结晶紫蚀斑法检测重组痘苗病毒艾滋病疫苗病毒滴度方法的建立

摘要

Objective To establish the crystal violet plaque assay for detection of virus titer of recombined Tiantan vaccinia AIDS vaccine,and provide more stable method of virus titration for rTV AIDS Vaccine.Methods Optimized the concentration of Vero cells,the time and temperature of virus adsorption,and the time of determination for CPE,then established the crystal violet plaque assay for virus titer of rTV.Counting and analysis the plaques by BioSpot Reader,then analyzed the relativity of plaques counted with BioSpot Reader and manual; Several lots rTV AIDS Vaccine and Tiantan vaccinia were titrated by the method of plaque formation-hemadsorption assay,neutral red and crystal violet plaque assay,then analyzed the relativity of the results of three methods ; meanwhile,the virus titer of samples were determine repeatedly by the crystal violet plaque assay,then calculated the coefficient of variation( CV),and verified the precision of the method; SPSS17.0 was used in statistical analysis of the experimental results.Results When the concentration of Vero cells was 5.0×105-9.O×105 cells/ml,virus been adsorbesd 2 h at 37℃,then cultivated 72 h after adding the culture medium containing methyl cellulose.Plaques counted by BioSpot Reader was highly related with counted by manual (r =0.985),so BioSpot Reader counting can objectively reflect the virus plaques with various size,and reduce the error by manual counting; compared the virus titration for different lots of rTV AIDS vaccine and Tiantan vaccinia with three methods,the crystal violet plaque assay was highly related with plaque formation-hemadsorption assay (r =0.997,P＜0.01 ) and neutral red plaque assay(r=0.980,P＜0.01 ).Conclusion Crystal violet plaque assay was established for virus titration of rTV AIDS Vaccine.%目的 建立重组天坛株痘苗病毒(rTV)艾滋病疫苗的结晶紫蚀斑病毒滴度检测方法,为rTV艾滋病疫苗病毒滴度测定提供更稳定的方法.方法 通过对Vero细胞浓度、病毒吸附时间及温度、病变判定时间等方面进行优化,建立rTV艾滋病疫苗病毒滴度的结晶紫蚀斑检测方法,并应用BioSpot Reader进行蚀斑计数及分析,比对仪器蚀斑计数和人工蚀斑计数的相关性；应用血球吸附法、中性红蚀斑、结晶紫蚀斑3种方法,对多批rTV艾滋病疫苗及天坛株痘病毒滴度进行检定,进行3种方法的相关性分析；采用结晶紫蚀斑法重复测定样品,计算变异系数(CV),对方法的精密性进行验证；采用SPSS17.0软件对实验数据进行统计分析.结果 确定Vero细胞浓度为5.0×105 ～9.0×105个/ml时,病毒37℃吸附2h后加入含甲基纤维素的维持液,培养72 h,应用BioSpot Reader进行蚀斑计数,与人工计数的相关系数r=0.985,能客观反映不同大小的病毒蚀斑,降低了人工计数引起的非客观因素误差；经过3种方法对不同批次的rTV艾滋病疫苗及天坛株痘病毒进行病毒滴度检定,结晶紫蚀斑与血吸附法的相关系数r=0.997,结晶紫蚀斑法与中性红蚀斑法相关系数r=0.980 (P＜0.01),具有高度相关性.结论 建立了可用于rTV艾滋病疫苗病毒滴度检测的结晶紫蚀斑方法.