Construction of Recombinant Protein of Botulinum Neurotoxin Light Chain and Analysis of Cleavage Effect on VAMP1 Protein in Brown Rat

In order to explore the feasibility of the new rodenticide of type D Botulinum toxin to prevent and control the population density of pests in farmland from a molecular perspective. This study aimed to analyze the feasibility of D-type botulinum toxin for the control of urban rodents from a molecular perspective, and to detect the cleavage effect of D-type botulinum neurotoxin light chain recombinant protein on the synaptic vesicle membrane protein VAMP-1 of brown house mouse. The genomic DNA of Clostridium botulinum D 8901 strain was used as a template. Based on the botulinum toxin D gene sequence reported in GenBank, specific primers were designed. Plasmids were constructed by gene synthesis and subcloned into the pET28a expression vector. The recombinant plasmid was transformed into BL21 (DE3) Rosetta competent cells, and induced expression by IPTG. The expression products were purified by Ni-NTA affinity chromatography, identified by SDS-PAGE and Western blotting, and the recombinant proteins were analyzed by SDS-PAGE and Western blotting. The cleavage of VAMP1 protein in SD brown house mouse. The pET-28a- BDLc expression plasmid was successfully constructed and transformed into E. coli BL21 (DE3) Rosetta. Western blot confirmed that the recombinant protein pET-28a- BDLc (residues Met 1-Met 94) obtained soluble expression, and obtained 5 mg, Recombinant protein with purity > 90%. It has good biological activity as determined by animal method. The recombinant protein can decompose VAMP1 protein into two fragments. Type D botulinum toxin protein can specifically cleave synaptic vesicle membrane protein (VAMP1) of brown house mouse, and type D botulinum toxin is feasible for urban rodent control.