基于稳定表达萤火虫荧光素酶Vero细胞系体外病毒中和活性检测方法的建立及应用
Development and application of in vitro antiviral neutralizing activity detection method based on Vero cell line stably expressing firefly luciferase
丁如霞 1李晓玉 1赵晨燕 1刘强 1姚佳维 2黄维金 1王佑春1
作者信息
- 1. 中国食品药品检定研究院,北京 102629
- 2. 北京百奥赛图基因生物技术有限公司,北京 102629
- 折叠
摘要
目的 建立一种基于稳定表达萤火虫荧光素酶(firefly luciferase,fluc)Vero细胞系的高通量检测方法,以评估样品的体外抗病毒中和活性.方法 基于成簇规律间隔短回文重复序列(clustered regularly interspaced short palin-dromic repeats,CRISPR)/Cas9靶向基因编辑技术,构建一种稳定表达fluc的Vero细胞系,在此基础上建立痘病毒和呼吸道合胞病毒(respiratory syncytial virus,RSV)感染细胞活性检测方法,利用该方法评估痘病毒免疫血清及RSV单抗的中和活性,并与噬斑抑制法及细胞活力检测(adenosine triphosphate,ATP)法检测结果进行比较.结果 成功构建了 Vero-fluc细胞系,其细胞量与表达的fluc相关性良好(R2=0.994 8).利用Vero-fluc细胞作为感染细胞验证的痘病毒中和活性检测方法具有良好的稳定性、可靠性[信噪比(signal/background ratio,S/B)=25],可满足高通量的检测需求(Z'=0.9),相比传统噬斑抑制法一致性强(R2=0.76),免疫血清的ID50值较噬斑法平均高出2倍.建立的RSV中和活性检测方法,检测的抗RSV单抗中和活性与ATP活性试剂盒检测结果一致,Vero-fluc细胞法在1∶103～1∶105的抗体滴度水平上抑制率显著高于ATP法.结论 建立的基于Vero-fluc细胞系的病毒中和活性检测方法与传统方法相比,具有良好的相关性,结果可靠,且具有更高的灵敏度及检测通量,是一种通用型体外抗病毒中和活性评价方法.
Abstract
Objective To develop a high-throughput detection method based on Vero cell line stably expressing firefly luciferase(flue)to evaluate the antiviral neutralizing activity of samples in vitro.Methods Based on CRISPR/Cas9 targeted gene editing technology,a Vero cell line stably expressing fluc was constructed,and a cell viability detection method for poxvirus and respiratory syncytial virus(RSV)infection was developed.This method was used to evaluate the neutralizing activity of serum immunized by vaccinia virus and RSV monoclonal antibodies,and the result was compared with that by plaque suppression method and cell viability(adenosine triphosphate,ATP)assay.Results Vero-fluc cell line was successfully constructed,and there was a good correlation between the cell quantity and the expression of fluc(R2=0.994 8).Vero-fluc cells were used as infected cells to test the neutralizing activity of poxvirus,which had good stability and reliability[signal/background ratio(S/B)=25],and met the requirements of high-throughput detection(Z'=0.9).Compared with traditional plaque suppression method,the consistency of this method was stronger(R2=0.76),and the ID50 value of immune serum was twice higher than that of plaque suppression method on average.The neutralizing activity of anti-RSV monoclonal antibodies detected by the developed method was consistent with that by ATP activity kit.The inhi-bition rate of Vero-fluc cell method was significantly higher than that of ATP method at the titer level of 1∶103 to 1∶105.Conclusion The antiviral neutralizing activity detection method based on Vero-fluc cells has good correlation with tradi-tional methods,and has higher sensitivity and detection throughput with reliable results,which is a universal in vitro antiviral neutralizing activity evaluation method.
关键词
萤火虫荧光素酶/Vero细胞/高通量检测/抗病毒活性/中和活性Key words
Firefly luciferase(fluc)/Verocell/High-throughput detection/Antiviral activity/Neutralizing activity引用本文复制引用
基金项目
新药创制重大专项(十三五)(2018ZX09101001)
出版年
2024
NETL
NSTL
万方数据