FLAG標(biāo)簽單克隆抗體的制備、鑒定與應(yīng)用研究
發(fā)布時(shí)間：2018-11-25 20:35
【摘要】： 隨著基因組學(xué)和蛋白質(zhì)組學(xué)的發(fā)展,融合蛋白的純化已成為蛋白質(zhì)組學(xué)的重要任務(wù),后期蛋白純化已占總成本的70%以上,因此建立快速、高效、方便、低廉的純化方法已成為其發(fā)展的瓶頸問(wèn)題。本研究通過(guò)應(yīng)用淋巴細(xì)胞雜交瘤技術(shù),獲得具有效價(jià)高、特異性強(qiáng)、反應(yīng)性好、親和力高等特點(diǎn)的抗FLAG單抗,在含F(xiàn)LAG的融合蛋白的親和層析純化中取得良好效果,建立可用于FLAG融合蛋白純化和檢測(cè)的方法,這將為后基因組時(shí)代大量涌現(xiàn)的新分子的結(jié)構(gòu)和功能研究提供重要的工具,有很高的實(shí)用價(jià)值。同時(shí),本研究方法可作為一種技術(shù)平臺(tái),為其他多種帶標(biāo)簽蛋白單克隆抗體的制備提供技術(shù)支持。探討不同方法、多種載體制備FLAG完全抗原進(jìn)而制備抗FLAG標(biāo)簽單克隆抗體的效果,以及抗FLAG標(biāo)簽單抗在融合蛋白純化中的作用。 利用牛血清白蛋白(BSA)、卵清白蛋白(OVA)和匙孔血藍(lán)蛋白(KLH)作為載體蛋白,通過(guò)碳化二亞胺法合成FLAG-BSA、FLAG-OVA和FLAG-KLH三種完全抗原,采用SDS-PAGE初步判斷偶聯(lián)效果,并將三種抗原作為免疫原通過(guò)腹腔、脾內(nèi)和皮下免疫BALB/c小鼠,取尾血后利用間接ELISA方法檢測(cè)血清效價(jià),以此最終確定偶聯(lián)效果,選最優(yōu)者利用雜交瘤技術(shù)制備抗FLAG標(biāo)簽單克隆抗體的雜交瘤細(xì)胞株,制備腹水,并對(duì)其純化、分析和鑒定。用Western-blot方法檢測(cè)mAb對(duì)融合蛋白的反應(yīng)性,并制備交聯(lián)抗FLAGmAb的親和層析柱,用以純化帶FLAG標(biāo)簽的融合蛋白,經(jīng)SDS -PAGE后薄層掃描分析純化后蛋白純度。 經(jīng)間接ELISA檢測(cè)小鼠尾血效價(jià),FLAG-KLH偶聯(lián)效果最好,小鼠免疫效價(jià)在104以上,共制備1株抗FLAG標(biāo)簽單克隆抗體雜交瘤細(xì)胞株,命名為5F-2,此單抗鑒定結(jié)果為:抗體屬于IgG,其亞類為IgG1,體外多次傳代培養(yǎng)后分泌抗體穩(wěn)定。5F2單抗的腹水效價(jià)達(dá)到106。間接ELISA法證實(shí)此單抗與其他抗原無(wú)交叉反應(yīng),特異性高。制備出酶標(biāo)抗體,活性達(dá)到104。融合蛋白經(jīng)親和層析純化后的純度85%以上,且可用于融合蛋白的Western-blot檢測(cè),建立了可用于帶FLAG標(biāo)簽融合蛋白的純化方法和檢測(cè)方法。 成功進(jìn)行了FLAG完全抗原的偶聯(lián),同時(shí)制備出抗FLAG標(biāo)簽的單克隆抗體,為帶FLAG標(biāo)簽的融合蛋白純化提供重要工具。
[Abstract]:With the development of genomics and proteomics, the purification of fusion proteins has become an important task in proteomics. Low-cost purification method has become the bottleneck of its development. In this study, monoclonal antibodies against FLAG were obtained by using lymphocyte hybridoma technique with high titer, specificity, reactivity and affinity. The monoclonal antibodies were successfully purified by affinity chromatography of fusion protein containing FLAG. A method for the purification and detection of FLAG fusion proteins has been established, which will provide an important tool for the study of the structure and function of a large number of new molecules emerging in the post-genomic era, and will be of great practical value. At the same time, this method can be used as a technical platform to provide technical support for the preparation of monoclonal antibodies against other labeled proteins. To study the effect of different methods on the preparation of FLAG complete antigen and then to prepare monoclonal antibody against FLAG label, and the role of anti FLAG tag monoclonal antibody in the purification of fusion protein. Using bovine serum albumin (BSA),) ovalbumin (OVA) and keyhole hemocyanin (KLH) as carrier proteins, three complete antigens, FLAG-BSA,FLAG-OVA and FLAG-KLH, were synthesized by carbonized diimide method. SDS-PAGE was used to determine the coupling effect, and three antigens were used as immunogens to immunize BALB/c mice through abdominal cavity, intrasplenic and subcutaneous. The titer of serum was detected by indirect ELISA method after taking tail blood, so as to determine the coupling effect. The best hybridoma cell lines with monoclonal antibody against FLAG label were prepared by hybridoma technique, and ascites were prepared, purified, analyzed and identified. The reactivity of mAb to the fusion protein was detected by Western-blot method, and the affinity chromatography column of cross-linked anti-FLAGmAb was prepared to purify the fusion protein with FLAG label. The purity of the purified protein was analyzed by TLC scanning after SDS PAGE. The titer of mouse tail blood was detected by indirect ELISA, and the FLAG-KLH coupling effect was the best. The immunological titer of mice was above 104. A hybridoma cell line with monoclonal antibody against FLAG label was prepared and named 5F-2. The result of identification of this monoclonal antibody was as follows: the antibody belongs to IgG,. The subclass of 5F2 McAb was that the antibody secreted by IgG1, was stable after several passages in vitro, and the ascites titer of 5F2 McAb reached 106. Indirect ELISA assay confirmed that the McAb had no cross-reaction with other antigens and had high specificity. The enzyme labeled antibody was prepared and its activity reached 104. The purity of the fusion protein was more than 85% after purification by affinity chromatography, and it could be used for the Western-blot detection of the fusion protein. A method for the purification and detection of the fusion protein with FLAG label was established. The FLAG complete antigen coupling was successfully carried out, and the monoclonal antibody against FLAG tag was prepared, which provided an important tool for the purification of fusion protein with FLAG tag.
