FLAG标签单克隆抗体的制备、鉴定与应用研究
发布时间：2018-11-25 20:35
【摘要】： 随着基因组学和蛋白质组学的发展,融合蛋白的纯化已成为蛋白质组学的重要任务,后期蛋白纯化已占总成本的70%以上,因此建立快速、高效、方便、低廉的纯化方法已成为其发展的瓶颈问题。本研究通过应用淋巴细胞杂交瘤技术,获得具有效价高、特异性强、反应性好、亲和力高等特点的抗FLAG单抗,在含FLAG的融合蛋白的亲和层析纯化中取得良好效果,建立可用于FLAG融合蛋白纯化和检测的方法,这将为后基因组时代大量涌现的新分子的结构和功能研究提供重要的工具,有很高的实用价值。同时,本研究方法可作为一种技术平台,为其他多种带标签蛋白单克隆抗体的制备提供技术支持。探讨不同方法、多种载体制备FLAG完全抗原进而制备抗FLAG标签单克隆抗体的效果,以及抗FLAG标签单抗在融合蛋白纯化中的作用。 利用牛血清白蛋白(BSA)、卵清白蛋白(OVA)和匙孔血蓝蛋白(KLH)作为载体蛋白,通过碳化二亚胺法合成FLAG-BSA、FLAG-OVA和FLAG-KLH三种完全抗原,采用SDS-PAGE初步判断偶联效果,并将三种抗原作为免疫原通过腹腔、脾内和皮下免疫BALB/c小鼠,取尾血后利用间接ELISA方法检测血清效价,以此最终确定偶联效果,选最优者利用杂交瘤技术制备抗FLAG标签单克隆抗体的杂交瘤细胞株,制备腹水,并对其纯化、分析和鉴定。用Western-blot方法检测mAb对融合蛋白的反应性,并制备交联抗FLAGmAb的亲和层析柱,用以纯化带FLAG标签的融合蛋白,经SDS -PAGE后薄层扫描分析纯化后蛋白纯度。 经间接ELISA检测小鼠尾血效价,FLAG-KLH偶联效果最好,小鼠免疫效价在104以上,共制备1株抗FLAG标签单克隆抗体杂交瘤细胞株,命名为5F-2,此单抗鉴定结果为:抗体属于IgG,其亚类为IgG1,体外多次传代培养后分泌抗体稳定。5F2单抗的腹水效价达到106。间接ELISA法证实此单抗与其他抗原无交叉反应,特异性高。制备出酶标抗体,活性达到104。融合蛋白经亲和层析纯化后的纯度85%以上,且可用于融合蛋白的Western-blot检测,建立了可用于带FLAG标签融合蛋白的纯化方法和检测方法。 成功进行了FLAG完全抗原的偶联,同时制备出抗FLAG标签的单克隆抗体,为带FLAG标签的融合蛋白纯化提供重要工具。
[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.
【学位授予单位】：河南工业大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392
本文编号：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.
【学位授予单位】：河南工业大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392
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