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HIV (human immunodeficiency virus) inhibitor screening model and preparation method and application thereof

An inhibitor screening and modeling technology, applied in the field of biochemistry, can solve the problems of unsuitable CFP, low fluorescence quantum yield, very sensitive environment, etc., and achieve the effect of strong screening signal, clear mechanism of action, and high sensitivity

Active Publication Date: 2016-08-31
SOUTHERN MEDICAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

CFP-YFP is currently the most widely used FRET donor-acceptor pair in the study of protein interactions, but the CFP-YFP pair also has certain shortcomings: such as low fluorescence quantum yield, low energy transfer efficiency, very sensitive to the environment, and CFP is not suitable for laser as excitation light, etc.

Method used

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  • HIV (human immunodeficiency virus) inhibitor screening model and preparation method and application thereof
  • HIV (human immunodeficiency virus) inhibitor screening model and preparation method and application thereof
  • HIV (human immunodeficiency virus) inhibitor screening model and preparation method and application thereof

Examples

Experimental program
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Effect test

preparation example

[0034] 1. Materials and instruments:

[0035] 1. The base sequences are SEQ ID NO.1~SEQ ID NO.3 synthesized by Shanghai Yingweijieji Guangzhou Branch

[0036] 2. Plasmid pEGFP-N3: ​​purchased from Clonetech, USA

[0037] 3. Plasmid pDsRed-N1: purchased from Clonetech, USA

[0038] 4. Tool enzymes XhoI, EcoI, PstI, KpnI were purchased from Takara Company

[0039] 5. Laser confocal microscope (Olympus FV1000Olympus Corp Japan)

[0040] 2. Method:

[0041] 3. Construction of fluorescent expression vectors for gp120, CD4, and CXCR4:

[0042] choose as figure 1 The indicated plasmid pEGFP-N3 expressing green fluorescent protein and the plasmid pDsRed-N1 expressing red fluorescent protein are used as the fluorescent expression vectors of gp120, CD4 and CXCR4, and then the restriction sites shown in Table 1 are used for CD4, CD4 and CXCR4 respectively. The coding genes of CXCR4, gp120 and the expression vector are subjected to corresponding restriction enzyme digestion to form ...

example 1

[0054] 1. Drugs to be screened

[0055] Chinese name: AMD3100, purchased from Sigma Company.

[0056] Chemical name: 1,1'-[1,4-phenylenebis(methylene)]bis[1,4,8,11-tetraazacyclotetradecane]

[0057] Features: AMD3100 has a large molecular weight, and the cations in the structure are not conducive to the molecules passing through the cell membrane, resulting in difficult absorption, low oral activity, and can cause toxic side effects such as abnormal heart rhythm.

[0058] Mechanism of action: AMD3100 is a small molecule CXCR4 inhibitor that acts on the early stage of HIV-1 infection, specifically blocking HIV-1 with CXCR4 as a co-receptor from entering cells, not acting on CCR5 receptors, nor on gp120 protein. take effect.

[0059] 2. Screening method

[0060] (1) Using the six recombinant plasmids gp120 / pDsRed-N1, gp120 / pEGFP-N3, CXCR4 / pDsRed-N1, CXCR4 / pEGFP-N3, CD4 / pDsRed-N1 and CD4 / pEGFP-N3 obtained in the preparation example, respectively HEK293T cells were transfected...

example 2

[0064] 1. Drugs to be screened

[0065] Name: anti-human CD4IgG, purchased from Biolegend Company.

[0066] Features: macromolecular protein, low oral availability.

[0067] Mechanism of action: CD4-specific neutralizing antibody, by specifically binding to CD4, inhibits HIV from entering cells.

[0068] 2. Screening method

[0069] (1) Using the six recombinant plasmids gp120 / pDsRed-N1, gp120 / pEGFP-N3, CXCR4 / pDsRed-N1, CXCR4 / pEGFP-N3, CD4 / pDsRed-N1 and CD4 / pEGFP-N3 obtained in the preparation example, respectively HEK293T cells were transfected to obtain 6 cells; then 6 cells were seeded in 24-well cell culture plates with coverslips, placed at 37°C and filled with 5% CO. 2 The cells were cultured in a gas cell incubator for 48 h, washed three times with PBS, fixed with 4% paraformaldehyde solution by volume for 15-20 min, and then fixed the cell-loaded coverslip on the slide and sealed with cover agent. 6 negative control cells were obtained;

[0070] (2) The screening ...

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Abstract

The invention relates to an HIV (human immunodeficiency virus) inhibitor screening model. The HIV screening model consists of a cell model I, a cell model II and a cell model III, wherein the cell model I is an HEK293T cell which is transfected by recombinant plasmids gp120 / pEGFP-N3 and CD4 / pDsRed-N1; the cell model II is an HEK293T cell which is transfected by recombinant plasmids gp120 / pDsRed-N1 and CXCR4 / pEGFP-N3; the cell model III is an HEK293T cell which is transfected by recombinant plasmids CD4 / pEGFP-N3 and CXCR4 / pDsRed-N1; the sequence of a gene gp120 which is inserted into the recombinant plasmids gp120 / pDsRed-N1 and gp120 / pEGFP-N3 is shown in SEQ ID NO.1; the sequence of a gene CD4 which is inserted into the recombinant plasmids CXCR4 / pEGFP-N3 and CXCR4 / pDsRed-N1 is shown in SEQ ID NO.2; the sequence of a gene CXCR4 which is inserted into the recombinant plasmids CXCR4 / pEGFP-N3 and CXCR4 / pDsRed-N1 is shown in SEQ ID NO.3. The HIV screening model has the advantages that the feature of FRET (fluorescence resonance energy transfer) is realized, and the anti-HIV drug can be screened.

Description

technical field [0001] The invention relates to biochemistry, in particular to a cell model modified by introducing foreign genes, and the system composed of the cell model can screen anti-AIDS drugs on a large scale. Background technique [0002] AIDS (acquired immunodeficiency syndrome, AIDS) is a major infectious disease caused by human immunodeficiency virus (human immunodeficiency virus, HIV) infection. In recent years, AIDS has spread rapidly around the world, increasingly threatening global public health, social, economic and political stability (Black RE. Lancet. 2013;382(9894):751-753). Since the most effective AIDS vaccine is still difficult to come out in a short period of time, the development of efficient, safe and inexpensive anti-HIV drugs is still a key measure to save the lives of AIDS patients, and it is also a top priority for the current AIDS prevention and treatment. [0003] So far, 26 anti-HIV monomeric drugs and 9 fixed-compatibility compound drugs h...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/85C12Q1/02
Inventor 马伟峰李百华段司沁范遥赵雪李雨静
Owner SOUTHERN MEDICAL UNIVERSITY
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