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目的:设计、表达两个A型肉毒毒素多表位串联体。方法:从文献报道的A型肉毒毒素(botulinum neuro-toxin type A,BoNT/A)的表位及生物信息学方法预测得到的B细胞表位中遴选表位,并加入适当的辅助性元件,设计多表位串联体A、B。对其基因进行优化后,经重叠PCR方法合成串联体A、B的全长基因。分别插入原核表达载体pQE-30,再转化E.coliM15[pREP4]感受态细胞中进行诱导表达,以金属螯合亲和层析法纯化重组蛋白,并进行鉴定。结果与结论:成功设计并构建了两个多表位串联体A、B,并在E.coli中以包涵体形式获得高效表达。Ni-NTA法纯化后分别获得纯度大于92.2%、99%的重组串联体A、B蛋白,并经透析复性法复性。Western印迹和间接ELISA显示纯化、复性后的重组蛋白与抗天然BoNT/A马血清有特异性结合反应。
Objective: To design and express two Botulinum Toxin A multi-epitope tandem bodies. METHODS: The selected epitopes were selected from epitopes predicted by the reported botulinum neuro-toxin type A (BoNT / A) and bioinformatics methods, and the appropriate accessory elements , Design of multi-epitope tandem A, B. After optimizing their genes, the full-length genes of A and B were synthesized by overlapping PCR. Were inserted into the prokaryotic expression vector pQE-30, and then transformed into E. coliM15 [pREP4] competent cells for induction of expression, metal chelate affinity chromatography purified recombinant protein, and identified. RESULTS AND CONCLUSION: Two multi-epitope concatemers A and B were successfully designed and constructed, and highly expressed in E.coli as inclusion bodies. After purification by Ni-NTA, the recombinant A, B proteins with purity over 92.2% and 99%, respectively, were obtained and refolded by dialysis refolding method. Western blotting and indirect ELISA showed that the purified and renatured recombinant protein reacted specifically with anti-native BoNT / A horse serum.