使用优化的信号肽和密码子在Expi293F细胞中高表达重链抗体可变区抗体

High expression of variable domain of heavy-chain antibodies in Expi293F cells with optimized signal peptide and codons

摘要:

重链抗体可变区抗体(variable domain of heavy-chain antibody, VHH)已被广泛用于药物治疗、诊断和研究。大肠杆菌是生产VHH最常用的表达系统，但可能会导致VHH的生物活性降低。哺乳动物细胞是目前最理想的VHH表达宿主之一。本研究通过优化VHH的信号肽(signal peptide, SP)和密码子，以期提高VHH在Expi293F细胞中的产量。VHH1-Fc被用于筛选SP，通过酶联免疫吸附试验(enzyme-linked immunosorbent assay, ELISA)筛选出的SP IFN-α2分泌效果最佳；通过提高基因的GC3和GC含量，VHH1的产量提高了约1倍，且对VHH1与A型塞内卡病毒(Senecavirus A, SVA)的结合活性无明显影响；其他5种重组VHHs分别连接SP IFN-α2并进行密码子优化，其平均产量大于191.6 mg/L。此外，这些VHHs在培养上清中具有高纯度和高回收率的优点。本研究证实SP IFN-α2和密码子优化可以在Expi293F细胞高效表达VHH，为VHH的大规模生产提供了参考。

Abstract:

The variable domain of heavy-chain antibody (VHH) has been developed widely in drug therapy, diagnosis, and research. Escherichia coli is the most popular expression system for VHH production, whereas low bioactivity occurs sometimes. Mammalian cells are one of the most ideal hosts for VHH expression at present. To improve the yield of VHH in Expi293F cells, we optimized the signal peptide (SP) and codons of VHH. Firstly, the fusion protein VHH1-Fc was used to screen SPs. The SP IFN-α2 showed the highest secretion as quantified by enzyme-linked immunosorbent assay (ELISA). Subsequently, codon optimization by improving GC3 and GC content doubled the yield of VHH1 and kept its binding activity to Senecavirus A (SVA). Finally, the mean yields of other 5 VHHs that fused with SP IFN-α2 and codon-optimized were over 191.6 mg/L, and these VHHs had high recovery and high purity in the culture supernatant. This study confirms that SP IFN-α2 and codon optimization could produce VHHs in Expi293F cells efficiently, which provides a reference for the large-scale production of VHHs.