人工改造的A型肉毒毒素重链Hc段基因在大肠杆菌中的可溶性表达研究

Soluble expression of reconstructed heavy chain fragment of botulinum neurotoxin serotype A in Escherichia coli

目的人工合成A型肉毒毒素Hc段基因,并在大肠杆菌中进行高效表达。方法人工合成Hc段基因,选择宿主细胞偏爱的同义密码子替换其原有稀有密码子,使AT含量降至57.3%,然后将其克隆入pQE-60表达载体,在大肠杆菌中进行可溶性表达。结果可溶性表达产物占细菌可溶性蛋白的11.5%左右,表达产物可被A型肉毒毒素马血清抗毒素识别。结论成功地表达并鉴定了A型肉毒毒素Hc蛋白,为进一步进行A型肉毒毒素的疫苗或抗体研究奠定了基础。

Objective To clone the reconstructed He gene of botulinum neurotoxin type A （BoNT/AHc） and to explore the soluble expression of the reconstructed gene in E. coll. 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 plasrnid 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 colunm. 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.