Polypeptide sequence for identifying spores of Bacillus anthracis, and application of polypeptide sequence
A technology of bacillus anthracis and polypeptide sequences, which is applied in the direction of microorganism-based methods, microorganism measurement/testing, lyase, etc., and can solve the problem of unknown plyG
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Embodiment 1
[0034] Acquisition of NVRTHQSWSGKYCPHRMLAEG polypeptide (SBD1) capable of recognizing anthrax spores:
[0035] The full-length sequence of the previously published plyG protein is as follows:
[0036] MEIQKKLVDPSKYGTKCPYTMKPKYITVHNTYNDAPAENEVSYMISNNNEVSFHIAVDDKKAIQGIPLERNAWACGDGNGSGNRQSISVEICYSKSGGDRYYKAEDNAVDVVRQLMSMYNIPIE NVRTHQSWSGKYCPHRRMLAEG RWGAFIQKVKNGNVATTSPTKQNIIQSGAFSPYETPDVMGALTSLKMTADDFILQSDGLTYFISKPTSDAQLKAMKEYLDRKGWWYEVK.
[0037] Through in vitro truncated plyG protein experiments, it was identified that the 21 amino acids above the protein can recognize Bacillus anthracis spores, and the underlined part of the bottom is the polypeptide sequence (SBD1) corresponding to the 21 amino acids in the full-length amino acid sequence of the phage lyase plyG position on the
[0038] SBD1 has the ability to bind to spores of Bacillus anthracis.
[0039] In order to further confirm the specificity and affinity of the polypeptide sequence for binding to the spores of Ba...
Embodiment 2
[0043] Verification of SBD1 polypeptide recognition of anthrax spores:
[0044] (1) Anthrax spore pretreatment:
[0045] In this experiment, formaldehyde-inactivated anthrax spores were used as experimental samples. The inactivated anthrax spores were blocked with PBSM (1×PBS, 5% skimmed milk) at 37°C for 2 h, washed 3 times with PBST (1×PBS, 0.05% Tween-20) by centrifugation (12000 g, 1 min), and dissolved in PBS.
[0046] (2) Staining and fluorescence microscope analysis of anthrax spores:
[0047] The treated anthrax spores were incubated with FITC-SBD and FITC at the same molar concentration for 30min-1h at room temperature, washed with PBST (1×PBS, 0.05% Tween-20) for 3 times by centrifugation (12000g, 1min), and dissolved in phosphate buffer. An appropriate amount of anthrax spores incubated with FITC-SBD was spread on a glass slide, and the fluorescence of Bacillus anthracis spores was observed with a fluorescence microscope. At the same time, using the anthracis s...
Embodiment 3
[0055] Polypeptides recognizing Bacillus anthracis spores containing longer plyG amino acid sequences were obtained:
[0056] On the SBD1 sequence, by adding more amino acid sequences on plyG, it is possible to obtain a better polypeptide capable of recognizing anthrax spores. In this embodiment, only one column is used for illustration. In this example, by fusing the green fluorescent protein (EGFP) gene with the plyG gene containing the polypeptide gene fragment of interest, and then expressing it in Escherichia coli, it becomes a polypeptide with a fluorescent protein marker. The polypeptide labeled with the fluorescent protein is used for the identification of bacillus anthracis spores.
[0057] This embodiment takes as an example the fusion protein EP6 which is fused with a polypeptide with a length of 60 amino acid sequences (106-165, p60) and EGFP.
[0058] MEIQKKLVDPSKYGTKCPYTMKPKYITVHNTYNDAPAENEVSYMISNNNEVSFHIAVDDKKAIQGIPLERNAWACGDGNGSGNRQSISVEICYSKSGGDRYYKAE DNAV...
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