O-GlcNAcase抗原片段的选择、优化表达和多克隆抗体的制备

Antigen selection, optimized expression and polyclonal antibody preparation of O-GlcNAcase

摘要:

为了探讨O-GlcNAc修饰的生物学作用和相关疾病的发病机理，需制备高效、专一的O-GlcNAcase (OGA) 抗体。通过对人源OGA蛋白进行序列分析发现，氨基端1~350 aa片段 (sOGA) 抗原性和亲水性较强，将该片段构建至原核表达载体pET-28a，并在大肠杆菌Escherichia coli BL21(DE3) 中进行诱导表达，通过优化IPTG浓度 (0.05 mmol/L) 和诱导时间 (10 h) 获得了高可溶性表达的重组蛋白酶。采用Ni-NTA亲和层析和分子筛层析对重组蛋白进行了纯化，SDS-PAGE检测分子量的大小 (45 kDa) 和纯度 (95％以上)。以4-MU-O-GlcNAc为荧光底物，检测到sOGA的糖苷酶活性为106 nmol/(min·mg)，表明该片段是OGA糖苷酶的活性区域。以此片段作为抗原免疫新西兰大白兔，以CNBr活化Sepharose 4B微珠纯化抗血清制备OGA特异性多克隆抗体。Western blotting和ELISA检测表明，该抗体可以特异识别含有OGA糖苷酶活性区域的多种变体，检测灵敏度为0.11 ng/mL (效价为1∶80 000)，可应用于O-GlcNAcase生物功能研究。

Abstract:

In order to probe the biological function of O-GlcNAc and the pathogenesis of associated diseases, it is essential to prepare a potent and specific O-G1cNAcase (OGA) antibody. Based on protein sequence analysis, we found N terminal 1?350 amino acids of OGA (sOGA) has high antigenicity and hydrophilicity and then constructed it into plasmid pET28a vector. First, we optimized the expression of sOGA in Escherichia coli BL21(DE3) (0.05 mmol/L IPTG, 10 hours) and purified it with the Ni-NTA affinity chromatography and size exclusion chromatography respectively. SDS-PAGE verified the molecular weight (45 kDa) and the purity (>95%) of sOGA and the purified protein was subjected to immunize New Zealand rabbits. Finally, we obtained OGA polyclonal antibody by affinity purifying the antiserum with CNBr-activated Sepharose 4B beads. Western blotting and ELISA assay showed that this antibody could recognize three OGA isoforms with high specificity and the sensitivity was 0.11 ng/mL (the titer was 1:80 000). These results indicated the prepared polyclonal antibody of OGA can be used for the biological function study of OGA.