Recombinant protein of methicillin-resistant staphylococcus aureus IsdB protein active segment, preparation method thereof and application thereof
A methicillin-resistant and recombinant protein technology, applied in the field of biotechnology and pharmaceuticals, can solve the problems of transient immune response and inability to cause immunity, and achieve high expression, maintain spatial conformation and immunogenicity, and mild purification conditions Effect
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0067] Example 1: Cloning of Methicillin-resistant Staphylococcus aureus iron ion surface determinant protein IsdB active fragment IsdB2
[0068] 1. Firstly, according to the full-length gene sequence of MRSA-252IsdB protein, use bioinformatics software for structural analysis. For the analysis results, see the attached Figure 6-9 , so as to determine the IsdB2 target gene fragment that needs to be amplified.
[0069] 2. According to the analysis results, the PCR method is used to amplify the IsdB2 target gene fragment from the MRSA-252 genome, and the amplification steps are as follows:
[0070] 1) Design the PCR primers as follows, which are SEQ ID NO: 5-6 (the base sequence of the restriction site is underlined)
[0071] isdB2-F: SEQ ID NO: 5
[0072] 5'-CGC GGATCC ATGAATGGCGAAGCAAAAGCAGC-3'
[0073] BamH Ⅰ
[0074] isdB2-R: SEQ ID NO: 6
[0075] 5'-TTTTCCTTTT GCGGCCGC CTATGTCATATCTTTTATTAGATTCTTC
[0076] -3'Not Ⅰ
[0077] In this embodiment, the nucleotide seq...
Embodiment 2
[0106] Example 2: MRSA-252 IsdB subunit active fragment IsdB2 induced expression, purification and expression form identification in prokaryotic expression system-Escherichia coli
[0107] 1. Induced expression of target protein
[0108] 1) Take 100 μL of the pGEX-6P-2-IsdB2 / XL-1blue bacterial solution that was correctly identified by double enzyme digestion and add it to 10 mL of Amp-resistant TB medium, cultivate overnight at 80 rpm at 37°C, take 400 μL of the overnight cultured bacterial solution and add 20 mL of Amp In the resistant TB medium (the rest of the bacterial solution is stored in a 4°C refrigerator for later use), culture at 37°C for 2~3h, rotate at 200rpm, and reactivate to OD600 of 0.6~0.8, add 40μL of IPTG to make the final concentration 200 μM, then placed on a shaking table to induce expression at 30°C for 3h, 25°C for 5h, and overnight induction at 18°C, 16°C, and 12°C.
[0109] 2) Take out the bacterial solution after induced expression, centrifuge at 60...
Embodiment 3
[0116] Embodiment 3: Preparation of IsdB2 antigen
[0117] 1. Scale up culture to obtain protein
[0118] Take 400 μL of the spare pGEX-6P-2-isdB2 / XL-1blue bacterial solution stored in the refrigerator at 4°C and add it to 20mL TB medium containing Amp resistance for one activation. Add the primary activated bacterial solution to 400mL TB medium containing Amp resistance for secondary activation, culture at 37°C for 3~4h until OD600 is 0.8, add 80μl IPTG (final concentration is 200uM) and place in a shaker at 16°C After overnight induction, centrifuge at 6000rpm for 15min to collect the bacteria, add 20mL of lysis buffer to resuspend the bacteria, then ultrasonically lyse the bacteria for 3min (200V), collect the supernatant and 800μL Glutathione Sepharose 4B gel for binding to GST fusion protein Beads (beads) binding treatment; then SDS-PAGE gel electrophoresis, the results are shown in Figure 4 , it can be seen that the target protein is expressed in large quantities in ...
PUM
Property | Measurement | Unit |
---|---|---|
molecular weight | aaaaa | aaaaa |
molecular weight | aaaaa | aaaaa |
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