Ocean bacillus protein having antitumor activity and preparation and application thereof
A marine bacillus, anti-tumor activity technology, applied in the fields of genetic engineering and biomedicine, can solve the problems of difficult drug source, large-scale preparation, low content, etc., achieve good research and application value, good technical support and material basis Effect
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0022] Example 1: Construction of recombinant bacteria
[0023] 1) Use specific primers to amplify the gene encoding the protein. Design NcoI and HindIII restriction sites on the primers while amplifying, the primers are:
[0024] Forward: 5’-CATGCCATGGATGCAAGTACAGGCAGTC-3’
[0025] Reverse: 5’-CCCAAGCTTATTTTATCTGTACCACGC-3’
[0026] The PCR amplification system is: 2×ESTaqMasterMix 25μL, upstream primer 2μL, downstream primer 2μL, DNA template 2μL, add FreeWater to 50μL. The PCR amplification conditions were: pre-denaturation at 90°C for 5 minutes, denaturation at 94°C for 30 seconds, annealing at 58°C for 30 seconds, extension at 73°C for 1.0 min, 30 cycles, and finally extension at 73°C for 10 minutes.
[0027] 2) Double digestion of PET22b plasmid and amplified product with NcoI and HindIII respectively, and recover the target product after 1% electrophoresis. Then use T4DNA ligase to ligate at 16°C overnight to ligate the target gene with the vector. Finally, the ligation produ...
Embodiment 2
[0028] 3) Identify the grown-up positive clones by bacterial liquid PCR, double enzyme digestion of plasmids and sequencing. Example 2: Fermentation and culture of recombinant bacteria and separation and purification of recombinant proteins
[0029] The recombinant strain was inoculated into the sterilized initial fermentation broth, and fermented at 37°C, 150r / min. When it was cultured to OD600=0.70, add IPTG with a final concentration of 1.02mM, 30°C, 1500r / min for induction 9h. Obtain fermentation broth. The initial fermentation broth, in terms of mass fraction, includes 1.2% tryptone, 1.0% sodium chloride, 0.5% yeast extract, and the balance is water.
[0030] Centrifuge the fermentation broth at 10000rpm, 10min, 4℃, take the supernatant, and then use the 3kd, 50kd ultrafiltration membrane to intercept; then use 30%-60% ammonium sulfate precipitation, re-dissolve the obtained precipitate, dialysis and desalination ; Then use QFastFlow cation exchange chromatography to get pur...
Embodiment 3
[0032] Inhibitory effect of active protein on tumor cells: In this example, the active protein inhibits the 7 tumor cell lines tested, confirming that the active protein has a broad-spectrum anti-tumor effect.
[0033] 1. Cell culture and active protein preparation:
[0034] Inoculate tumor cells in a multi-well plate, and use DMEM medium containing 10% fetal bovine serum in 5% CO 2 Incubate in an incubator at 37°C for different time, 0.25% trypsin digestion and passage. The sample to be tested is diluted with PBS buffer into multiple concentration gradients and sterilized by filtration with a 0.22μm sterile filter membrane.
[0035] 2. MTT method detects the inhibitory effect of active protein on the growth of a variety of cancer cells
[0036] Take the tumor cells in the logarithmic growth phase, trypsinize them into a single cell suspension, collect the cell suspension, and count the cells. Inoculate 180μL per well in a 96-well cell culture plate, adjust the cell density so that t...
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