蜂毒素基因的突变与表达及其构效关系研究

Design and Expression in Pichia pastoris of Melittin and Research of Antibacterial Activity Increasing of Melittin

为获得保留有抗菌活性而降低溶血活性作用的蜂毒素,对其分子结构进行了改造.其中一条序列缺失Lys-7,另一条缺失Leu-9.用PCR技术获得两条新的蜂毒素基因,将其克隆到酵母表达载体pPICZα-A,获得重组质粒pPICZα-A-Mel1和pPICZα-A-Mel2.将2个重组质粒分别转化毕赤酵母菌GS115.筛选获得重组酵母菌.对重组酵母菌培养条件进行优化,在YEPD中含甘油2%,pH为7,重组酵母菌培养48h后,0.5%的甲醇诱导72h,表达目的蛋白通过Tricion-SDS-PAGE确定分子质量为5.4kD.杀菌效价分别达0.947、1.085.结果表明两条蜂毒素基因成功的在毕赤酵母中表达,经突变后表达的蜂毒素降低了溶血活性,同时保留了抗菌活性.

In order to keep activity of melittin and reduce the hemolysis activity, the molecular structure was reformed. Lys of the seventh site was deleted in one sequence and Leu of the ninth site was deleted in another sequence. The two reformed melittin genes obtained by PCR were cloned into pPICZα- A, producing the recombinant expression plasmids pPICZα-A-Mel 1 and pPICZα-A-Mel 2, which were then transformed into Pichia pastoris GS115 to acquire the recombinant saccharomycetes. The culturing conditions of the recombinant saccharomycetes were optimized through a 48-hours culturing by the recombinant saccharomycetes in the 2.0% glucose contained YEPD, pH 7, and after which a 72 hours inducing by 0.5% methanol. The molecular mass of the expression target protein was 5.4 kD, determined by the Tricion-SDS-PAGE. And the antibacterial effects reach to the amount of 0. 947 and 1. 085 each, which illustrated that the two melittin genes achieved the expression in Pichia pastoris. The hemolysis activity of melittin, expressed after mutation, was depressed, albeit with the antibacterial activity maintained.