A型肉毒毒素保护性抗原在酵母细胞中的表达与抗原性分析

目的：在酵母系统中实现A型肉毒毒素(BoNTa)Hc基因的可溶性分泌表达。方法：将Hc基因克隆入表达载体pPIC9K，用SacⅠ将重组质粒线性化，电转化巴氏毕赤酵母(Pichia pastoris)GS115细胞，筛选G418抗性阳性整合子，进行Mut^ 表型株甲醇快速利用诱导。结果：经过体外高拷贝整合子的筛选和诱导表达条件的优化，毒素Hc在pH6．5培养基BMMY中实现稳定的分泌表达，最终筛选到蛋白表达量占上清蛋白10％的分泌高表达株。免疫印迹和酶联免疫检测结果显示，重组BoNTa Hc可以识别特异抗肉毒马血清并具良好的特异结合活性，可以诱导小鼠产生特异性抗体。结论：酵母系统来源的具有良好免疫原性的重组BoNTa保护性抗原可以作为有效的候选重组疫苗分子。

Secretion expression of protective fragment of botulinum toxin A in Pichia pastoris

Objective: To express the protective fragment Hc of botulinum toxin A in Pichia pastoris. Methods:The Hc fragment of botulinum toxin A was cloned into yeast expression vector pPIC9K. Recombinant plasmid was lineared by SacI and transformed into Pichia pastoris GS115. Positive integrated clone was screened and expressed by methanol inducing. Results: The Hc fragment of botulinum toxin A was expressed at level of 10% of the supernatant protein after optimizing expression condition in pH 6.5 BMMY. Recombinant product was identified by Western blot and the specific binding activity to antiserum was showed in ELISA. Mice immunized by recombinant Hc could produce specific antibody to toxin. Conclusion: The protective fragment Hc of botulinum toxin A with good immunity could be a candidate of recombinant botulinum toxin vaccine.