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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。
OBJECTIVE: To investigate the physiological and pathological effects of OGT on O-GlcNAc glycosyltransferase OGT, it is necessary to prepare antibodies that can efficiently and specifically detect OGT. METHODS: The human OGT gene sequence was searched in the NCBI database. According to the structural characteristics of OGT, a polypeptide sequence (amino acids 464-949) in the C-terminal catalytic domain of OGT was selected as the antigen. First of all, a recombinant expression vector pET30-a-OGT-C containing the C-terminal catalytic domain (464-949 amino acid residues) of OGT was constructed and transformed into E. coli BL21 (DE3) competent cells. IPTG induced the expression of His tag OGT-C protein, Ni + bead affinity chromatography purification of OGT-C protein. The recombinant protein of OGT-C was used as antigen to immunize Wistar rats to prepare polyclonal antibody. The titer of OGT antibody was detected by indirect ELISA and the specificity of antibody was identified by Western blotting. Results: The multivalent anti-titers reached 1: 80000. In the western blotting, the polyclonal antibody could detect the recombinant antigens efficiently and identify the two OGT subtypes ncOGT and mOGT endogenously expressed by the cultured cells. CONCLUSION: The experimental results show that the high titer and specificity of OGT polyclonal antibody can be used to detect the expression of ncOGT and mOGT in the biological research of OGT.