对两株NSP3区插入外源基因的重组轮状病毒遗传稳定性的研究
Genetic stability analysis of two recombinant rotaviruses harboring foreign gene insertions within the NSP3 region
李杉 1刘夏飞 2余俊杰 1李丹地 3段招军3
作者信息
- 1. 甘肃中医药大学公共卫生学院,兰州 730000;中国疾病预防控制中心病毒病预防控制所,北京 102206
- 2. 河南中医药大学第一临床医学院,郑州 450046;中国疾病预防控制中心病毒病预防控制所,北京 102206
- 3. 中国疾病预防控制中心病毒病预防控制所,北京 102206
- 折叠
摘要
目的 分析2株插入和表达外源基因的重组轮状病毒rLLR/NSP3-NLuc和rLLR/NSP3-CoV2/RBD在MA104细胞上连续传代增殖的遗传稳定性.方法 将2株重组轮状病毒滴度测定后,按照MOI0.01传至P2代,随后按1∶100稀释并活化上一代病毒裂解液,连续18代(P20)感染MA104细胞,用间接免疫荧光法测定P1,P5,P10,P15,P20代细胞裂解液病毒滴度,进行RT-PCR鉴定以及dsRNA-PAGE银染实验,并检测rLLR/NSP3-NLuc的荧光素酶活性.结果 重组轮状病毒rLLR/NSP3-NLuc在20代内均未发现插入片段丢失,重组病毒滴度为3.85～5.16×106 FFU/ml,各代次均有较强的荧光素酶信号.重组轮状病毒rLLR/NSP3-CoV2/RBD在第6代出现了插入片段的丢失,该重组病毒前5代的感染性滴度为:2.6～3.36×106 FFU/ml.结论 NSP3基因末端插入582 bp NLuc的重组轮状病毒具有很好的遗传稳定性,而NSP3基因末端插入885 bp RBD的重组轮状病毒仅在前5代稳定,提示重组轮状病毒NSP3基因末端可以插入和表达外源基因,但其稳定性需要更深入研究.
Abstract
Objective To analyze the genetic stability of two recombinant rotaviruses(rLLR/NSP3 NLuc)and(rLLR/NSP3 CoV2/RBD)that are inserted and express exogenous genes for continuous passage and proliferation on MA 104 cells.Methods After measuring the titers of two recombinant rotavirus strains,they were transferred to the P2 generation according to MOI0.01.Subsequently,the previous generation of virus lysate was diluted and activated at 1∶100,and MA 104 cells were continuously infected for 18 generations(P20).The virus titers of the P1,P5,P10,P15,and P20 generation of cell lysate were measured using indirect immunofluorescence,and RT-PCR identification and dsRNA PAGE silver staining were performed.The luciferase activity of rLLR/NSP3-NLUc was also detected.Results No inserted fragment loss was found in the recombinant rotavirus rLLR/NSP3 NLuc within 20 generations,with recombinant virus titers ranging from 3.85～5.16 × 106 FFU/ml,with strong luciferase signals in each generation.The recombinant rotavirus rLLR/NSP3 CoV2/RBD showed loss of inserted fragments in the 6th generation,with infectivity titers ranging from 2.6 to 3.36 in the first 5 generations of the recombinant virus × 106 FFU/ml.Conclusions The recombinant rotavirus with 582 bp NLuc inserted at the end of the NSP3 gene has good genetic stability,while the recombinant rotavirus with 885 bp RBD inserted at the end of the NSP3 gene was only stable in the first 5 generations,indicating that foreign genes can be inserted at the end of the NSP3 gene of the recombinant rotavirus,and the insert can express,but its stability requires more in-depth research.
关键词
重组轮状病毒/MA104细胞/遗传稳定性Key words
Recombinant rotavirus/MA104 cells/Genetic stability引用本文复制引用
出版年
2024
NETL
NSTL
万方数据