蜂毒素分子的改造及其基因在毕赤酵母中的表达

为获得保留有抗菌活性而降低溶血作用的蜂毒素,对蜂毒素的分子结构进行了改造。将第5位的Val变为Arg,第15位Ala变为Arg,删除了第16位的Leu。用PCR技术获得了改造后的蜂毒素基因,将其克隆入酵母表达载体pPICZa-A,获得重组表达质粒pPICZa-A-MEA。该质粒转化毕赤酵母菌GS115,甲醇诱导下表达,发酵上清液经抑菌活性、溶血活性测定及亲和层析纯化,结果表明,蜂毒素基因成功地在毕赤酵母中表达,经改造后表达的蜂毒素保留了抗菌活性且溶血活性显著降低,经纯化后用Bradford法测定表达蜂毒素的含量约为0.29mg／ml。

Design,Synthesis and Expression in Pichia pastoris of Melittin

In order to keep activity of melittin, and reduce the hemolysis activity, the molecular structure of melittin was reformed. Arg substituted Val and Ala on both the fifth and the fifteenth sites, respectively. Leu of the sixteenth site was deleted. The reformed melittin gene gotten by PCR was cloned into pPICZa-A, producing the recombinant expression plasmid pPIZa-A-MEA. Then, the vectors were transformed into Pichia pastoris GS115, and the clones were induced by methanol to express melittin. The supernatant of the fermentation was measured for antibacterial activity and hemolysis activity. The expression product was purified by Ni-NTA Purification. The results showed that melittin gene was expressed in Pichia pastoris successfully, and the reformed melittin kept antibacterial activity and decreased the hemolysis activity.The purified expression product in GS115 was measured by Bradford and the content of melittin was about 0.29mg/ml.