Production of recombinant human erythropoietin/Fc fusion protein by genetically manipulated chickens
Production of recombinant human erythropoietin/Fc fusion protein by genetically manipulated chickens
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DOI：
10.1007/s11248-009-9310-z
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发表时间：
2010-04-01
影响因子：
3
通讯作者：
Kamihira, Masamichi
中科院分区：
文献类型：
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作者：
Penno, Carlos Alberto;Kawabe, Yoshinori;Kamihira, Masamichi
我们之前报道过使用转基因 (GM) 鸡生产人类促红细胞生成素 (hEpo)。重组hEpo是在转基因鸡的血清和蛋清中产生的，并且血清来源的hEpo的寡糖链结构比蛋清来源的hEpo更有利。本研究将编码hEpo与人免疫球蛋白G Fc区融合蛋白（hEpo/Fc）表达盒的逆转录病毒载体注射到发育中的鸡胚胎中，目的是通过母体抗体的卵黄积累机制从蛋黄中回收血清来源的hEpo。孵化出来的转基因鸡不仅在血清和蛋清中稳定产生 hEpo/Fc 融合蛋白，而且在蛋黄中也如预期的那样产生了 hEpo/Fc 融合蛋白。凝集素印迹分析显示，转基因鸡的血清和鸡蛋中产生的大量 hEpo/Fc 寡糖链以半乳糖封端，并且血清和蛋黄来源的 hEpo/Fc 的寡糖链掺入了唾液酸残基。此外，使用 Epo 依赖性细胞的生物活性评估表明，蛋黄来源的 hEpo/Fc 表现出与血清和 CHO 来源的 hEpo/Fc 相当的性能。这些结果表明，将 Fc 融合蛋白从鸡的血液循环转运至蛋黄代表了使用转基因鸡生物反应器生产药用糖蛋白的有效策略。
We previously reported the production of human erythropoietin (hEpo) using genetically manipulated (GM) chickens. The recombinant hEpo was produced in the serum and egg white of the GM chickens, and the oligosaccharide chain structures of the serum-derived hEpo were more favorable than those of the egg white-derived hEpo. In the present study, a retroviral vector encoding an expression cassette for a fusion protein of hEpo and the Fc region of human immunoglobulin G (hEpo/Fc) was injected into developing chicken embryos, with the aim of recovering the serum-derived hEpo from egg yolk through the yolk accumulation mechanism of maternal antibodies. The GM chickens that hatched stably produced the hEpo/Fc fusion protein not only in their serum and egg white, but also in the egg yolk as expected. Lectin blot analyses revealed that significant amounts of the oligosaccharide chains of hEpo/Fc produced in the serum and eggs of GM chickens terminated with galactose, and that the oligosaccharide chains of the serum- and yolk-derived hEpo/Fc incorporated sialic acid residues. Moreover, biological activity assessment using Epo-dependent cells revealed that the yolk-derived hEpo/Fc exhibited a comparable performance to the serum- and CHO-derived hEpo/Fc. These results indicate that transport of Fc fusion proteins from the blood circulation to the yolk in chickens represents an effective strategy for the production of pharmaceutical glycoproteins using transgenic chicken bioreactors.