抗小鹅瘟病毒截短型 IgY 抗体表达及其中和活性检测

Expression of truncated IgY antibody against gosling plague virus and its neutralizing activity detection

摘要

为获得针对小鹅瘟(GP)的本动物源治疗性工程抗体,将试验室制备的pET27b-tHC10、pET27b-tHC11、pET27b-L 3种表达载体,经大肠杆菌表达系统包涵体表达,获得1种轻链蛋白和2种截短型重链蛋白,将截短型重链和轻链按照3:2 质量比稀释复性,获得截短型抗体IgY(ΔFc)-10 和IgY(ΔFc)-11。蛋白质印迹法和ELISA结果表明：2种IgY(ΔFc)均可特异性结合小鹅瘟病毒(GPV)VP2蛋白和完整GPV颗粒。细胞水平阻断试验结果表明：IgY(ΔFc)-10 和IgY(ΔFc)-11最低有效抑制质量浓度分别为10、5 mg·L^-1。在鹅胚攻毒保护试验中,以100 ELD₅₀(每0.2 mL的EID₅₀为10^-4.625)的GPV攻击时,100 μg的IgY(ΔFc)-10和IgY(ΔFc)-11对鹅胚的攻毒保护率分别为40%、60%,且存活胚胎的生长发育状态与生理盐水对照组无差异。此外,通过建立基于实时荧光定量PCR的GPV中和活性评价体系,确定Ct=22.12为细胞病变临界值,为抗体中和活性评估提供新方法。可知,截短型IgY抗体可有效阻断GPV感染,为小鹅瘟治疗性抗体开发奠定基础。

Abstract

To obtain host-derived therapeutic engineered antibodies against gosling plague (GP), the pET27b-tHC10, pET27b-tHC11, and pET27b-L expression vectors prepared in the laboratory were used to express light chain proteins and two truncated heavy chain proteins in an E. coli expression system. The truncated heavy chains and light chains were diluted and renatured at a mass ratio of 3:2 to produce truncated IgY(ΔFc)-10 and IgY(ΔFc)-11. Western blotting and ELISA results demonstrated that both IgY(ΔFc) antibodies could specifically bind to GPV VP2 protein and intact GPV particles. Cell-based blocking assays showed that the minimum effective inhibitory mass concentrations of IgY(ΔFc)-10 and IgY(ΔFc)-11 were 10 and 5 µg·mL^-1, respectively. In the goose embryo challenge protection test, when embryos were challenged with 100 ELD₅₀ (where 0.2 mL of EID₅₀ corresponds to 10^-4.625) of GPV, the protection rates provided by 100 μg of IgY(ΔFc)-10 and IgY(ΔFc)-11 were 40% and 60%, respectively. Moreover, the growth and development status of surviving embryos showed no significant differences compared with the physiological saline control group. Additionally, a novel evaluation system for GPV neutralizing activity was established based on real-time quantitative PCR, with Ct=22.12 determined as the critical value for cytopathic effects. This provided a new method for assessing antibody neutralizing activity. It indicated that truncated IgY antibodies could effectively block GPV infection, laying a foundation for the development of therapeutic antibodies against gosling plague.

关键词

鹅细小病毒 / 截短型IgY抗体 / 治疗性抗体 / 中和活性试验 / 攻毒保护性试验

Key words

goose parvovirus / truncated IgY antibody / therapeutic antibody / neutralizing activity test / challenge protection test

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基金资助

黑龙江省重点研发计划(创新基地)项目(JD24B009)