Pichia pastoris genetic engineering hybrid antibacterial peptide CC29 and preparing method thereof
A technology of hybrid antimicrobial peptides and Pichia pastoris, which is applied in genetic engineering and biological fields, can solve the problems of large molecular weight and low content of antimicrobial peptides, which are difficult to meet large-scale production, and achieve high antibacterial activity.
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[0051] ② Preparation of Competent Cells: Use TakaRa’s Competent Cell Preparation Kit to prepare Escherichia coli DH5α competent cells. The specific method is as follows:
[0052] a Strain purification: use LB plate medium. Pick Escherichia coli DH5α (preserved in glycerol at -80°C) with a sterile inoculation needle, grade and streak on the plate medium, place it upside down in a constant temperature incubator at 37°C, and cultivate overnight, so as to be able to grow a single colony.
[0053] b bacteria culture:
[0054] a Take 20mL of φb×broth and transfer it into a 100mL Erlenmeyer flask, plug it with a cotton plug, sterilize it under high temperature and high pressure, and then cool it to room temperature; pick a single colony on the streaked plate, and inoculate it into the sterilized and cooled medium above; Shaking (120rmp) culture at 37°C;
[0055] b Measure the OD600 value, when the OD600 value reaches 0.35-0.5 (about 5 hours of cultivation), place it on ice to stop ...
Embodiment 1
[0110] Bacteriostatic Activity of Hybrid Antimicrobial Peptides by Zone of Inhibition Method
[0111] The antibacterial activity of the target protein was preliminarily detected by the agar hole diffusion method. The strains used in the experiments included Escherichia coli, Staphylococcus aureus, Salmonella gallinarum, Streptococcus dysgalactiae, Lactobacillus brevis, Lactobacillus delbrueckii and Lactobacillus crispatus. Inoculate the above-mentioned various bacteria on appropriate solid medium respectively, and after a single colony grows, carry out shaking culture in the corresponding liquid medium. Using the plate dilution method, dilute the above-mentioned suspensions of various strains in the logarithmic growth phase to 108 / μL, inoculate them into a suitable solid medium at 1%, mix well and invert the plate. After the plate was condensed, holes were punched with an autoclaved punch (3 mm), and 20 μL of liquid was added dropwise to each hole. After incubating at 37°C f...
Embodiment 2
[0114] Determination of minimum inhibitory concentration (MIC)
[0115] Pick a single colony of Escherichia coli, Staphylococcus aureus, Streptococcus dysgalactiae, Salmonella, Lactobacillus brevis, Lactobacillus delbrueckii and Lactobacillus crispatus in the plate and culture them overnight at 37°C and 200rpm air shaking in a suitable liquid medium. The 6 hybrid antimicrobial peptides were diluted stepwise into 8 gradients: 200, 100, 50, 25, 12.5, 6.25, 3.125 and 1.5625 mg / mL. Dilute the overnight cultured bacteria to 2×105-7×105 CFU / mL, add the diluted test bacteria solution to the 96-well culture plate respectively, 50 μL per well for 1-12 wells in each row, and then add dropwise to each well 50 μL of hybrid antimicrobial peptides. Three replicates were set for each sample, and no test bacteria was used as a negative control, and Amp was used as a positive control; cultured at a constant temperature at 37°C with a rotation speed of 100rpm for 24h; the absorbance was measur...
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