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

【摘要】：FKBP是生物中廣泛存在的一個(gè)蛋白質(zhì)家族。目前在酵母中已經(jīng)發(fā)現(xiàn)了FKBP家族的四個(gè)成員：Fpr1, Fpr2, Fpr3, Fpr4。Fpr3是核仁蛋白,可以參與核糖體的裝配,指導(dǎo)核糖體或者相關(guān)蛋白質(zhì)穿越核孔復(fù)合體。Fpr3還對細(xì)胞周期具有調(diào)控作用,當(dāng)細(xì)胞周期出現(xiàn)異常情況時(shí),Fpr3就會發(fā)揮作用,使細(xì)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(jié)構(gòu),但是Fpr3很難結(jié)晶。單克隆抗體是一種專門針對一種抗原決定簇的抗體。當(dāng)單獨(dú)的蛋白質(zhì)不能結(jié)晶時(shí),往往能與其抗體共同結(jié)晶�？贵w與相應(yīng)的蛋白質(zhì)結(jié)合后,可以穩(wěn)定蛋白質(zhì)的構(gòu)象,有利于提高蛋白質(zhì)結(jié)晶的成功率。此時(shí),抗體的作用就相當(dāng)于分子伴侶。分子伴侶可以與不穩(wěn)定的蛋白結(jié)構(gòu)結(jié)合,通過控制結(jié)合和釋放來幫助其折疊,組裝,轉(zhuǎn)運(yùn)和降解,而不構(gòu)成蛋白質(zhì)結(jié)構(gòu)執(zhí)行功能時(shí)的組份。本實(shí)驗(yàn)通過將pEGX-5X-1-fpr3轉(zhuǎn)入大腸桿菌E.coliBL21中,IPTG誘導(dǎo)表達(dá)GST-Fpr3融合蛋白,分離純化蛋白后免疫小鼠,通過細(xì)胞融合技術(shù)獲得了2株可以穩(wěn)定分泌Fpr3抗體的細(xì)胞株,純化后的單克隆抗體,通過計(jì)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(jià)檢測和亞類的鑒定,最終得到2株細(xì)胞株分泌的抗體的亞類均為IgG1,效價(jià)分別為1.5×104,1.0x105。同時(shí)還通過Western blot的方法鑒定單克隆抗體的特異性,最終結(jié)果證明這2株雜交瘤細(xì)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(jié)合,對我們以后研究Fpr3蛋白的結(jié)構(gòu)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(xué)位授予單位】：華中師范大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R392

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