| 张迪 张瑜 陈丹阳 陈博雅 修玉宗 于文功 顾玉超△.OGT 多克隆抗体的制备及特异性分析[J].,2016,16(3):414-418 |
| OGT 多克隆抗体的制备及特异性分析 |
| Preparation and Characterization of a Polyclonal Antibody Against OGT |
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| DOI： |
| 中文关键词: OGT 多克隆抗体 ELISA Western blotting |
| 英文关键词: OGT Polyclonal antibody ELISA Western blotting |
| 基金项目:国家自然科学基金项目（81272264；81101505；81172013）；国家大学生创新创业训练计划（201310423053） |
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| 中文摘要: |
| 目的：为了探讨O-GlcNAc 糖基转移酶OGT 的生理和病理作用，需制备能高效特异性检测OGT 的抗体。方法：在NCBI数
据库中，查找人源OGT 基因序列，根据OGT 的结构特点，选取OGT 的C 末端催化结构域中的一段多肽序列（464-949 位点氨基
酸）做抗原。首先，构建OGT 的C 末端催化结构域（464-949 位点氨基酸）的重组表达载体pET30-a-OGT-C，转化至大肠杆菌BL21
(DE3)感受态细胞中，IPTG诱导表达融合His标签的OGT-C 蛋白，Ni+珠亲和层析法纯化提取OGT-C蛋白。再以OGT-C 重组蛋
白作为抗原，免疫Wistar 大鼠制备多克隆抗体，并用间接ELISA法检测OGT 抗体的效价，Western blotting 鉴定抗体特异性。结
果：多抗效价达1：80000；在免疫印迹实验中，此多抗可以高效的检测重组抗原，并可以特异性识别培养细胞内源表达的ncOGT
和mOGT这2 种OGT 亚型。结论：实验结果表明，获得高效价、高特异性的OGT 多克隆抗体，在OGT 的生物学研究中可以用于
检测ncOGT 和mOGT的表达。 |
| 英文摘要: |
| Objective:To discuss the physiological and pathological role of OGT (O-linked N-acetylglucosamine transferase), the
efficient and specific polyclonal antibodies against the OGT are needed to prepared.Methods:To find the DNA sequence of human OGT
from the database of NCBI, the C-terminal of OGT (464-949 amino acid) was selected as the antigen. First, the DNA sequence corresponding
the C-terminal of OGT (464-949 amino acid) was inserted into the expression vector pET30-a. Recombinant proteins
named as OGT-C were expressed. They were purified by Ni+ affinity chromatography. Sera were obtained from Wistar rats immunized
with OGT-C. The titer and specificity of anti-OGT-C polyclonal antibodies were detected by ELISA and Western blotting.Results:The
titer of polyclonal antibody was approximately 1:80000, and the polyclonal antibody could sensitively detect the recombinant OGT-C by
Western blotting. The antibody could specifically recognize ncOGT and mOGT protein in the lysate of H1299.Conclusion:Our data
suggest that this antibody has high specificity and titer aginst OGT, which may be used to measure the expression of ncOGT and mOGT
in the biology study of OGT. |
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