蜂毒肽类似物的基因表达与构效关系研究

根据先前建立的蜂毒肽QSAR模型预测，为获得溶血活性相对最低，保留或提高抑菌活性的蜂毒肽类似物。设计了六条蜂毒肽类似物基因，采用P. pastoris表达系统，通过构建 pPICZα-A-Mel重组质粒，电击转化酵母。以α-factor为分泌信号，在GS115中分泌表达。以MDH、MMH筛选，诱导培养，6×His亲和层析纯化表达产物, Tricine-SDS-PAGE电泳证实。新设计的六条类似物基因表达后抑菌活性与天然序列蜂毒肽相比，四条增强，两条减弱。类似物【C】抑菌效价(U)为1.29，是重组天然序列蜂毒肽的1.6倍左右，活性最强，而重组天然序列蜂毒肽为0.7977。类似物【C】溶血活性比天然序列蜂毒肽降低至1/25。类似物【B】的表达量最高，为0.31mg/mL，其抑菌活性仅次于【C】。两种类似物的溶血活性降低至天然序列蜂毒肽的1/25左右，两种降低至1/20左右，还有两种降低至1/15左右。对蜂毒肽分子结构的优化设计合理，达到了保留或提高抑菌活性，降低溶血活性的目的。

Research on Gene Expression and Structure-Activity Relationship of Melittin Anologues

According to the prediction by the QSAR Model established previously for melittin, in order to obtain the melittin analogues with relatively lower hemolytic activity and with inhibitory activity retained or elevated, six genes of the melittin analogues were designed. The P. pastoris was applied as the expression system. The recombinant plasmid of pPICZα-A-Mel was constructed, and transformed into the yeast by electric shock for the secretory expression in the P. pastoris GS115 when α-factor is used as the secretory signal. The recombinants were screened with MDH and MMH, and induced. The expression products were purified by 6×His affinity chromatography, and confirmed by Tricine-SDS-PAGE. The result shows that, among the expression products of the six newly designed genes, four of them shows stronger inhibitory activity on bacteria than that of melittin with natural sequence, and two of them shows weaker activity than that. The Inhibition titer(U)of Analogue【C】is 1.29, which is 1.6 times of that of melittin with natural sequence, as the strongest one, whereas the melittin with recombinated natural sequence is 0.7977. Its hemolytic activity reduced to 1/25 of that of the melittin with natural sequence. The expression level of Analogue【B】is the highest, at 0.31mg/mL, whose inhibitory activity is only second to【C】. The hemolytic activity of two analogues reduces to 1/25 of that of the melittin with natural sequence, that of another two anologues reduces to 1/20, and that of the other two anologues reduces to 1/15 approximately. To sum up, the optimization of the melittin molecular design is reasonable, which has achieved the target of retaining or elevating the inhibitory activity, and decreasing the hemolytic activity.