Fpr3的原核表达及其单克隆抗体的制备

发布时间：2018-04-11 04:25

本文选题：单克隆抗体 + Fpr3　；参考：《华中师范大学》2011年硕士论文

【摘要】：FKBP是生物中广泛存在的一个蛋白质家族。目前在酵母中已经发现了FKBP家族的四个成员：Fpr1, Fpr2, Fpr3, Fpr4。Fpr3是核仁蛋白,可以参与核糖体的装配,指导核糖体或者相关蛋白质穿越核孔复合体。Fpr3还对细胞周期具有调控作用,当细胞周期出现异常情况时,Fpr3就会发挥作用,使细胞周期停滞在G2期。正是由于Fpr3重要的生理功能,我们希望了解其空间结构,但是Fpr3很难结晶。单克隆抗体是一种专门针对一种抗原决定簇的抗体。当单独的蛋白质不能结晶时,往往能与其抗体共同结晶。抗体与相应的蛋白质结合后,可以稳定蛋白质的构象,有利于提高蛋白质结晶的成功率。此时,抗体的作用就相当于分子伴侣。分子伴侣可以与不稳定的蛋白结构结合,通过控制结合和释放来帮助其折叠,组装,转运和降解,而不构成蛋白质结构执行功能时的组份。本实验通过将pEGX-5X-1-fpr3转入大肠杆菌E.coliBL21中,IPTG诱导表达GST-Fpr3融合蛋白,分离纯化蛋白后免疫小鼠,通过细胞融合技术获得了2株可以稳定分泌Fpr3抗体的细胞株,纯化后的单克隆抗体,通过计算得到其分子量为152kDa,这与抗体分子量的理论值相符。对腹水做效价检测和亚类的鉴定,最终得到2株细胞株分泌的抗体的亚类均为IgG1,效价分别为1.5×104,1.0x105。同时还通过Western blot的方法鉴定单克隆抗体的特异性,最终结果证明这2株杂交瘤细胞分泌的单克隆抗体可以特异性的识别Fpr3蛋白。利用抗原和抗体特异性的结合,对我们以后研究Fpr3蛋白的结构和功能起着非常重要的作用。
[Abstract]:FKBP is a family of proteins widely present in organisms.Up to now, four members of the FKBP family: FPR1, FPR2, FPR3, Fpr4.Fpr3 are found in yeast, which can participate in ribosomal assembly and direct ribosomes or related proteins to pass through nuclear pore complex. FPR3 can also regulate cell cycle.When the cell cycle is abnormal, FPR3 will play a role in cell cycle arrest in G2 phase.Because of the important physiological function of Fpr3, we want to know its spatial structure, but it is difficult to crystallize Fpr3.A monoclonal antibody is an antibody that is specific to an antigenic determinant.When a single protein is unable to crystallize, it is often co-crystallized with its antibodies.When the antibody binds to the corresponding protein, it can stabilize the conformation of the protein and improve the success rate of protein crystallization.At this point, antibodies act as molecular chaperones.Molecular chaperones can bind to unstable protein structures by controlling their binding and release to help them fold assemble transport and degrade without forming components of protein structures that perform functions.In this experiment, pEGX-5X-1-fpr3 was transferred into E. coli E.coliBL21 to induce the expression of GST-Fpr3 fusion protein. The purified protein was isolated and immunized to mice. Two cell lines which could secrete Fpr3 antibody stably were obtained by cell fusion technique, and the purified monoclonal antibody was obtained.The molecular weight of the antibody was calculated to be 152 kDa, which is in agreement with the theoretical value of antibody molecular weight.The titer and subclass identification of ascitic fluid showed that the antibodies secreted by the two cell lines were IgG1, and the titers were 1.5 脳 10 ~ (4) C ~ (-1) 脳 10 ~ (5) 脳 10 ~ (5) respectively.At the same time, the Western blot method was used to identify the specificity of the monoclonal antibody. The final results showed that the monoclonal antibodies secreted by the two hybridoma cells could specifically recognize the Fpr3 protein.The specific binding of antigens and antibodies plays an important role in the study of the structure and function of Fpr3 proteins.
【学位授予单位】：华中师范大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392

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