青蛙皮肤活性肽QUB2984的重组表达及体外活性评价  

Recombinant expression and in vitro activity identification of a bioactive peptide QUB2984 from skin secretion of Agalychnis callidryas

作　　者：唐子琰 顾顺强 陈小玲 王蕾 马成帮 周玫 陈天豹[3] 杜丽娜 金义光 Tang Ziyan;Gu Shunqiang;Chen Xiaoling;Wang Lei;Ma Chengbang;Zhou Mei;Chen Tianbao;Du Lina;Jin Yiguang(Institute of Radiation Medicine,Academy of Military Medicine,Academy of Military Science of Chinese PLA,Beijing 100850,China;Department of Emergency,Ningxia Armed Police Corps Hospital,Yinchuan 750000,Ningxia Hui Autonomous Region,China;Natural Drug Discovery Group,School of Pharmacy,Queen’s University Belfast,Belfast BT71NN,UK)

机构地区：[1]军事科学院军事医学研究院辐射医学研究所,北京市100850 [2]武警宁夏总队医院急诊科,宁夏回族自治区银川市750000 [3]英国贝尔法斯特女王大学药学院天然药物研究组,英国贝尔法斯特BT71NN

出　　处：《中国组织工程研究》2024年第17期2675-2681,共7页Chinese Journal of Tissue Engineering Research

摘　　要：背景:青蛙活性肽有丰富活性,如抗菌、抗肿瘤,有望解决抗生素耐药性问题。目的:从红眼树蛙皮肤分泌物中发现活性肽QUB2984,采用生物信息学手段模拟其结构和性质,合成、纯化、鉴定该肽并考察其生物学功能。方法:采用电刺激得到红眼树蛙皮肤分泌物,分离出mRNA构建cDNA文库,测序得到肽序列。用生物大分子序列搜索工具进行肽序列比对,蛋白质结构模型和功能预测工具开展蛋白二级结构模拟。通过固相合成法合成多肽、反向高效液相进行纯化、基质辅助激光解吸电离飞行时间质谱进行验证。所获得的纯肽用于生物活性评价,采用最小抑菌浓度法评价其抗菌作用,MTT法检测其细胞毒作用,溶血实验考察其安全性。结果与结论:①所得活性肽QUB2984的结构预测为α-螺旋,有完整的疏水面,对生物膜有一定破坏能力,其序列上第3个氨基酸位置由W组成,并具有G-X-G结构;②活性肽QUB2984对革兰阳性金黄色葡萄球菌的最小抑菌浓度为2μmol/L,对革兰阴性大肠杆菌的最小抑菌浓度为2μmol/L,对真菌白色念珠菌的最小抑菌浓度为8μmol/L;③活性肽QUB2984在10^(-5)mol/L浓度下对人非小细胞肺癌细胞NCI-H838具有明显抑制作用,在64μmol/L浓度下对马红细胞的溶血作用为50%;④结果显示,QUB2984具有抗菌抑癌活性,其带+3正电荷,有利于与细菌或细胞接触。BACKGROUND:Frog active peptides have rich activities,such as antibacterial and anti-tumor,and are expected to solve the problem of antibiotic resistance.OBJECTIVE:The active peptide QUB2984 was discovered in the skin secretions of Agalychnis callidryas.Its structure and properties were simulated by bioinformatics.The peptide was synthesized,purified,and identified and its biological functions were investigated.METHODS:Agalychnis callidryas skin secretions were collected by electrostimulation.The sequence of QUB2984 was obtained through constructing a cDNA library with isolated mRNA.BLAST was used for peptide sequence alignment.Besides that,Iterative Threading ASSEmbly Refinement(I-TASSER)and HeliQuest tools were used for protein secondary structure simulation.It was synthesized by solid-phase peptide synthesis,purified by reverse-phase high-performance liquid chromatography,and structurally confirmed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.The purified peptide was used to evaluate its biological activity.Its antibacterial effect was evaluated by the minimum inhibitory concentration method.Its cytotoxic effect was detected by MTT assay.Its safety was investigated by a hemolysis test.RESULTS AND CONCLUSION:(1)Peptide QUB2984 had basicallyα-spiral structure,with a relatively intact hydrophobic surface,and a certain destructive ability to biofilm.The third amino acid position of QUB2984 was composed of W and had a G-X-G structure.(2)The minimum inhibitory concentration of QUB2984 against gram-positive Staphylococcus aureus was 2μmol/L,the minimum inhibitory concentration against gram-negative Escherichia coli was 2μmol/L,and the minimum inhibitory concentration against the fungus Candida albicans was 8μmol/L.(3)The active peptide QUB2984 had obvious inhibitory effect on human non-small cell lung cancer cells NCI-H838 at 10^(-5) mol/L concentration,and the hemolytic effect on horse red cells at 64μmol/L concentration was 50%.(4)The results showed that QUB2984 had anti-bacte

关 键 词：生物活性肽 生物合成 结构模拟 构效关系 分离纯化 抗菌 抑癌 溶血评价 

分 类 号：R459.9[医药卫生—治疗学] R318.08[医药卫生—临床医学] R931