结核分枝杆菌环七肽配体分子的筛选和鉴定

Screening and identification of C7C peptide ligands of Mycobacterium tuberculosis

目的 基于噬菌体展示随机环七肽库,通过亲和筛选获得结核分枝杆菌（MTB）环七肽配体分子,初步鉴定其与分枝杆菌的结合能力,以优化纳米检测.方法 以MTB标准株H37Rv灭活菌体为靶分子,卡介苗（BCG）为反筛分子,对噬菌体展示随机环七肽文库进行筛选.4轮淘选后,挑选H37Rv和BCG洗脱单噬菌体进行测序,ELISA检测单噬菌体与其亲和力将高亲和力单噬菌体所展示环肽进行体外合成,并标记荧光,荧光显微镜与流式细胞仪检测合成环肽的结合活性,并与前期获得的线性结合肽进行比较.结果 经过4轮亲合淘选,能与靶分子结合的噬菌体明显富集.单噬菌体测序共获得16种共同序列.ELISA检测显示,单噬菌体SB1、SB5、SB8、SB26与H37Rv和BCG的亲和力均较高,其吸光度值（A450）与阴性对照吸光度值（A450）比≥2.1,与3种非分枝杆菌不结合,确定为阳性克隆.流式细胞仪检测结果显示,H37Rv与环肽SB1、SB5、SB24、SB26结合的阳性细胞率分别为（73.2±6.3）％、（63.2±5.3）％、（32.9±3.1）％、（89.4±7.0）％;BCG与这4种环肽结合的阳性细胞率分别为（65.6±6.1）％、（48.6±4.5）％、（10.3±1.8）％、（86.6±7.9）％,H37Rv和BCG与4种环肽结合的阳性细胞率均高于H8[（4.0±1.0）％、（5.5±1.2）％].荧光显微镜观察结果显示,环肽SB1、SB5、SB24、SB26荧光肽均与H37Rv发生结合,荧光强度较强,与其他18种分枝杆菌均有一定的亲和力,但荧光强度弱于H37Rv,与3种非分枝杆菌均不结合.结论 本研究以环七肽库替代线性七肽库,成功筛选获得新的MTB配体分子,其与分枝杆菌的亲和力提高.

Objective To obtain the C7C peptide ligands of Mycobacterium tuberculosis by affinity screening based on the phage-displayed random C7C peptide library,and preliminarily identify the binding capacity of the peptide to Mycobacterium.Methods Inactive Mycobacterium tubercalosis reference strain H37Rv was used as the target molecule to screen the Ph.D.-C7C peptide library,and Mycobacterium boris,BCG was used for reverse screening.After 4 rounds of affinity screening,single phages eluted by H37Rv and BCG were selected for DNA sequencing.ELISA was used to detect the binding affinities of different single phage clones.The cyclic peptides displayed by the phage clones showing the highest appetency were synthesized in vitro with fluorescent markers.Fluorescence microscopy and flow cytometer was used to detect the binding affinities of synthesized cyclic peptides,comparing with linear binding peptides obtained before.Results After 4 rounds of biopanning,phages that could bind with target molecules were remarkable enriched.16 common sequences were obtained by sequencing analysis of single phages.With ELISA,phage SB1,SB5,SB8 and SB26 all showed higher affinity with H37 Rv and BCG,the ratio to negative control of which were ≥ 2.1,but could not bind to the 3 nonmycobacteria,which were identified as the positive clones.Based on the results of flow cytometer detection,the affinities to H37 Rv of 4 cyclic peptides SB1,SB5,SB24,SB26 were （73.2±6.3）％,（63.2±5.3）％,（32.9±3.1）％,（89.4±7.0）％,and to BCG were （65.6 ±6.1）％,（48.6 ±4.5）％,（10.3 ± 1.8）％,（86.6 ±7.9）％,separately,which were all higher than H8 （（4.0 ± 1.0）％,（5.5 ± 1.2）％）.From the results of fluorescence microscopy observation,all of the fluorescent labeled cyclic peptides SB1,SB5,SB24,5B26 could bind to H37Rv and showed higher fluorescence intensities,which also had certain affinities to other 18 mycobacteria,but the fluorescence intensities were lower than H37Rv,and didn＇t bind to 3 non-mycobacteria.Conclusion Based on the replacement of linear 7 peptide library with C7C peptide libra,,new ligands of Mycobacterium tuberculosis were achieved successfully,which showed significantly higher binding affinities to mycobacteria.