本文選題：單克隆抗體 + Fpr3　； 參考：《華中師范大學》2011年碩士論文

【摘要】：FKBP是生物中廣泛存在的一個蛋白質家族。目前在酵母中已經發(fā)現(xiàn)了FKBP家族的四個成員：Fpr1, Fpr2, Fpr3, Fpr4。Fpr3是核仁蛋白,可以參與核糖體的裝配,指導核糖體或者相關蛋白質穿越核孔復合體。Fpr3還對細胞周期具有調控作用,當細胞周期出現(xiàn)異常情況時,Fpr3就會發(fā)揮作用,使細胞周期停滯在G2期。正是由于Fpr3重要的生理功能,我們希望了解其空間結構,但是Fpr3很難結晶。單克隆抗體是一種專門針對一種抗原決定簇的抗體。當單獨的蛋白質不能結晶時,往往能與其抗體共同結晶。抗體與相應的蛋白質結合后,可以穩(wěn)定蛋白質的構象,有利于提高蛋白質結晶的成功率。此時,抗體的作用就相當于分子伴侶。分子伴侶可以與不穩(wěn)定的蛋白結構結合,通過控制結合和釋放來幫助其折疊,組裝,轉運和降解,而不構成蛋白質結構執(zhí)行功能時的組份。本實驗通過將pEGX-5X-1-fpr3轉入大腸桿菌E.coliBL21中,IPTG誘導表達GST-Fpr3融合蛋白,分離純化蛋白后免疫小鼠,通過細胞融合技術獲得了2株可以穩(wěn)定分泌Fpr3抗體的細胞株,純化后的單克隆抗體,通過計算得到其分子量為152kDa,這與抗體分子量的理論值相符。對腹水做效價檢測和亞類的鑒定,最終得到2株細胞株分泌的抗體的亞類均為IgG1,效價分別為1.5×104,1.0x105。同時還通過Western blot的方法鑒定單克隆抗體的特異性,最終結果證明這2株雜交瘤細胞分泌的單克隆抗體可以特異性的識別Fpr3蛋白。利用抗原和抗體特異性的結合,對我們以后研究Fpr3蛋白的結構和功能起著非常重要的作用。
[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.
【學位授予單位】：華中師范大學
【學位級別】：碩士
【學位授予年份】：2011
【分類號】：R392

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