Soluble expression of reconstructed heavy chain fragment of botulinum neurotoxin serotype A in Escherichia coli / 解放军医学杂志

ABSTRACT

Objective To clone the reconstructed Hc gene of botulinum neurotoxin type A(BoNT/AHc)and to explore the soluble expression of the reconstructed gene in E.coli.Methods The gene of Hc fragment was synthesized by replacing rare codons with frequent ones in E.coli,while the components of amino acids didn't change,and the contents of AT decreased from 76.4 % to 57.3%.The reconstructed gene was then cloned into the prokaryotic expression vector pQE-60.The recombinant plasmid pQE-60Hc was introduced into E.coli Origami(DE3)that was induced to express the protein.The soluble expression products were then detected by Western Blot.Finally the expressed product of recombination plasmid pQE-60Hc was analyzed with SDS-PAGE after purification through Ni-NTA column.Results The reconstructed Hc gene of BoNT/AHc was amplified by PCR.The expression vector pQE-60Hc was constructed successfully with BamH Ⅰ and Nco Ⅰto ingest both BoNT/AHc and vector pQE-60.Reconstructed gene could be expressed effectively in E.coli in soluble form.The molecular weight of expressed product of recombination plasmid pQE-60Hc analyzed by SDS-PAGE was 52 000,which was the same as anticipated.And the soluble expression product accounted for to 11.5 % of the total bacterial protein.Western blot assay showed that the expression product could bind to specific antibody agent BoNT/A.Conclusion The expression vector has been constructed and the reconstructed gene was expressed successfully and effectively in E.coli,which may provide a foundation for further study on antitoxin and vaccine.