Silurus asotus growth hormone (saGH)-cell penetrating peptide trans-activating transcriptional activator (TAT) fusion protein and its preparation method and use
A fusion protein, Escherichia coli technology, applied in biochemical equipment and methods, microorganism-based methods, applications, etc., to achieve the effect of large expression, low cost, and promoting fish growth
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0079] Embodiment 1: prepare catfish growth hormone gene, its steps are:
[0080] 1.1 Extraction of RNA from catfish pituitary gland
[0081] Follow the operation of the TIANGEN RNA extraction kit with minor changes, the specific steps are as follows:
[0082] Sampling: Cut open the fish skull, take out the pituitary gland with tweezers, weighing about 100mg;
[0083] Put the pituitary gland into an Eppendorf tube with 1ml of TRNzol, and mash it with a homogenizer;
[0084] Place the homogenized sample at 20°C for 5 minutes to completely separate the nucleic acid-protein complex;
[0085] Centrifuge at 12000rpm at 4°C for 10min, and take the supernatant;
[0086] Add 0.2ml of chloroform, cover the tube cap, shake vigorously for 15 seconds, and place at room temperature (20-25°C, the same below) for 3 minutes;
[0087] Centrifuge at 12000rpm at 4°C for 15min, the sample will be divided into three layers: yellow organic phase, middle layer and upper colorless aqueous phase, ...
Embodiment 2
[0171] Example 2: PCR amplification of saGH / saGH-TAT gene fragment and construction of expression vector
[0172] Primers were designed according to the sequence of saGH (GenBank: AY157496.1):
[0173] Upstream primer: 5'GGACTG CATATG TTCGAGAACCAGCGTCTCT 3' (the underline is the Nde I restriction site,)
[0174] Downstream primer: 5′CAT CTCGAG CAGGGTGCAGTTGGAATCC 3' (the underline is the Xho I restriction site)
[0175] In addition, according to the base sequence of 11 amino acids in TAT PTD, it was connected to the saGH fragment by PCR extension method, and the primers were designed as follows:
[0176] Upstream primer: 5'-GGACTGCATATGTTCGAGAACCAGCGTCTCT-3'
[0177] Downstream primer 1: 5'-TTTCTTACGGCCATACAGGGTGCAG-3'
[0178] Downstream primer 2: 5'-ACGACGCTGACGACGTTTCTTACGG-3'
[0179] Downstream primer 3: 5'-CATCTCGAGACGACGACGCTGACGACGT-3'
[0180] The above primers were synthesized by Sunny Company.
[0181] Using the extracted pMD18T-saGH as a template, a 50 μl...
Embodiment 3
[0195] Example 3: Construction of genetically engineered bacteria and induced expression and purification of fusion proteins
[0196] The transformation method is the same as the JM109 transformation method.
[0197] 1) Pick a single colony transformed with the plasmid, inoculate it in 5 ml of selective LB liquid medium, and cultivate overnight at 37° C. with shaking at 250 rpm / min.
[0198] 2) The next day, re-inoculate 200 μl of the cultured bacteria solution overnight into 20ml (1:100) selective 2×YT liquid medium (17g peptone, 10g yeast extract, 5g NaCl, add water to 1000ml), 37°C, 250rpm / min shaking culture to optical density (OD 600 =0.6), take a 1ml sample as the pre-induction sample, centrifuge at 10000g for 1min to collect the bacterial pellet, and freeze it at -20°C for later use.
[0199] 3) Add 1mol / L IPTG to the bacterial solution to make the final concentration of IPTG 1mM, 37°C, 250rpm / min
[0200] 4) Culture with shaking for 4 hours. Take a 1ml sample as t...
PUM
Property | Measurement | Unit |
---|---|---|
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