Qiao Xiangjin, Li Wenxin, Bai Lijuan, Hu Wei, Nan Huaiyan
Lanzhou Lanshi Energy Equipment Engineering Institute CO., LTD, Lanzhou 730300, Gansu, China.
Northwest Institute of Eco-environment and Resource, Chinese Academy of Sciences, Lanzhou 730000, Gansu, China.
Sheng Wu Gong Cheng Xue Bao. 2018 Jun 25;34(6):1002-1011. doi: 10.13345/j.cjb.170506.
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.
根据人血管活性肠肽（VIP）的序列，并依据巴斯德毕赤酵母的密码子偏好性，设计了VIP的PCR引物，通过SOE-PCR获得了VIP序列。然后将VIP基因克隆到巴斯德毕赤酵母分泌表达载体中，构建了细胞分泌系统GS115-pPICZαA-vip。用甲醇诱导重组菌株96小时，收集上清液并通过质谱鉴定VIP。VIP的分子量与理论分子量一致。最终结果表明目标肽VIP成功表达。琼脂糖凝胶扩散实验研究表明，重组表达的修饰VIP对大肠杆菌ATCC25922和金黄色葡萄球菌ATCC25923具有较强的抗菌活性。VIP对大肠杆菌ATCC25922和金黄色葡萄球菌ATCC25923的最小抑菌浓度（MIC）分别为8 mmol/L和16 mmol/L。进一步的细胞毒性和溶血实验表明，重组VIP对正常细胞NCM460和IPEC-J2无毒，对SD大鼠红细胞溶血活性很小。同时，通过透射电子显微镜观察发现，VIP主要通过破坏细胞膜来抑制细菌。这些实验为抗菌肽VIP的进一步研究、应用和大规模生产建立了一个有用的系统。
