基于噬菌体展示结合肽和纳米探针的结核杆菌检测新方法分析-微生物学专业论文.docxVIP

优秀毕业论文 IV IV 精品参考文献资料 Research on new detection method of Mycobacterium tuberculosis based on the phage displayed binding peptide and nanoprobe Abstract Objective: To obtain the Mycobacterium tuberculosis(MTB) binding peptide with phage-displayed random peptide libraries，and to analyze the binding capacity of the peptides to MTB. Conjugating magnetic nanoparticles （MNPs）and Quantum dots (QDs) separately with MTB binding peptide to establish a new detection method of MTB based on immunomagnetic beads combined with functionalized fluorescent QDS. Method: MTB reference stain H37Rv was used as the target to screen the phage-displayed random C7C peptide libraries, with the biopanning process of absorption, elution and amplification. BCG was used as the counter selection molecule during the second and fourth selection. After 4 rounds of screening, 28 and 27 single phages separately eluted by H37Rv and BCG were randomly selected from various direction of the titration detection plates and amplified. Single-stranded DNA was extracted and sued for sequencing. Different phage clones were identified with indirect enzyme linked immunosorbent assay (ELISA). The peptides displayed by positive phage clones were synthesized in vitro and labeled with fluorescent molecules. The binding affinity and specificity of the peptide to H37Rv, BCG and 19 mycobacterium standard strains were observed with fluorescence microscopy and detected by flow cytometry. Using wet chemical preparation of magnetic nanoparticles (MNPs) and quantum dots (QDs), the H37Rv binding peptide H8 and purchased anti-MTB polyclonal antibody (Pab) were individually coupled with MNPs to obtain immunomagnetic beads MNP-H8 and MNP-Pab; the peptide H8, anti-MTB monoclonal antibody (Mab) and anti-heat shock protein 65 of MTB monoclonal antibody (Mabc) were separately conjugated with QDs to get three kinds of functionalized fl QDs uorescent QDs: QD-H8, QD-Mab and QD-Mabc. Immunomagnetic beads c