Antibacterial peptide, preparation method therefor and applications
A technology of antimicrobial peptides and precursors, applied in the field of biopharmaceuticals, to achieve the effect of broad application prospects
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Embodiment 1
[0047] Embodiment 1 Obtaining of the coding sequence of the antimicrobial peptide of the present invention
[0048] Using molecular biology techniques, amplified by nested PCR (see Goode et al., (2002) Nested RT-PCR .Methods in Molecular Biology.Volume 193, II, 65-79) to obtain the cDNA clone of the gene. The specific design of the experiment is to first extract the total RNA of Microsporum canis, and then use the dT3AP primer (CTGATCTAGAGGTACCGGATCCTTTTTTTTTTTTTTTTT-3 (SEQ ID NO: 4)); and then carry out reverse transcription according to the following method: reverse transcription conditions are 10 μl total RNA, 1 μl dT3AP ( 100 μM), immediately placed on ice after incubation at 70°C for 5 minutes; then added to the RT-PreMix reaction tube. 42°C for 60 minutes to synthesize cDNA synthesis, and finally 94°C for 5 minutes to inactivate reverse transcriptase. The reverse transcription kit RT-PreMix was provided by Beijing Saibaisheng Gene Technology Co., Ltd., and reverse t...
Embodiment 2
[0056] The chemical synthesis of embodiment 2 antimicrobial peptides of the present invention
[0057] Using the classic chemical synthesis method, we artificially synthesized the linear peptide of the antimicrobial peptide, that is, the precursor of the antimicrobial peptide (synthesized by Shanghai Qiangyao Biotechnology Co., Ltd.). Then, air oxidative folding was carried out under alkaline conditions (100-200mM Tris-HCl, pH8.0-8.3). The antimicrobial peptides with high purity and uniformity were obtained by separating and purifying the folded products by reversed-phase high-performance liquid chromatography (RP-HPLC). details as follows:
[0058] (1) Test instruments and materials
[0059] 1. Raw materials: Resin: 2-CL-Trt Resin with a degree of substitution of 0.3 (purchased from Tianjin Nankai Hecheng Technology Co., Ltd.); amino acid raw materials: various amino acids protected by Fmoc (the sulfhydryl group of the cysteine side chain is Trt protected amino acids)....
Embodiment 3
[0066] Confirmation of embodiment 3 antimicrobial peptides of the present invention
[0067] The antimicrobial peptides prepared in Example 2 were identified by matrix-assisted laser desorption tandem time-of-flight mass spectrometry (MAIDI-TOF), as Figure 3a As shown, its molecular weight is 4055.7Da, which is completely consistent with the theoretically calculated molecular weight. This result confirmed that the oxidation product formed three pairs of intramolecular disulfide bonds.
[0068] And the antimicrobial peptide prepared in Example 2 is measured by a nuclear magnetic resonance instrument (Bruker Avance 600nmr spectrometer, International Equipment Trading Ltd USA), and the nuclear magnetic resonance two-dimensional spectrum (nuclear Overhauser effect spectrum) is as follows Figure 3b shown, and according to the data of NMR two-dimensional spectrum, by CYANA (Combined assignment and dynamics algorithm for NMR applications) software (Güntert, P. (2004). Automated ...
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