军事医学2023，Vol.47Issue(12)：907-912,6.DOI:10.7644/j.issn.1674-9960.2023.12.005
B型肉毒毒素轻链荧光表达细胞模型的构建及应用
Construction and application of a fluorescent expression cell model of botulinum toxin type B light chain
摘要
Abstract
Objective To construct a fluorescent expression cell model of botulinum toxin type B light chain(BoNT/B,BLC),and evaluate the effect of small molecule compounds with this model.Methods The BLC gene was inserted into the fluorescent expression vector pEGFP-N1 to construct a recombinant plasmid before being transfected into the neural cell line Neuro-2a cells for expression.The fluorescence expression level of BLC-EGFP protein in the cells was observed under a fluorescence microscope,and the enzyme digestion activity and stability of BLC-EGFP in the cells were detected by Western blotting.Furthermore,the model was used to evaluate the effect of SRC kinase inhibitor KX2-391 on the intracellular stability of BLC-EGFP protein.Results The recombinant expression plasmid pEGFP-N1-BLC was constructed.It was found that the expression level of BCL-EGFP protein in Neuro-2a cells gradually increased over time,and that the intracellular substrate vesicle-associated membrane protein-2(VAMP-2)was cleaved after plasmid transfection.CHX was added to terminate protein synthesis after the plasmid was transfected for 12 h,and the intracellular level of BLC-EGFP did not change significantly within 72 h.Twenty-four hours of treatment with KX2-391 could significantly promote the intracellular degradation of BLC-EGFP protein.Conclusions A cell model of fluorescent expression of botulinum toxin type B light chain has been established,which provides a technical reserve for the subsequent study onthe intracellular persistence mechanism and intracellular antidote screening of botulinum toxin type B light chain.关键词
B型肉毒毒素轻链/基因重组/重组荧光质粒/Neuro-2a细胞Key words
botulinum toxin type B light chain/genetic recombination/recombinant fluorescent plasmid/Neuro-2a cells分类
医药卫生引用本文复制引用
谭玲,朱晨思,王保刚,李涛,王慧..B型肉毒毒素轻链荧光表达细胞模型的构建及应用[J].军事医学,2023,47(12):907-912,6.基金项目
全国重点实验室自主面上课题(SKLPBS2223) （SKLPBS2223）
