A kind of antibacterial peptide and its preparation method and application
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, through nested PCR amplification (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 test design is first to extract Microsporum canis total RNA, then use dT3AP primer (CTGATCTAGAGGTACCGGATCCTTTTTTTTTTTTTTTTT-3 (SEQ ID NO: 4)); then carry out reverse transcription according to the following method: the reverse transcription condition is to get 10 μ l total RNA, 1 μ dT3AP (100 μ M), Immediately place on ice after 5 min incubation at 70°C; then add to RT-PreMix reaction tube. Synthesize cDNA at 42°C for 60 minutes, and finally inactivate reverse transcriptase at 94°C for 5 minutes. The reverse transcription kit RT-PreMix was provided by Beijing Saibaisheng Gene Technology Co., Ltd., and reverse transcription was performed according t...
Embodiment 2
[0057] The chemical synthesis of embodiment 2 antimicrobial peptides of the present invention
[0058] 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 folded products were separated and purified by reversed-phase high-performance liquid chromatography (RP-HPLC) to obtain high-purity and uniform antimicrobial peptides. details as follows:
[0059] (1) Test instruments and materials
[0060] 1. Raw materials: resin: 2-CL-TrtResin 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 (wherein the sulfhydryl group of the cysteine side chain is protected by Trt of amino acids).
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Embodiment 3
[0067] Confirmation of embodiment 3 antimicrobial peptides of the present invention
[0068] The antimicrobial peptides prepared in Example 2 were identified by matrix-assisted laser resolution 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.
[0069] And the antibacterial peptide that embodiment 2 is made is measured by nuclear magnetic resonance instrument (BrukerAvance600nmrspectrometer, InternationalEquipmentTradingLtdUSA), and nuclear magnetic resonance two-dimensional spectrum (nuclear Overhauser effect spectrum) is as follows Figure 3b Shown, and according to the data of nuclear magnetic resonance two-dimensional spectrum, by CYANA (Combined assignment and dynamics algorithm for NMR applications) software (Güntert, P. (200...
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