Selecting and affinity analysis of DNA aptamers to MPT64 protein from Mycobacterium tuberculosis

• Published in: Zhonghua jiehe he huxi zazhi Vol. 31; no. 6; p. 453
• Main Authors: Qin, Lian-Hua, Ma, Zhan-Zhong, Zheng, Rui-Juan, Liu, Zhong-Hua, Lu, Jun-Mei, Hu, Zhong-Yi
• Format: Journal Article
• Language: Chinese
• Published: China 01.06.2008
• Subjects: Antigens, Bacterial - genetics; Antigens, Bacterial - isolation & purification; Aptamers, Nucleotide - biosynthesis; Bacterial Proteins - genetics; Bacterial Proteins - isolation & purification; Base Sequence; Gene Library; Mycobacterium tuberculosis - genetics; Mycobacterium tuberculosis - isolation & purification; Nucleic Acid Conformation; SELEX Aptamer Technique - methods
• ISSN: 1001-0939

To obtain DNA oligonucleotide aptamers which can specifically bind to MPT64 protein from Mycobacterium tuberculosis by SELEX technology. An in vitro synthesized 78 per random DNA library was subjected to 12 rounds of selection by SELEX (Systematic evolution of ligands by exponential enrichment) method against MPT64 protein. Binding of the aptamers to the protein was examined by biotin-streptavidin-horseradish peroxidase system. DNAMAN package was employed to analyze the sequences and the second structures of the aptamers. Moreover, target protein was bound to one aptamer and another aptamer modified with biotin together forming a sandwich-like complex, which was captured in microwell, to be tested in negative group including BCG and reference strains from nontuberculous mycobacteria, and positive group including H37Rv, Mycobacterium bovis reference strain, and clinical strains from Mycobacterium tuberculosis. After 12 rounds of selection, high-affinity aptamers to MPT64 was obtained. The OD value at 450 nm of