With the sequence of the vasoactive intestinal peptiepeptide (VIP) from humans and according to the condon bias of Pichia pastoris, we designed PCR primers of VIP and obtained the sequence of VIP by SOE-PCR. Then VIP gene was cloned into Pichia pastoris secretory expression vector and the cell secretary system GS115-pPICZαA-vip was constructed. The recombinant strain was induced by methanol for 96 hours, and we collected the supernatant and identified the VIP by mass spectrometry. The molecular weight of VIP was consistent with theoretical molecular weight. The final result showed that the target peptide VIP was successfully expressed. The experimental investigations of agarose gel diffusion revealed that the recombinant expression modified VIP had relatively strong antibacterial activity to E. coli ATCC25922 and S. aureus ATCC25923. The minimal inhibitory concentration (MIC) of VIP to E. coli ATCC25922 and S. aureus ATCC25923 was 8 mmol/L and 16 mmol/L. Further cytotoxicity and hemolytic experiments indicated that recombinant VIP was non-toxic to normal cells NCM460 and IPEC-J2, had little hemolysis activity to SD rat erythrocytes. Meanwhile, by transmission electron microscopy, we found that VIP mainly inhibited bacteria by disrupting the cell membrane. These experiments established a useful system for further studies, application and mass production of antimicrobial peptide VIP.
基于人抗菌肽VIP (Vasoactive intestinal peptide) 基因序列，按照毕赤酵母密码子偏好性设计引物；用SOE-PCR 法扩增目的基因；然后将目的基因克隆至毕赤酵母分泌型表达载体pPICZαA 上，构建VIP 分泌表达菌株GS115-pPICZαA-vip。用甲醇诱导96 h 收集上清，用质谱进行鉴定，结果显示分泌表达产物与人抗菌肽VIP理论值 (3 326.82 Da) 完全一致，表明人抗菌肽VIP 成功得到分泌表达。琼脂糖凝胶扩散法实验结果显示，重组VIP 对大肠杆菌Escherichia coli ATCC25922 和金黄色葡萄球菌Staphylococcus aureus ATCC25923 都有很强的抗菌活性，MIC (Minimal inhibitory concentration) 分别为8 mmol/L 和16 mmol/L。进一步细胞毒性和溶血性实验结果显示，重组VIP 对正常细胞NCM460 和IPEC-J2 没有毒性，其对SD 大鼠红细胞不具有溶血活性。通过透射电镜观察了VIP 的抗菌机制，结果显示VIP 主要通过破坏细胞膜的方式抑杀细菌。本研究为人抗菌肽VIP 的开发应用和大量生产奠定了基础。.
Keywords: Pichia pastoris; antibacterial activity; antimicrobial peptide; gene cloning; secretory expression.