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Model for filtering active regulator of human lipoprotein receptor in high density

A technology of high-density lipoprotein, activity regulator, applied in the direction of biochemical equipment and methods, determination/inspection of microorganisms, etc.

Inactive Publication Date: 2007-02-21
MEDICINE & BIOENG INST OF CHINESE ACAD OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although many data indicate that elevated plasma levels of high-density lipoprotein (HDL) are beneficial for lowering cholesterol, so far there is no selective HDL-targeted therapy

Method used

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  • Model for filtering active regulator of human lipoprotein receptor in high density
  • Model for filtering active regulator of human lipoprotein receptor in high density
  • Model for filtering active regulator of human lipoprotein receptor in high density

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1 Preparation of recombinant baculovirus Bacmid-CLA-1 and successful expression in insect cell Sf9 in CLA-1

[0038] 1. Acquisition of CLA-1 cDNA

[0039] 1) Extraction of total RNA

[0040] Using Promega's SV total RNA kit, from human liver cancer BEL-7402 cells (about 1×10 6 cells) to extract total RNA. The extraction method was carried out according to the recommended steps of the kit. Total RNA was dissolved in 100 μl nuclease-free water and stored at -70°C.

[0041] 2) RT-PCR amplification of the gene encoding the target protein

[0042] cDNA was synthesized by reverse transcription using the total RNA obtained in step 1) as a template and Oligo dT as a primer. According to the sequence of human CLA-1 cDNA, primers for amplifying the fragment encoding human CLA-1 were designed. A BglII restriction site and two protective bases were introduced at the 5' and 3' primer ends. The primer sequences are as follows:

[0043] P1: 5' end primer: 5'-AA AGATCT...

Embodiment 3

[0062] Example 3 Relation between the Time of Recombinant Baculovirus Infected Insect Cells Expressing CLA-1 and the Fluorescence Readout of CLA-1 Binding to Fluorescent Ligands

[0063] In the screening model constructed with DiI-AcLDL as the fluorescent ligand, the experimental results of the time of recombinant virus infection of insect cells and the expression level of CLA-1 are shown in Figure 8. It can be seen from Figure 8 that, compared with the blank group, the combination of DiI-AcLDL reached the highest peak after 72 hours of recombinant virus infection. And the fluorescence readings between the two groups were p<0.01. After 96 hours of infection, the expression level of CLA-1 was the highest, but as the infection time prolongs, the state of the cells becomes worse and worse, and some cells no longer adhere to the wall. At 120 hours, some cells appear to be lysed. At this time, the insect cells The ability to bind fluorescent ligands is very poor, so three days of ...

Embodiment 4

[0065] Embodiment 4 The influence of the incubation time of the fluorescent ligand and the recombinant insect cell on the fluorescence reading

[0066] In the screening model constructed with DiI-AcLDL as the fluorescent ligand, we selected the concentration of DiI-AcLDL as 1.5 μg / ml, and kept other conditions unchanged, but the incubation times were 2, 4, 6, 8, 10, and 12 hours. The results See Figures 9A and 9B. After incubation for 2 and 4 hours, p>0.05 was compared between the control group and the blank group, and there was no significant difference between the two groups. After incubation for 6, 8, 10, and 12 hours, the comparison between the control group and the blank group was p<0.01, and there was a significant difference between the two groups. Fluorescence readings did not increase significantly after 12 hours of incubation.

[0067] In the screening model constructed with DiI-HDL as the fluorescent ligand, we selected the concentration of DiI-HDL as 0.5 μg / ml. W...

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Abstract

This invention relates to a model for in vitro high throughput screening of human high-density lipoprotein receptor CLA-1 activity regulators. This invention also relates to a method for utilizing the model to screen CLA-1 activity regulators. Besides, CLA-1 activity regulators, especially CLA-1 stimulator and CLA-1 antagonist screened by this method are also disclosed.

Description

field of invention [0001] The present invention relates to an in vitro high-throughput screening model of a CLA-1 activity regulator targeting human high-density lipoprotein receptor (CLA-1). The present invention also relates to a screening model for human high-density lipoprotein A method for a lipoprotein receptor activity regulator, and a human high-density lipoprotein receptor activity regulator obtained by screening the model, especially a human high-density lipoprotein receptor agonist and a human high-density lipoprotein receptor antagonist . Background of the invention [0002] Atherosclerosis (AS) cardiovascular disease is one of the common diseases that endanger human health. Although many data show that the increase of high-density lipoprotein (HDL) level in plasma is beneficial for lowering cholesterol, there is still no selective treatment for HDL so far. [0003] The high-density lipoprotein receptor was confirmed late. In 1996, Krieger et al. (Acton, S.Rigo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/02
Inventor 司书毅洪斌田德峰张忠兵王娟张月琴解云英姜威王丽非巫晔翔
Owner MEDICINE & BIOENG INST OF CHINESE ACAD OF MEDICAL SCI
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