结核分枝杆菌H37Rv结合肽的筛选研究

目的 应用噬菌体展示随机7肽文库,筛选MTB标准株H37Rv的结合肽,并初步鉴定其与MTB的结合能力.方法 以H37Rv灭活菌体为靶分子,应用噬菌体展示随机7肽文库进行筛选,并于第2～4轮的筛选中加入耻垢分枝杆菌灭活菌体进行反筛,经4轮生物淘选后,随机选取单噬菌体进行测序,间接酶联免疫吸附测定(ELISA)法鉴定阳性克隆.将亲和性最强的阳性单噬菌体所展示的短肽进行体外合成及荧光标记,观察其与16种分枝杆菌标准株及3种非分枝杆菌(铜绿假单胞菌、金黄色葡萄球菌和白色念珠菌)的结合活性.结果 通过4轮生物淘选,能与靶分子特异性结合的噬菌体得到明显富集.单噬菌体测序分析共获得5种共同序列.间接ELISA检测出5个单噬菌体均为阳性克隆,其中单噬菌体H8与H37Rv及耻垢分枝杆菌的亲和性均较强.荧光显微镜下观察到,荧光标记H8可与H37Rv结合,与包括耻垢分枝杆菌在内的其他15种分枝杆菌有一定亲和力,而与3种非分枝杆菌均不结合.结论 利用噬菌体展示随机肽库技术淘选可获得MTB标准株H37 Rv 的结合肽,与MTB的结合活性及特异度较高,这为以标记肽为基础探索新的MTB体外检测方法提供了思路.

Abstract：

Objective To screen the Mycobacterium tuberculosis H37Rv binding peptide using phagedisplayed random peptide libraries, and to analyze the binding capacity of the peptide with Mycobacterium tuberculosis. Methods lnactive Mycobacterium tuberculosis H37 Rv was used for screening of the binding peptide from the Ph. D. -7 peptide library, and Mycobacterium smegmatis was used for reverse screening during the 2nd to 4th rounds of screening. After 4 rounds of screening, single phages were randomly selected for DNA sequencing. The selected clones were tested by indirect enzyme linked immunosorbent assay (ELISA). The peptide of positive clone, which showed the highest affinity, was synthesized in vitro with fluorescent markers. The specific combination of the peptide with 16 mycobacterium standard strains and 3 other microbes ( Pseudomonas aeruginosa, Staphylococcus aureus and Candida albicans) were observed by fluorescence microscopy. Results After 4 rounds of biopanning, remarkable enrichment of the phages that specifically bind with target molecules were observed. Single phages were randomly selected for sequencing analysis and 5 sequences were obtained. Five phages with different sequences were detected using indirect ELISA and all of them were found to be positive clones. Phage 8 showed the highest affinity with target molecule. The peptide of phage H8 was synthesized in vitro with fluorescent markers, and it was confirmed that the peptide could bind with H37Rv and other 15 mycobacterium including Mycobacterium smegmatis, but not with 3 other microbes. Conclusions By using phage-displayed random peptide libraries, we obtained the binding peptide of H37 Rv. It was shown that the peptide could bind with Mycobacterium tuberculosis specifically, which provided a new way for the detection of Mycobacterium tuberculosis in vitro.

Screening of the binding peptides of Mycobacterium tuberculosis H37 Rv

Objective To screen the Mycobacterium tuberculosis H37Rv binding peptide using phagedisplayed random peptide libraries, and to analyze the binding capacity of the peptide with Mycobacterium tuberculosis. Methods lnactive Mycobacterium tuberculosis H37 Rv was used for screening of the binding peptide from the Ph. D. -7 peptide library, and Mycobacterium smegmatis was used for reverse screening during the 2nd to 4th rounds of screening. After 4 rounds of screening, single phages were randomly selected for DNA sequencing. The selected clones were tested by indirect enzyme linked immunosorbent assay (ELISA). The peptide of positive clone, which showed the highest affinity, was synthesized in vitro with fluorescent markers. The specific combination of the peptide with 16 mycobacterium standard strains and 3 other microbes ( Pseudomonas aeruginosa, Staphylococcus aureus and Candida albicans) were observed by fluorescence microscopy. Results After 4 rounds of biopanning, remarkable enrichment of the phages that specifically bind with target molecules were observed. Single phages were randomly selected for sequencing analysis and 5 sequences were obtained. Five phages with different sequences were detected using indirect ELISA and all of them were found to be positive clones. Phage 8 showed the highest affinity with target molecule. The peptide of phage H8 was synthesized in vitro with fluorescent markers, and it was confirmed that the peptide could bind with H37Rv and other 15 mycobacterium including Mycobacterium smegmatis, but not with 3 other microbes. Conclusions By using phage-displayed random peptide libraries, we obtained the binding peptide of H37 Rv. It was shown that the peptide could bind with Mycobacterium tuberculosis specifically, which provided a new way for the detection of Mycobacterium tuberculosis in vitro.