Novel natural antibacterial peptides, and coding sequence and uses thereof
A technology for sequences and polynucleotides, which is applied in the fields of biotechnology and medicine, and can solve the problems of high cost of chemical synthesis methods.
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Embodiment 1
[0098] Example 1. Construction of Antimicrobial Peptide Expression Plasmids
[0099] The present inventors selected a prokaryotic expression vector for intein-mediated purification, namely pTWIN1 expression vector (purchased from NEB Company). Its advantage is that protein cleavage is a post-translational modification event, which cuts out the intein (Intein) inserted in the middle of the precursor protein, and connects the protein polypeptide chains on both sides with normal peptide bonds. In this process, no coenzymes or cofactors are required, and only four intramolecular reactions are required. Intein and its flanking sequences can be mutated to produce highly specific self-cleavage for protein purification, protein ligation and protein cyclization reactions. The pTWIN 1 vector has a chitin binding domain (CBD) fused to the N-terminus of Intein, which allows the Intein fusion protein to be adsorbed on chitin beads. When the target protein is fused to the C-terminus of In...
Embodiment 2
[0105] Example 2. Transformation and Identification of Antimicrobial Peptide Expression Plasmids
[0106] Take 200 μl of competent cell E.coli ER2566 strain (New England Biolabs) suspension from the -70°C refrigerator, thaw it at room temperature, and put it on ice immediately after thawing. Add plasmid DNA solution (content not more than 50ng, volume not more than 10μl), shake gently, and place on ice for 30 minutes. Heat shock for 90 seconds in a water bath at 42°C or 5 minutes in a water bath at 37°C. After the heat shock, quickly place on ice to cool for 3-5 minutes. Add 1ml of LB liquid medium (without Amp) to the tube, mix well and incubate with shaking at 37°C for 1 hour to restore the normal growth state of the bacteria and express the antibiotic resistance gene (Amp) encoded by the plasmid. Shake the above-mentioned bacterial solution, take 100 μl and spread it on the screening plate containing Amp, place it face up for half an hour, after the bacterial solution is c...
Embodiment 3
[0108] Embodiment 3. the preparation of antimicrobial peptide protein
[0109] Pick a single clone from the plate, inoculate it into a 3ml LB test tube containing 0.1μg / ml, incubate overnight at 37°C 200rpm, take 1ml of bacteria, transfer to a shaker flask containing 100ml LB, incubate at 37°C 200rpm for 3hrs, measure OD600 To about 0.5-0.7, add 0.5mM IPTG at 25°C 200rpm to induce culture overnight.
[0110] Centrifuge 100ml of bacterial solution cultured overnight at 12000rpm at 4°C for 10min to discard the supernatant, add 10ml of lysis buffer B1 (20mM Tris-HCl, 50mM Nacl, 1mM EDTA, pH 8.5) and 0.15% Tween20 and 20μM PMSF to the pellet, and ultrasonically break , sonication time 8s, interval 15s, energy 300W, total time 10min, after 2 times of sonication, centrifuge at 12000rpm 4°C for 15min to take the supernatant.
[0111] Add 10ml of the supernatant to a column containing 4ml of chitin filler. The column is first washed with 20ml of buffer B1 to equilibrate. After washi...
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