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A HIV inhibitor screening model and its preparation method and application

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

Inactive Publication Date: 2019-09-13
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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  • A HIV inhibitor screening model and its preparation method and application
  • A HIV inhibitor screening model and its preparation method and application
  • A HIV inhibitor screening model and its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

preparation example

[0034] 1. Materials and instruments:

[0035] 1. The base sequence is SEQ ID NO.1~SEQ ID NO.3 synthesized by Shanghai Yingwei Jieji 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 and 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] select as figure 1 The 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 the following table 1 are used for CD4, CD4, and CXCR4, respectively. The coding genes of CXCR4 and gp120 and the expression vector are subjected to corresponding restriction enzyme digest...

example 1

[0054] 1. Drugs to be screened

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

[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 passage of molecules 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, which acts on the early stage of HIV-1 infection, specifically blocks the entry of HIV-1 with CXCR4 as a co-receptor, does not act on CCR5 receptors, and does not affect gp120 protein take effect.

[0059] 2. Screening method

[0060] (1) Use the 6 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 Transfect HEK293T cells to obtain...

example 2

[0064] 1. Drugs to be screened

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

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

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

[0068] 2. Screening method

[0069] (1) Use the 6 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 Transfect HEK293T cells to obtain 6 cells; then inoculate 6 cells in 24-well cell culture plates with coverslips, place at 37°C and fill with a volume concentration of 5% CO 2 Cultivate in an air-conditioned cell culture box for 48 hours, then wash with PBS three times, fix the cells with a volume concentration of 4% paraformaldehyde solution for 15-20 minutes, then fix the coverslips loaded with cells on glass slides, and seal them with a capping agent. slices to obtai...

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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 introduced with foreign gene modification, 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, society, economy and political stability (BlackRE.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 cheap anti-HIV drugs is still the key measure to save the lives of AIDS patients, and it is also the top priority of the current AIDS prevention and control. [0003] So far, 26 anti-HIV monomer drugs and 9 fixed-compatibility compound drugs have b...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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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