Analysis of Glycosylation Modification of Human Erythropoietin Expressed in Pernyi Pupae

Abstract

Recombinant human erythropoietin(rhEPO)was expressed in pernyi pupae using DNA recombination technology and gel purified. The purified components were separated by SDS-PAGE(sodium dodecyl sulfate polyacrylamide gel electrophoresis), and the rhEPO gel strips were cut and its glycosylation modification was detected by electrospray ionization tandem mass spectrometry(ESI-MS/MS). The mass spectrometry results showed that the glycosylation modification sites of rhEPO expressed in pernyi silkworm pupae were consistent with EPO expressed in humans, with three N-glycosylation sites and one O-glycosy-lation site. Although the specific sugar chains at the glycosylation sites cannot be determined based on ESI-MS/MS, its results combined with lectin experiments can help determine the specific sugar chains at the glycosylation modification sites. According to the results, the overall sugar chain lacks sialic acid modification. The results of cell experiments showed that rhEPO expressed by pernyi pupae had certain biological activity, with a specific activity of 1190 U/μg. Therefore, pernyi pupae can express rhEPO without sialic acid modification with certain biological activity, and low sialylated EPO can play an important role in the treatment of central nervous system diseases after nasal administration. The results provide a basis for further studying the glycosylation and biological activity of exogenous proteins after expression in the pernyi pupae-Anthraea pernyi nucleopolyhedrorirus(ApNPV)host vector expression system.

利用DNA重组技术在柞蚕蛹中表达重组人促红细胞生成素(Recombinant human erythropoietin， rhEPO)， 经过亲和层析纯化后， 用SDS-PAGE分离纯化各组分， 并通过电喷雾电离串联质谱技术(ESI-MS/MS)检测其糖基化修饰. 结果显示， 在柞蚕蛹中表达的rhEPO比活性约为1190 U/μg， 其糖基化修饰位点与人体表达的EPO一致， 有3个N-糖基化位点和1个O-糖基化位点. 凝集素杂交实验结合质谱结果表明， 柞蚕蛹表达的rhEPO的糖链中缺乏唾液酸修饰, 而缺少唾液酸修饰的EPO通过鼻腔给药后在多种神经系统疾病的治疗中发挥着重要的作用. 所得结果为进一步研究外源蛋白在柞蚕蛹-柞蚕核型多角体病毒(Anthraea pernyi nucleopolyhedrorirus, ApNPV)宿主载体表达系统表达后的糖基化与生物活性提供了依据.