance and behavior, including learning and memory. In addition, the morphogenesis and plasticity of dendritic spine is an intrinsic phenotype of many neurodevelopmental disorders and neuropathic disorders. Therefore, in recent years, the molecular mechanism of regulating the plasticity of dendritic spine and related pathological molecular mechanism has been a hot topic. A series of recent studies have confirmed that Kalirin-7 is an important regulatory factor in the development of dendritic spine, and is also an important regulatory factor in structural and functional plasticity of dendritic spine, which gives us a better understanding of the dynamic changes of dendritic spine. A molecular regulatory model for structural plasticity of dendritic spine is provided, which illustrates the role of Kalirin-7 in synaptic pathology of some dysfunctional diseases, including schizophrenia and Alzheimer's disease. We synthesized 19 amino acids at the C-terminal of Kalirin-7 and a short peptide consisting of 20 amino acids with cysteine residues. The 20 amino acid short peptide was hapten. The peptide was cross-linked with KLH (keyhole chirin) by BMS (m-maleimidobenzoy-N-hydoxysuccinimide ester) to prepare the antigen and immunize female Balb/c mice. On the 9th day after the third immunization, the titer of 3 mice was the highest (1: 6.4x104). B lymphocytes from the spleen of mouse No. 3 were fused with SP2/0 cells. After cell cloning, two hybridoma cell lines, 3C4 and 4G7, which secreted antibodies, were obtained and the corresponding antibodies were prepared. The results of antibody identification were as follows: (1) the cell culture medium of two monoclonal cells against Kalirin-7 antibody was identified. 3C4 subclass was IgG1,4G7 subclass: IgG2a,. Their subtypes were Kappa; (2) the ascites titer of anti-Kalirin-7 antibody 3C4 mice was 1:4 脳 104G7 and 1:2 脳 104by indirect ELISA detection. (3) the affinity constant of 3C4 and 4G7 was 2.25x 1011 and 3.64 脳 10 ~ (9) by indirect ELISA detection. (4) Immunohistochemistry showed that the affinity constant of monoclonal antibody against Kalirin-7 was good, and the affinity constant of 4G7 was 3.64 脳 10 ~ (9). (4) Immunohistochemical analysis showed that the affinity constant of 3C4 and 4G7 was good.
【学位授予单位】：陕西师范大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392.1

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