Preparation method and antibacterial application of a kind of P. shigella-like phage lyase
A technology of phage lysing enzymes and Pseudomonas, applied in the field of bioengineering, can solve problems such as eye and respiratory tract irritation, complicated production, and pulmonary edema, and achieve the effect of short action time, good water solubility, and wide action spectrum
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0020] Extraction of phage genome
[0021] (1) Crude phage particles
[0022] (1) Preparation of host bacteria: pick Plesiomonas Shigelloides. from solid medium [1] A single colony was inoculated in 5mL LB liquid medium, and cultured with shaking at 37°C for 6-8h.
[0023] (2) Preparation of pure phage culture solution: pick a single phage plaque, inoculate it in 5mL logarithmic phase host bacteria culture solution, culture it with shaking at 37°C for 4-6h, then centrifuge the lysate at 10000rpm for 10min, and the supernatant is ready For the pure culture medium of bacteriophage.
[0024] (3) Preparation of crude phage particles: Transfer the overnight culture of P. shigella-like to 100 mL liquid LB medium, the inoculum size is 1%, and the expansion culture is reached to the logarithmic phase (OD 600 About 0.4), add 5 mL of P. shigella-like phage pure culture solution, shake culture at 37° C. for 6-8 hours to obtain phage lysate. Add DNase I and RNase A to the lysate to a ...
Embodiment 2
[0035] Cloning of lyase gene gp2 and construction of expression vector
[0036] (1) Design a pair of specific primers according to the sequence encoded by the gp2 gene:
[0037] gp2F:5'-CGG GGTACC ATG CAA CCA TCG CGA GCG TG-3', see Seq ID NO.3; gp2R: 5'-CCC AAGCTT CTG GCG GCG GTG GAT TTT TG-3', see Seq ID NO.4; use the phage genome DNA as a template to amplify the gp2 gene with the above primers, electrophoresis on 1% agarose, and identify the size of the amplified fragment.
[0038](2) The PCR product was directly digested with restriction endonucleases KpnI and HindIII, 37 ° C water bath for 2 hours, 1% agarose electrophoresis, and the gel recovery kit was used to purify and recover the fragments, and the fragments were combined with KpnI and HindIII before The double digested and purified pQE30 vector was ligated overnight at 16°C, and transformed into E. coli M15 / pREP4 competent the next day. The transformation mixture was added to an appropriate amount of LB medium, ...
Embodiment 3
[0042] Induced expression and purification of lyase gp2 protein
[0043] Inoculate the bacteria HeL1 containing the recombinant plasmid into the LB medium containing ampicillin (100 μg / mL) and kanamycin (25 μg / mL), and cultivate overnight at 37°C with shaking; the next day, transfer to In 100mL LB medium, shake culture at 37°C until OD 600 When the value is about 0.6, add IPTG to a final concentration of 0.5mmol / L, and induce at 16°C for 16h. Bacteria were collected, cells were disrupted by ultrasonic waves, centrifuged at 10,000 rpm / min at 4°C for 10 min, the supernatant was collected, and the supernatant was filtered through a 0.22 μm filter membrane, and the protein expression in the lysed supernatant was analyzed by SDS-PAGE. The filtered lysed supernatant was purified with a His affinity chromatography nickel column (GE Healthcare, Sweden), specifically according to the kit instructions, and the obtained protein was named lyase Gp2.
[0044] The results of SDS-PAGE anal...
PUM
Login to view more Abstract
Description
Claims
Application Information
Login to view more - R&D Engineer
- R&D Manager
- IP Professional
- Industry Leading Data Capabilities
- Powerful AI technology
- Patent DNA Extraction
Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.
© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap