基于天蚕素A和胡蜂毒素的杂合肽设计及活性鉴定

Design and Activity Identification of Hybrid Peptide Based on Cecropin A and Mastoparan

为了进一步改善胡蜂毒素(mastoparan,MP)的抗菌、抗肿瘤活性并减少细胞毒性,本研究通过分子杂合方法对其进行改造,并初步鉴定设计的杂合多肽的生物活性。以天蚕素A的N-端1-8序列片段和胡蜂毒素的保守序列为模板进行杂合设计,获得一条新型杂合肽KL-21。以胡蜂毒素MP-L作为参照,采用生物信息学方法、微量倍比稀释法、杀菌动力学方法、溶血试验、CCK-8法分别对KL-21和MP-L进行理化参数分析、结构预测、最小抑菌浓度(MIC)和最小杀菌浓度(MBC)测定、杀菌动力学研究、溶血活性测定及肿瘤细胞毒性试验。结果表明,KL-21可以在短时间内迅速杀灭细菌,对革兰氏阳性菌(金黄色葡萄球菌)和阴性菌(大肠杆菌)的MIC均为4~8μg/mL,MBC均为8~16μg/mL;KL-21对真菌(白色念珠菌)的最小MIC为32μg/mL,MBC为64μg/mL。兔红细胞溶血试验表明,KL-21在128μg/mL时溶血活性仅为20%,较MP-L的溶血活性明显降低。细胞毒性试验表明,KL-21对肺癌细胞A549具有良好的抑制作用,16μg/mL的体外抑制率达86%。研究表明,新型杂合肽KL-21较MP-L的抗菌和抗肿瘤活性有较大提高,细胞毒性显著降低,可以作为先导肽进一步研究和开发。

In order to further improve mastoparan’s antibacterial and antitumor activities and reduce its cytotoxicity,the molecular modification was carried out by molecular hybridization,and the biological activity of the designed peptide was identified.A novel hybrid peptide KL-21 was obtained by using the N-terminal 1-8 sequences fragment of cecropin A and the conserved sequence of mastoparan.The physicochemical parameters and structures of KL-21 were analyzed by bioinformatics method.The antibacterial activity of KL-21 was studied with MP-L as reference.In addition,the hemolysis rate of the two peptides and the activity of anti-tumor cell A549 were measured.The results showed that KL-21 could quickly kill bacteria in a shorter time.The minimum inhibitory concentration(MIC)of KL-21 to Gram-positive bacteria(S.aureus)and Gram-negative bacteria(E.coli)was 4-8μg/mL,and the minimum bactericidal concentration(MBC)was 8-16μg/mL.The MIC and MBC of KL-21 to fungus(C.albicans)were 32 and 64μg/mL,respectively.The hemolytic activity of KL-21 was only 20%at 128μg/mL,which was lower than that of MP-L.KL-21 could inhibit lung cancer cell A549 with inhibition rate in vitro about 86%at 16μg/mL.These results indicated that KL-21 had higher antibacterial and antitumor activity and lower cytotoxicity than MP-L,which could be used as a leading peptide for further research and development.