溶瘤病毒选择增殖活性检测方法的建立及优化
10.13200/j.cnki.cjb.004335
- VernacularTitle:Establishment and optimization of a detection method for selective proliferative activity of modified herpes simplex oncolytic virus
- Author:
WANG Guangyu
- Publication Type:Journal Article
- Keywords:
Oncolytic virus;
Selective proliferative activity;
qPCR;
Recombinant human herpes simplex virus-1(rhHSV-1);
Proliferation ratio
- From:
Chinese Journal of Biologicals
2024;37(11):1349-1353
- CountryChina
- Language:Chinese
-
Abstract:
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. Afteracertainperiodofculture,theviralnucleicacidwasextractedandusedastemplateforqPCRamplification. 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. 000 1, 0. 000 01) 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. 000 1, 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
