Construction and application of a fluorescent expression cell model of botulinum toxin type B light chain
10.7644/j.issn.1674-9960.2023.12.005
- VernacularTitle:B型肉毒毒素轻链荧光表达细胞模型的构建及应用
- Author:
Ling TAN
1
;
Chensi ZHU
;
Baogang WANG
;
Tao LI
;
Hui WANG
Author Information
1. 军事科学院军事医学研究院微生物流行病研究所病原微生物生物安全国家重点实验室,北京 100071
- Keywords:
botulinum toxin type B light chain;
genetic recombination;
recombinant fluorescent plasmid;
Neuro-2a cells
- From:
Military Medical Sciences
2023;47(12):907-912
- CountryChina
- Language:Chinese
-
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.