【學(xué)位授予單位】：河南工業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R392
本文編號(hào)：2357306
[Abstract]:With the development of genomics and proteomics, the purification of fusion proteins has become an important task in proteomics. Low-cost purification method has become the bottleneck of its development. In this study, monoclonal antibodies against FLAG were obtained by using lymphocyte hybridoma technique with high titer, specificity, reactivity and affinity. The monoclonal antibodies were successfully purified by affinity chromatography of fusion protein containing FLAG. A method for the purification and detection of FLAG fusion proteins has been established, which will provide an important tool for the study of the structure and function of a large number of new molecules emerging in the post-genomic era, and will be of great practical value. At the same time, this method can be used as a technical platform to provide technical support for the preparation of monoclonal antibodies against other labeled proteins. To study the effect of different methods on the preparation of FLAG complete antigen and then to prepare monoclonal antibody against FLAG label, and the role of anti FLAG tag monoclonal antibody in the purification of fusion protein. Using bovine serum albumin (BSA),) ovalbumin (OVA) and keyhole hemocyanin (KLH) as carrier proteins, three complete antigens, FLAG-BSA,FLAG-OVA and FLAG-KLH, were synthesized by carbonized diimide method. SDS-PAGE was used to determine the coupling effect, and three antigens were used as immunogens to immunize BALB/c mice through abdominal cavity, intrasplenic and subcutaneous. The titer of serum was detected by indirect ELISA method after taking tail blood, so as to determine the coupling effect. The best hybridoma cell lines with monoclonal antibody against FLAG label were prepared by hybridoma technique, and ascites were prepared, purified, analyzed and identified. The reactivity of mAb to the fusion protein was detected by Western-blot method, and the affinity chromatography column of cross-linked anti-FLAGmAb was prepared to purify the fusion protein with FLAG label. The purity of the purified protein was analyzed by TLC scanning after SDS PAGE. The titer of mouse tail blood was detected by indirect ELISA, and the FLAG-KLH coupling effect was the best. The immunological titer of mice was above 104. A hybridoma cell line with monoclonal antibody against FLAG label was prepared and named 5F-2. The result of identification of this monoclonal antibody was as follows: the antibody belongs to IgG,. The subclass of 5F2 McAb was that the antibody secreted by IgG1, was stable after several passages in vitro, and the ascites titer of 5F2 McAb reached 106. Indirect ELISA assay confirmed that the McAb had no cross-reaction with other antigens and had high specificity. The enzyme labeled antibody was prepared and its activity reached 104. The purity of the fusion protein was more than 85% after purification by affinity chromatography, and it could be used for the Western-blot detection of the fusion protein. A method for the purification and detection of the fusion protein with FLAG label was established. The FLAG complete antigen coupling was successfully carried out, and the monoclonal antibody against FLAG tag was prepared, which provided an important tool for the purification of fusion protein with FLAG tag.
【學(xué)位授予單位】：河南工業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R392
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