OGT 多克隆抗体的制备及特异性分析

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。

Preparation and Characterization of a Polyclonal Antibody Against OGT

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.