Vector for recombinant expression of luteinizing hormone of panda, expression system and preparation method thereof
The technology of a luteinizing hormone and an expression system is applied to the vector, expression system and preparation field of recombinant expression of giant panda luteinizing hormone, which can solve the problems of adverse reactions of the body, affecting long-term effects, etc., achieves wide application prospects, is beneficial to The effect of high breeding quality and safety
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0050] This example provides the construction method of pCDH+LHβ-T2A-LHα expression vector. The build materials used are as follows:
[0051] Strains: CHO-K1 cells and HEK293T cells, purchased from ATCC, USA; vector pcDNA3.1(+), purchased from Invitrogen, USA; vector pCDH-CMV-MCS-EF1-Puro, purchased from SBI, USA; lentiviral system plasmid psPAX2 +pMD2.G was purchased from Invitrogen, USA.
[0052] Reagents: The first gene, the second gene and T2A sequence were synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd., and the primers were synthesized by Beijing Liuhe Huada Gene Technology Co., Ltd.; restriction enzymes and ligases were selected from Baoriyi Biotechnology Co., Ltd. (Beijing) Co., Ltd., and the transfection reagent was purchased from Daktronics.
[0053] Build method:
[0054] 1. Enzyme digestion reaction of vector and insert fragment.
[0055] The α and β subunit genes of giant panda LH and the T2A sequence connecting them were directly synthesized by Jinwei...
Embodiment 2
[0089] This example provides a method for constructing the CHO-K1 cell expression system.
[0090] Recombinant virus packaging and target cell infection, screening experiments.
[0091] (1) Culture HEK293T cells in a 6-well plate, and transfect when the cells are 80% full, and the transfection steps are performed according to the instructions of the kit.
[0092] (2) Add pCDH-LH plasmid (1.4μg) and packaging plasmid psPAX2 (1.4μg) and pMD2.G (0.7μg) to 200μl buffer, vortex for 10 seconds and centrifuge briefly.
[0093] (3) Add 3μl For the transfection reagent, vortex for 10s, centrifuge briefly, and let stand at room temperature (22-26°C) for 10min.
[0094] (4) Add 200 μl transfection mixture dropwise to the 6-well plate, and shake to distribute the reagent evenly.
[0095] (5) Change the medium after 4 hours, use growth medium containing antibiotics and incubate at 37°C, 5% CO 2 incubator culture growth.
[0096] (6) At 48 o'clock after transfection, collect the cul...
Embodiment 3
[0100] This example provides the pGL3-CRE-luciferase luciferase reporter system for detecting the recombinantly expressed LH active protein in Example 2.
[0101] So far, the sequence BLAST in the giant panda genome (Panda release 92:ailMel1) has suggested that there is a NNNN deletion in the 5-terminal sequence of the giant panda LHR gene, that is, there is a deletion in the genome sequence containing the start codon of the giant panda LHR gene, indicating that the giant panda genome Sequences await further annotation. In view of the fact that LH relies on binding to its specific receptor (LHR) to exert its biological effects, this embodiment clones the mouse LHR gene, and combines the giant panda LHR and the mouse LHR to encode a receptor protein with 690 amino acids, and It has a high amino acid sequence identity (87%), suggesting that the mouse LHR can be used to express and construct a eukaryotic expression vector (pcDNA3.1-LHR) to detect the activity of the recombinant L...
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