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Fat body and application thereof in evaluating interaction between to-be-detected protein and lipid droplets

A fat body and protein technology, applied in the biological field, can solve problems such as the inability to study the impact and the complexity of the research system

Active Publication Date: 2020-04-24
维康平生(北京)生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the complexity of the in vivo research system, the knockout or overexpression of the protein may cause the compensation of other proteins, and it is impossible to study the effect of phosphorylation, a single factor, on the activity of ATGL

Method used

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  • Fat body and application thereof in evaluating interaction between to-be-detected protein and lipid droplets
  • Fat body and application thereof in evaluating interaction between to-be-detected protein and lipid droplets
  • Fat body and application thereof in evaluating interaction between to-be-detected protein and lipid droplets

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0076] Embodiment 1, the preparation of fat body

[0077] 1. Preparation of fat body armor

[0078] Using vortex and two-step centrifugation to prepare fat body A (disclosed in Chinese invention patent document CN 105483076B), the specific steps are as follows:

[0079] 1. Add phospholipid 1 into a microcentrifuge tube, and dry the solvent with high-purity nitrogen.

[0080] 2. After completing step 1, add 100 μL of buffer B and neutral lipid to the microcentrifuge tube, vortex for 4 minutes (vortex for 10 s, stop for 10 s), and obtain a milky white lipid mixture 1. Dilute lipid mixture 1 with 20,000 g Centrifuge for 5min. After centrifugation, the bottom of the microcentrifuge tube is the precipitated component 1, and the liquid phase system presents two layers of stratification (the upper layer is the white band 1, and the part below the white band 1 is the solution 1).

[0081] The mass ratio of phospholipid 1 to neutral lipid is (0.25-3):5.

[0082] Phospholipid 1 is b...

Embodiment 2

[0111] Example 2, Application of Fat Body in Evaluating the Structure, Shape and Density of Proteins on Lipid Droplets

[0112] 1. Comparison of the morphology of endogenous ADRP in lipid droplets and the morphology of fat bodies coated with ADRP protein

[0113] 1. In order to observe the distribution of endogenous ADRP in cells, the inventors of the present invention constructed the C2C12 cell line (i.e. GFP-KI-ADRP cells) in which the EGFP gene was knocked in at the C-terminus of ADRP, and GFP-KI- ADRP cells were planted in Confocal dishes, treated with 100 μM OA for 12 hours, and images were taken with a confocal laser microscope Olympus FV1000.

[0114] The construction steps of GFP-KI-ADRP cells are as follows: use mouse ADRP mRNA as a template to design gRNA, construct the gRNA sequences at both ends of ADRP into the pX260a vector, and obtain the pX260a recombinant plasmid. The nucleotide sequence of the ADRP gene was constructed into the pQCXIP vector, and the GFP seq...

Embodiment 3

[0146] Example 3, the influence of phosphatidylinositol on the localization of ADRP in fat body

[0147] 1. The density of ADRP on the surface of fat body B containing phosphatidylinositol is reduced

[0148] Because it is very difficult to change the orientation of phospholipids on the surface of lipid droplets in vivo, and artificial lipid droplets can well simulate real lipid droplets, and can precisely change the composition of phospholipids, so the inventors of the present invention constructed fat body B with different phospholipid compositions, To study the regulation of phospholipids on ADRP localization; neutral lipids are all triglycerides, and the mass ratio of phospholipids to neutral lipids is 2:5. The phospholipids on the surface of lipid droplets are mainly composed of phosphatidylcholine (such as DOPC), phosphatidylethanolamine (such as DOPE) and phosphatidylinositol (PI). The research focuses on the regulation of phosphatidylethanolamine and phosphatidylinosit...

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Abstract

The invention discloses a fat body and application thereof in evaluating interaction between a to-be-detected protein and lipid droplets. The preparation method comprises the following steps: mixing triglyceride and phospholipid in a vortex manner, and purifying by using a two-step method to prepare the fat body consisting of a triglyceride core and a single-layer phospholipid membrane; wherein the phospholipid is 'DOPC and PI', 'DOPC and PA', 'DOPC and PS', 'DOPC, PI and DOPE', 'DOPC, PA and DOPE' or 'DOPC, PS and DOPE'. Meanwhile, it is found that the fat body is specifically used as follows: detecting the structure, the form and / or the density of the to-be-detected protein on the lipid droplets; evaluating the interaction between the to-be-detected protein and the lipid droplets, detecting the affinity of the to-be-detected protein and the lipid droplets, detecting ATGL enzyme activity, analyzing the influence of the position and / or type of the amino acid residue in the to-be-detected protein on the positioning of the to-be-detected protein on the lipid droplets, and analyzing the influence of the phospholipid component on the positioning of the to-be-detected protein on the lipid droplets. The invention has important application value.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a fat body and its application in evaluating the interaction between a test protein and a lipid droplet. Background technique [0002] Lipid droplet is a multifunctional organelle with a single-layer phospholipid membrane covering a neutral lipid core and a protein-coated surface. It is involved in the synthesis, storage, metabolism and transport of lipids, the storage and degradation of proteins, and the modification and synthesis of lipid signaling molecules and hormones. , and interact dynamically with other organelles. The changes of protein and lipid components of lipid droplets may lead to the abnormal function of lipid droplets, and then lead to the occurrence of human metabolic diseases. Therefore, the importance of research on lipid droplet biology has become increasingly prominent. [0003] With the help of proteomics and lipidomics techniques, the protein and...

Claims

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Application Information

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IPC IPC(8): C07K14/47G01N33/68A61K47/24A61K47/14
CPCC07K14/47G01N33/68A61K47/24A61K47/14
Inventor 刘平生马学婧支泽仑周畅
Owner 维康平生(北京)生物科技有限公司
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