抗菌肽模式肽PGYa的分子设计、克隆及在毕赤酵母中的分泌表达

在α-螺旋抗菌肽序列比较和两亲性分析的基础上，提取序列模板，计算机辅助（螺旋轮法）设计出新型抗菌肽模式肽PGYa(Peptide以Gly开头，以Tyr-NH2结尾)，然后选用毕赤酵母偏爱密码子，设计合成了PGYa基因（rPCR法）。所合成的基因全长为94bp，并在其N端引入kex2裂解位点，以保证表达抗菌肽具有天然N端。基因克隆入pPICZα-A质粒，构建分泌型酵母表达载体pPICZα-A-PGYa。在AOX1 (醇氧化酶)启动子调控下，PGYa蛋白获得分泌表达，其表达量达到132 mg/L。初步抑菌（E.coli DH5α）活性显示：PGYa有较好的抗菌活性。

Molecular Design and Cloning of Model Antimicrobial Peptide PGYa and Its Secreted Expression in Pichia pastoris

Based on sequence analogy and amphipathicity analyse of α-helical antimicrobial peptides, a sequence template was extracted. The model Antimicrobial Peptide PGYa(peptide beginning with Gly and ending with Tyr-NH2) was designed by virtue of the template and computer-aided design subsequently. Using the optimal condon of Pichia pastoris, PGYa gene with 94bp length were achieved through a recursive PCR strategy. Especially a Kex2 signal cleavage site was fused in 5’end of the antibacterial peptide gene to make sure the expression protein with nature N-terminal. The modified antibacterial peptide gene was then cloned into the pPICZα-A vector to construct the recombinant expression vector pPICZα-A- PGYa, and transformed into yeast host strain X-33. Under the control of the promoter AOX1(alcohol oxidase 1), the PGYa protein with a molecular weight of 2.8 KD was expressed in supernatant and the amount of secreted pertein can reach 132 μg/mL. Preliminary antibacterial assays showed that PGYa has a potent antibacterial activity against E.coli DH5α.