Rescue of enterovirus group A type 71 VP1 protein mutant strain and its effect on virulence
10.3760/cma.j.cn112866-20220506-00109
- VernacularTitle:肠道病毒A组71型VP1蛋白突变株的拯救及其对病毒毒力的影响
- Author:
Mengyu DU
1
;
Xiaoying XU
;
Mengting CHEN
;
Zequn WANG
;
Hongling WEN
Author Information
1. 山东大学齐鲁医学院公共卫生学院微生物检验学系 山东省"十三五"高等学校感染性疾病防控重点实验室，济南 250012
- Keywords:
Enterovirus;
VP1 protein;
Mutant strain;
Virulence
- From:
Chinese Journal of Experimental and Clinical Virology
2022;36(6):701-706
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To rescue enterovirus group A type 71 (EV-A71) VP1 protein mutant strain (Val147→Ala) and explore the effect of this locus on viral virulence.Methods:The SDLY107 constructed plasmid pMD19-T-EGFP-107 was used as template, the recombinant plasmid pMD19-T-EGFP-107 (VP1-1) was constructed by site-directed mutation and reverse genetics. The recombinant plasmid was transfected into BSR-T7/5 cells, and the transfection products were subcultured in RD cells for three times. The mutant strain SDLY-EGFP-107(VP1-1) was successfully saved. The viral replication was detected by qRT-PCR, and the cell damage caused by the virus was detected by cell proliferation (CCK-8) and lactate dehydrogenase (LDH) tests.Results:The target fragment was amplified by overlapping fusion PCR, and the size was about 3.4 kb. The recombinant plasmid was identified by double enzyme digestion and the mutation was verified by sequencing. Obvious fluorescence was observed 24 hours after transfection of BSR-T7/5 cells with recombinant plasmid and 24 hours after inoculation into RD cells. The replication ability of mutant strain SDLY107 (VP1-1) in RD cells was weaker than that of virulent strain SDLY107 ( t=9.58, P<0.001) by qRT-PCR. CCK-8 and LDH tests showed that the cytotoxicity of mutant strain SDLY-EGFP-107(VP1-1) in RD cells ( t=106.60, P<0.001; t=39.88, P<0.001), SH-SY5Y cells ( t=18.72, P<0.001; t=19.09, P<0.001) were significantly weaker than that of virulent strain SDLY107. Conclusions:The mutant strain SDLY-EGFP-107(VP1-1) was successfully saved, which confirmed that the mutation of the 147th amino acid site of VP1 protein could reduce the replication and cell damage of the virus, providing a basis for further study of the role of VP1 protein in the pathogenesis of EV-A71.
