Establishment method of high-throughput drug screening model based on icam-1 signaling pathway

A technology of ICAM-1 and signaling pathway, which is applied in the field of high-throughput drug screening model establishment, and can solve problems such as adverse reactions and damage

Active Publication Date: 2021-07-16
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although these two types of anti-inflammatory drugs have certain clinical anti-inflammatory effects, long-term large-scale use will also produce a series of adverse reactions, such as gastric mucosa, liver, kidney damage, etc.

Method used

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  • Establishment method of high-throughput drug screening model based on icam-1 signaling pathway
  • Establishment method of high-throughput drug screening model based on icam-1 signaling pathway
  • Establishment method of high-throughput drug screening model based on icam-1 signaling pathway

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Example 1: Plasmid vector construction of engineered yeast

[0057] (1) Using your own plasmid pCTCON-wGFP as a template, use primers wGFP (5,-ATCGAATTCTACTTCATACATTTTCAAATTAAGATGGCTAGCGTGAGCAAGGGCGAGGAG-3, sequence-specific primers plus an ECORI site) and wGFP (5,-GTTCGGATCCAGTGATCCCGGCGGCGTTC-3 ,, sequence-specific primers plus BamHI site) to amplify the wGFP fragment (720bp). PCR reaction conditions: pre-denaturation at 94°C for 3 min; 35 cycles of 94°C for 30 sec, 54°C for 30 sec, and 72°C for 1 min; extension at 72°C for 10 min. The amplified wGFP gene PCR product was inserted between the ECORI and BamHI restriction sites behind the YS2H vector promoter GAL1, and positive clones were screened and sequenced to obtain the gene wGFP. We named this plasmid pDV3-intra-wGFP

[0058](2) On the basis of the above pDV3-intra-wGFP vector, the applicant inserted the human LFA-1αL I domain gene (wt, D137A, F265S / F292G) between the NcoI and SalI restriction sites. wt was ampl...

Embodiment 2

[0063] Example 2: Introduction of engineered yeast vector into yeast and expression of induced protein

[0064] The plasmid vector pDV3-intre-huαL Idwt of the above-mentioned three dual-channel protein expression engineering yeast of wGFP and LFA-1αL I domain gene (sequence see SEQ ID NO: 1 and SEQ ID NO: 2, 3, 4, 5) -wGFP, pDV3-intre-huαL Id D137A-wGFP and pDV3-intre-huαL Id F265S / F292G-wGFP with PEG / LiAc method (Ito H, Fukuda Y, Murata K, et al. Transformation of intact yeast cells treated with alkali cations [J].Journal of bacteriology,1983,153(1):163-168.) and a plasmid vector corresponding to a yeast cell were respectively introduced into the yeast cell EBY100 to obtain recombinant yeast cells, cultivate the recombinant yeast cells and induce the Expression of recombinant vector (specific steps follow: Hu X, Kang S, Chen X, et al. Yeast surface two-hybrid for quantitative in vivo detection of protein-protein interactions via the secretory pathway[J]. Journal of Biological...

Embodiment 3

[0070] Example 3: Verification of protein expression in engineered yeast

[0071] After the recombinant yeast is induced, the expressions of I domain and GFP can be checked respectively by flow cytometry. The expression level of Idomain was detected with Flag tagged protein by flow cytometry. Specific steps are as follows:

[0072] (1) Aspirate 3 μl of yeast cells expressing wGFP and LFA-1αL I domain proteins into a 96-well V-shaped plate, take yeast cells that do not express proteins as a negative control, and add 100 μl of labeling buffer A pH=7.4 to each well (Recipe: PBS+0.5%BSA+10mM MgCl 2 ), mix well and centrifuge (4°C, 3min, 3000rpm) to pellet the cells, and remove the supernatant. Aspirate buffer A, add 20 μl of buffer A containing 10 μg / ml primary antibody (mouse anti-flag antibody (Santa Cruz Biotechnology, USA)) to each well, and incubate at 30°C for 30 minutes with shaking.

[0073] (2) Add 100 μl of buffer A to wash the yeast cells, centrifuge (4°C, 3min, 300...

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Abstract

The invention belongs to the technical field of drug molecular model building, and in particular relates to a method for building a high-throughput drug screening model based on the ICAM-1 signaling pathway. ICAM‑1 is an important immune system component and an important characterization molecule in many diseases. The present invention focuses on the ICAM-1 molecule and its regulatory network, uses engineered yeast as a medium to construct a novel drug screening system, displays the ICAM-1 high-affinity receptor LFA-1 insertion domain on the surface of the yeast and expresses green in its cells fluorescent protein. If the drug acts on ICAM-1 and its signaling pathway, it will affect the number of yeast cells and unit animal cells. Drugs can be quickly screened through the difference in fluorescence intensity. The fluorescence intensity of yeast can be used to achieve efficient, simple, and efficient detection of ICAM-1 expression. Economical high-throughput drug screening. The present invention has high plasticity, and respective drug screening platforms can be established according to animal cells of different disease types.

Description

technical field [0001] The invention belongs to the technical field of drug molecular model building, and in particular relates to a method for building a high-throughput drug screening model based on the ICAM-1 signaling pathway. Background technique [0002] Inflammation is the body's defense response to various inflammatory factors and the damage they cause, but excessive inflammatory responses can also cause damage to the body (Kleiman A and Tuckermann JP, 2007; Cohen J, 2002). There are two main types of anti-inflammatory drugs commonly used in clinical practice: non-steroidal anti-inflammatory drugs and steroidal anti-inflammatory drugs. Although these two types of anti-inflammatory drugs have certain clinical anti-inflammatory effects, long-term large-scale use will also produce a series of adverse reactions, such as gastric mucosa, liver, and kidney damage. Natural medicines have the characteristics of wide sources, low cost, and small side effects. With the deepeni...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/071C12N15/81C12N15/65C12Q1/02
CPCC12N5/069C12N15/65C12N15/81C12N2503/02G01N33/5064G01N2500/10
Inventor 胡学博张启云
Owner HUAZHONG AGRI UNIV
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