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an antibacterial protein
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An antibacterial protein and strain technology, applied in the field of biochemistry, can solve the problems of inconsistent raw material quality and chemical residues.
Active Publication Date: 2016-04-13
CHANGCHUN XINYUERAN SCI & TECH CO LTD
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Problems solved by technology
For chemical disinfectants, there are still problems of chemical residues, and for plant-extracted disinfectants, the quality of raw materials cannot be guaranteed, and chemical solvents are used in the extraction process, and there are also problems of certain chemical residues
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Embodiment 1
[0053] Example 1: LYS-ABP gene acquisition
[0054] Design the polypeptide sequence of LYS-ABP gene according to Genbank, and then design the LYS-ABP gene sequence according to the principle of optimal password, synthesize the LYS-ABP fusion protein gene sequence through the whole gene, and add EcoRI-HindIII enzyme digestion at both ends site, and the gene sequence is shown in SEQ ID NO:4.
Embodiment 2
[0055] Embodiment 2: the preparation of antimicrobial protein MBP-LYS-ABP
[0056] 1. Construction and transformation of recombinant plasmid pMAL-p2x / LYS-ABP
[0057] The LYS-ABP fusion protein gene fragment synthesized by the whole gene is double-cut with EcoRI-HindIII, and recovered as the fragment to be inserted.
[0058] The expression vector pMAL-p2x was purchased from NEB Company. The pMAL-p2x plasmid was double-cut with EcoRI-HindIII and the large fragment was recovered as a vector. It was ligated with the above-mentioned LYS-ABP fragment with T4 ligase, and then CaCl 2 Transformed into E.ColiDH5a, the specific steps were carried out according to the method in the "Molecular Cloning" manual. The positive clones were screened by enzyme digestion, and those with a band of about 490 bp were plasmids inserted into the target gene, named pMAL-p2x / LYS-ABP.
[0059] The screened recombinant plasmid was transformed into expression strain BL21(DE3)pLysS to obtain recombinant e...
Embodiment 3
[0067] Embodiment 3: the preparation of antibacterial protein LYS-ABP
[0068]Prepare 25mMpH8.0 Tris-HCl buffer solution, add Lys-ABP pure product containing MBP into the solution, the final protein concentration is 1 mg per ml, add appropriate amount of thrombin, place at 25°C for 5 hours, and cut to obtain Lys-ABP and MBP The mixture was stored in a refrigerator at 4°C for later use.
[0069] Ion exchange chromatography purification: chromatography medium: QSepharoseFastFlow; chromatography column specifications: inner diameter 50cm, height 40cm, medium height about 10cm, medium volume about 200mL; monitoring: 280nm; equilibrium liquid is 25mM pH8.0 Tris-HCl buffer. First, equilibrate the chromatographic column with more than 5 column volumes with the balance solution, and the flow rate is 20mL / min, then dilute the DNA-digested bacterial liquid sample 3 times with the balance solution, and then load the sample, the flow rate is 20mL / min, after loading the sample, continue E...
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Abstract
The invention belongs to the field of biochemistry, and discloses an antibacterial protein LYS-ABP and an antibacterial protein MBP-LYS-ABP having an LYS-ABP protein N terminal added with MBP, and preparation methods thereof, wherein the antibacterial protein LYS-ABP and the antibacterial protein MBP-LYS-ABP have no stimulation to human bodies and have good antibacterial effect. The antibacterial protein LYS-ABP and the antibacterial protein MBP-LYS-ABP have no stimulation to the human bodies and have good antibacterial effect, the antibacterial effect is better than single muramidases and antibacterial peptide proteins, and the biological activity stability is good. The antibacterial protein MBP-LYS-ABP is obtained by in-vitro recombinant expression, and is easy to prepare and relatively low in cost; and the antibacterial protein MBP-LYS-ABP has high soluble expression yield and is suitable for large-scale preparation. The antibacterial protein LYS-ABP is obtained by enzyme digestion of a recombinant expression-prepared MBP-LYS-ABP fusion protein with thrombin, and is easy to prepare and relatively low in cost.
Description
technical field [0001] The invention belongs to the field of biochemistry, in particular to an antibacterial protein. Background technique [0002] Disinfection refers to the method of killing pathogenic microorganisms, but not necessarily bacterial spores. Substances used for disinfection are called disinfectants. The disinfectant, as the name suggests, is a liquid disinfectant. An ideal disinfectant should have a broad bactericidal spectrum, strong bactericidal ability, fast action speed, good stability, non-toxic, no residue, non-corrosive, non-irritating, soluble in water, safe for humans and animals, and cheap and easy to use. and low environmental pollution. [0003] At present, disinfectant products are mainly divided into environmental disinfection, conventional hand and foot disinfectant and wound disinfectant. Environmental disinfection disinfectant refers to products mainly used in sanitation and epidemic prevention and conventional disinfection in disaster ar...
Claims
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