摘要目的:通过聚乙二醇化获得多价抗西尼罗病毒单链抗体（single-chain variable fragment，scFv），以提高其抗原结合能力以及中和活性。方法:在抗西尼罗病毒scFv的C端引入半胱氨酸序列构建携带C端半胱氨酸残基的单链抗体（single-chain variable fragment carrying a C-terminal cysteine residue，scFvC）。采用马来酰亚胺活化的聚乙二醇靶向半胱氨酸的硫醇基，完成scFvC的多聚化。ELISA检测scFvC多聚体的抗原结合活性，假病毒中和试验评估scFvC多聚体的体外中和活性。统计学分析采用单因素方差分析。结果:聚乙二醇化scFvC的抗原结合能力较单体增强。假病毒中和试验显示，与未加抗体的对照组比较，scFvC单体及聚乙二醇化的多聚体均表现出良好的中和活性（ P均<0.000 1），且scFvC多聚体展现出更强的阻断假病毒感染靶细胞的能力（ P<0.05），提示scFvC多聚体能够通过亲合力效应增强其体外功能。 结论:本研究成功构建了抗西尼罗病毒scFvC并通过聚乙二醇化获得多价scFvC，提高了亲本单链抗体的抗原结合力以及中和活性。

abstractsObjective:To obtain a polyvalent single-chain variable fragment（scFv）against West Nile virus through PEGylation in order to improve its antigen-binding ability and neutralizing activity.Methods:A scFv carrying a C-terminal cysteine residue（scFvC）was constructed by introducing Cys into the C-terminal of scFv against West Nile virus. Then the multimerization of scFvC was achieved by targeting the thiol group of Cys with maleimide-activated polyethylene glycol. ELISA was used to detect the antigen-binding activity of the multivalent scFvC. Pseudovirus-based neutralization assay was used to evaluate the neutralizing activity of the multivalent scFvC in vitro. One-way analysis of variance was used for statistical analysis. Results:The PEGylated scFvC multimers showed higher antigen-binding ability than the monomeric scFvC. In the pseudovirus-based neutralization assay，both monomeric scFvC and PEGylated scFvC multimers showed good neutralizing activity compared with the control group（ P<0.000 1）. Moreover，the PEGylated scFvC multimers showed a more effective ability to block the pseudovirus infection in target cells（ P<0.05），suggesting that the PEGylated scFvC multimers could enhance their function in vitro through avidity effect. Conclusion:In this study，a scFvC targeting West Nile virus is successfully constructed and its polyvalent form is generated through PEGylation，which improves the antigen-binding and neutralizing activity of the parental scFv.