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

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。

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.