Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Insulin receptor protein expression cell strain and application thereof

A technology of cell lines and human insulin, applied in the application, cells modified by introducing foreign genetic material, using vectors to introduce foreign genetic material, etc., can solve the problems of cumbersome operation, large differences, and low sensitivity of the method, and achieve repeatability Good, low cost, easy standardization effect

Pending Publication Date: 2022-07-01
LUNAN PHARMA GROUP CORPORATION
View PDF1 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method requires the induction of preadipocytes in vitro, which involves the dissection of rats, the extraction and primary culture of preadipocytes, the operation is cumbersome, the sensitivity of the method is not high, and the difference is large

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Insulin receptor protein expression cell strain and application thereof
  • Insulin receptor protein expression cell strain and application thereof
  • Insulin receptor protein expression cell strain and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1 Synthesis and construction of expression INSR vector

[0032] 1. Amplification and identification of the INSR gene

[0033] The sequence of human INSR protein is shown in SEQ ID NO: 1, with a full length of 1382 amino acids. The sequence optimization and full-length DNA synthesis were commissioned to the outside world, and a Hind III restriction enzyme cleavage site was added to the N-terminal of the nucleotide sequence encoding the human INSR protein. and kozak sequence, adding a terminator TGA and an ECORI restriction site at the C-terminus, and the optimized full-length nucleotide sequence is shown in SEQ ID NO: 2. The full sequence gene was synthesized and then ligated into PUC-57 vector to construct PUC57-INSR vector.

[0034] Both the PUC57-INSR plasmid and the vector PGS2 were digested with restriction enzymes HindIII and ECORI, and the target fragment was recovered, ligated with T4 ligase, ligated at 16°C overnight, and the ligated product was transfo...

Embodiment 2

[0038] Example 2 Analysis of expression of INSR activity in CHO-K1-INSR-6B3, CHO-K1-INSR-2A4 and CHO-K1-INSR-3B1 cells

[0039] Insulin and its analogs, after binding to insulin receptors, phosphorylate at Tyr1150 / 1151 of downstream β receptors, and the kit provided by CISbio can specifically detect the phosphorylated β receptors by sandwich ELISA method, an antibody Label the Eu3+ hole, another antibody labels d2, which is phosphorylated after insulin and analogs bind to the receptor. The higher the activity, the higher the phosphorylation level. After cell lysis, the phosphorylated β receptor is released, and the Eu3+ hole is labeled. The antibody (donor) and the D2-labeled antibody (acceptor) can specifically bind to the phosphorylated β-receptor, when the two antibodies bind to the phosphorylated β-receptor, when the dyes are in close proximity , the light source (laser or flash lamp) excites the donor to emit fluorescence resonance energy transfer (FRET) to the acceptor, ...

Embodiment 3

[0044] The detection of embodiment 3 insulin biological activity

[0045] The IR beta phosphor-Y1150 / 1151 kit was used for detection, and CHO-K1-INSR-6B3 cells were suspended in PBS at 5×10 5 1 ml, 50 μL / well, added to a 96-well plate, starved for 2 hours. 2-fold dilution of different insulins in PBS (insulin aspart (300 units / 3ml / vial), insulin glargine (300 units / 3ml / vial), insulin detemir (300 units / 3ml / vial)), 10 μL / well, added to The treated cells were starved for 20 min. After stimulation, immediately add the lysis working solution mixed with blocking reagent and 4× lysis solution at a ratio of 1:24, 20 μL / well, lyse at room temperature for 30 min, 150 rpm / min. After the lysis is completed, mix well, transfer 16 μL to a 96 shallow-well plate, and then add 4 μL of the pre-mixed antibody working solution (the two working antibodies and the detection buffer are mixed at 1:19, and the two diluted working antibodies are used before use. Mix again at 1:1), and incubate at r...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention belongs to the technical field of drug biological activity detection, and particularly relates to establishment of a genetic engineering cell strain of insulin receptor protein and a detection method for determining insulin biological activity based on change of insulin receptor phosphorylation level after the cell strain is stimulated by insulin. Specifically, the cell strain expressing the INSR protein is CHO-K1-INSR-6B3, after insulin is combined with an insulin receptor, the insulin receptor is phosphorylated, the activity of the insulin and the amount of the phosphorylated receptor have a dose-effect relationship, and the activity of the insulin can be quantitatively detected by detecting the phosphorylation level of the insulin receptor. The detection method is easy to standardize and good in repeatability, and has the characteristics of low cost, convenience and accuracy, thereby having a good application prospect.

Description

technical field [0001] The invention belongs to the technical field of drug biological activity detection, and in particular relates to the establishment of a genetically engineered cell line of insulin receptor protein, and the detection of insulin biological activity based on the change of the phosphorylation level of insulin receptor after the cell line is stimulated by insulin method. Background technique [0002] Insulin was first discovered in 1921 by Canadians F.G. Banting and C.H. Best. It began to be used clinically in 1922, saving diabetic patients who had died in the past. Until the early 1980s, almost all insulin used for clinical use was extracted from pig and bovine pancreas. Human insulin consists of two peptide chains, A and B. The A chain has 11 kinds of 21 amino acids, and the B chain has 15 kinds of 30 amino acids, making up a total of 16 kinds of 51 amino acids. Among them, the sulfhydryl groups in the four cysteines A7(Cys)-B7(Cys), A20(Cys)-B19(Cys) ...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
IPC IPC(8): C12N5/10C12N15/85C12N15/12G01N33/74C12R1/91
CPCC07K14/72C12N15/85G01N33/74
Inventor 张贵民刘忠赵丽丽朱中松魏永永马鲁南曹宇
Owner LUNAN PHARMA GROUP CORPORATION
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

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