Preparation method of exfoliative toxin C (ExhC) proteins of staphylococcus sciuri
A technology of Staphylococcus squirrel and protein is applied in the field of bioengineering to achieve the effect of simple operation and low cost
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0031] Example 1 Construction of ExhC prokaryotic expression vector
[0032] According to the instructions of the bacterial genomic DNA extraction kit, the genome of Staphylococcus squirrel was extracted. According to the published ExhC gene nucleotide sequence (accession number: AF515455) in GenBank, the primers are designed as follows: upstream primer (ExhC-F) 5'-CCG CCATGG CTATGCATTCAAAACTATTAAGTAAAT-3′, downstream primer (ExhC-R) 5′-ATA GCGGCCGC TTTAATTAATTGTTTGAGATCTCTAATGAG-3′, the upstream and downstream primers respectively introduced NcoI and NotI restriction sites (underlined) and corresponding protective bases. PCR amplification was carried out using the extracted genomic DNA as a template. The reaction program was: 94°C for 4min, 94°C for 30s, 55°C for 30s, 72°C for 1min, cycled 30 times, and the final extension at 72°C for 10min to amplify the full length of ExhC ( figure 1). The PCR products were gel-cut and recovered, digested with NcoI and NotI, and cloned...
Embodiment 2
[0033] Example 2 Expression of recombinant plasmid pET-28a(+)-ExhC in Escherichia coli
[0034] The correctly identified recombinant plasmid pET-28a(+)-ExhC was transformed into E.coli BL 21. Single-picked colonies were inoculated in LB liquid medium (containing 50 μg / ml kanamycin), and cultured overnight at 37°C with shaking. Inoculate in 50ml LB liquid medium according to 1%, shake culture at 37°C until OD 600 0.5-0.7. Add IPTG to a final concentration of 1 mM, collect the cells after induction at 30°C for 6 hours, and set E.coli BL 21 containing only the expression vector pET-28a(+) as a control. The bacterial lysate was separated by 12% SDS-PAGE, stained with Coomassie brilliant blue, and decolorized to obtain a fusion protein of about 32kD in size ( image 3 ). After the proteins were separated by SDS-PAGE, they were electrotransferred to nitrocellulose membrane (NC membrane). Block overnight at 4°C with 5% skim milk powder, incubate with mouse anti-6×His monoclonal ...
Embodiment 3E
[0035] The purification of embodiment 3ExhC protein
[0036] The solubility of the recombinant 6×His fusion protein should be analyzed before protein purification. For the method, refer to the instructions for use of the Bio-Scale Mini IMAC Cartridge. Protein purification is briefly described as follows: Induce 200ml of recombinant bacteria, 8000rpm, centrifuge at 4°C for 10min to collect the bacteria, and use 10ml of engineered bacteria lysate (50mM Tris-HCl, 50mM NaCl, 10% glycerol, 10mM DTT, 100μg / ml lysozyme, pH 8.0) Resuspend the bacterial pellet, inoculate at 4°C for 1 hour, then sonicate the cells in an ice bath, centrifuge at 10,000 rpm, and 4°C for 20 minutes, and the precipitate is the inclusion body. Wash the inclusion bodies with 10ml of engineering bacteria washing solution (50mM Tris-HCl, 50mM NaCl, 10% glycerol, 0.1mMDTT, 1% Triton X-100, pH 8.0), centrifuge at 10000rpm, 4°C for 20min, collect the precipitate, and repeat the washing once. The inclusion bodies w...
PUM
Abstract
Description
Claims
Application Information
- 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, Popular Technical Reports.
© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com