溶瘤病毒选择增殖活性检测方法的建立及优化  

Establishment and optimization of a detection method for selective proliferative activity of modified herpes simplex oncolytic virus

作　　者：王光裕[1] 于雷[1] 史新昌[1] 周勇[1] WANG Guangyu;YU Lei;SHI Xinchang;ZHOU Yong(National Institutes for Food and Drug Control,NHC Key Laboratory of Research on Quality and Standardization of Biotech Products,Bejing 100050,China)

机构地区：[1]中国食品药品检定研究院国家卫生健康委生物技术产品检定方法及其标准化重点实验室,北京100050

出　　处：《中国生物制品学杂志》2024年第11期1349-1353,共5页Chinese Journal of Biologicals

基　　金：中国医学科学院中央级公益性科研院所基本科研业务费专项资金(2023-PT350-01)。

摘　　要：目的以重组人单纯疱疹病毒-1(recombinant human herpes simplex virus-1,rhHSV-1)作为溶瘤病毒,建立溶瘤病毒选择增殖活性测定方法,并进行优化及验证,以期用于溶瘤病毒相关制品的质量评价。方法将rhHSV-1按相同的MOI分别感染正常细胞及肿瘤细胞,培养一段时间后,提取病毒核酸,以其为模板进行qPCR扩增,获得病毒基因组拷贝数,计算病毒增殖比值,以其作为选择增殖活性的评价指标。优化方法中的细胞种类(正常细胞为MRC-5及HEK-293,肿瘤细胞为Hep3B及Fadu)、MOI(0.1、0.01、0.001、0.0001、0.00001)、培养时间(2 h及2、3、4 d),并验证方法的精密性及专属性。采用优化的方法检测溶瘤病毒rhHSV-1工作种子批的选择增殖活性。结果以Hep3B作为肿瘤细胞、MRC-5作为正常细胞时,rhHSV-1的增殖效果最佳,最适MOI为0.0001,最佳培养时间为72 h。采用优化的方法重复3次检测rhHSV-1的增殖比值分别为260.68、336.65及259.14,均>100,CV为15.52%;采用优化的方法检测rhHSV-1可见明显扩增,检测人巨细胞病毒(human cytomegalovirus,HCMV)和5型腺病毒(adenovirus 5,Ad5)未见明显扩增。溶瘤病毒rhHSV-1工作种子批的增殖比值为727。结论建立的用于检测rhHSV-1选择增殖活性的方法具有良好的精密性及专属性,可用于溶瘤病毒选择增殖活性的评价。Objective To establish,optimize and validate a method for determining the selective proliferative activity of oncolytic virus using recombinant human herpes simplex virus-1(rhHSV-1)as the oncolytic virus,so as to apply it to the quality evaluation of oncolytic virus-related products.Methods Normal cells and tumor cells were infected with rhHSV-1 at the same MOI respectively.After a certain period of culture,the viral nucleic acid was extracted and used as template for qPCR amplification.The copy number of virus genome was obtained,and the virus proliferation ratio was calculated,which was used as an evaluation index for selective proliferative activity.The cell types(normal cells:MRC-5,HEK-293;tumor cells:Hep3B,Fadu),MOI(0.1,0.01,0.001,0.0001,0.00001)and culture time(2 h and 2,3,4 d)were optimized,and the precision and specificity of the method were verified.The selective proliferative activity of oncolytic virus rhHSV-1 working seed lot was detected by using the established method.Results Using Hep3B as tumor cells and MRC-5 as normal cells,rhHSV-1 had the best proliferation effect with the optimal MOI of 0.0001,and the optimal culture time was 72 h.The proliferation ratios of rhHSV-1 in three repeated tests by the optimized method were 260.68,336.65 and 259.14 respectively,with all of them more than 100 and a CV of 15.52%.Using the optimized method,rhHSV-1 had significant amplification,but human cytomegalovirus(HCMV)and adenovirus 5(Ad5)showed no obvious amplification.The proliferation ratio of oncolytic virus rhHSV-1 working seed lot was 727.Conclusion The established method for determining the selective proliferative activity of rhHSV-1 has good precision and specificity,and can be used to evaluate the selective proliferative activity of oncolytic viruses.

关 键 词：溶瘤病毒 选择增殖活性 qPCR法 重组人单纯疱疹病毒-1 增殖比值 

分 类 号：R979.14[医药卫生—药品]