Application of CBX4 used as HIV-1 latent infection activation target

A latent infection, HIV-1 technology, applied to medical preparations containing active ingredients, antiviral agents, pharmaceutical formulas, etc., can solve the problems of undisclosed CBX4 and other issues

Active Publication Date: 2018-01-19
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

CBX4 is a novel HIV-1 lat...

Method used

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  • Application of CBX4 used as HIV-1 latent infection activation target
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  • Application of CBX4 used as HIV-1 latent infection activation target

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Knocking down the expression of CBX4 gene can effectively promote the transcriptional activity of HIV-1 LTR, the specific experimental method is as follows:

[0019] (1) Take well-grown TZM-bl cells and seed them in 24-well plates. The medium used is a complete medium: high-glucose DMEM, 10% fetal bovine serum and 1% double antibody, and the culture condition is 5% carbon dioxide, 37°C;

[0020] (2) After 24 hours of adherence, co-transfect the pcDNA3.1-Tat plasmid and siRNA-CBX4 or siRNA-NC, respectively, and continue to culture under the conditions of 1);

[0021] (3) After 48 hours, collect the cell pellet, take a part of it and use the Trizol method to extract RNA, reverse to obtain cDNA, use CBX4-specific Q-PCR primers to detect the mRNA expression level of CBX4, and determine the knockdown efficiency of siRNA-CBX4;

[0022] (4) The remaining cells were taken, and after lysing, the transcriptional activity of HIV-1 LTR in different transfection treatment groups wa...

Embodiment 2

[0026] Inhibition of CBX4 in J-lat 10.6 HIV-1 latently infected cells effectively activates the HIV-1 latent infection reservoir, the specific experimental method is as follows:

[0027] (1) Synthesize the specific base sequence, anneal to construct the shRNA targeting CBX4, PLKO.1-CBX4, and confirm the correct sequence by sequencing.

[0028] (2) Take the well-grown human epi-renal cell line 239T cells and inoculate them in a 10cm flat-bottomed plate. The medium used is a complete medium: high-glucose DMEM, 10% fetal bovine serum and 1% double antibody, and the culture condition is 5% carbon dioxide, 37°C;

[0029] (3) After 24 hours of adherence, transfect PLKO.1-CBX4 or PLKO.1-NC and psPAX2, VSV-G into the cells, and continue to culture as in 2);

[0030] (4) After 48 hours, collect the supernatant, use PEG-6000 to concentrate the virus particles, and use the ELISA P24 kit to detect virus P24;

[0031] (5) Take the well-grown J-lat10.6 cells, count them, and infect them w...

Embodiment 3

[0038] Overexpression of CBX4 protein in TZM-bl cells can inhibit the transcriptional activity of LTR of HIV-1, the specific experimental method is as follows:

[0039] (1) Order the CBX4 cDNA plasmid template, design appropriate primers, and construct the CBX4 overexpression plasmid, pcDNA3.1-CBX4-FLAG;

[0040] (2) Spread well-grown TZM-bl cells in a 6-well transparent plate, 1×106 cells per well, the medium used is complete medium: high-glucose DMEM, 10% fetal bovine serum and 1% double antibody, The culture conditions are 5% carbon dioxide, 37°C;

[0041] (3) After 24 hours of attachment, transfect the pcDNA3.1-CBX4-FLAG plasmid, and continue to culture as in 2);

[0042] (4) After 48 hours, collect the cells, use RIPA lysate to lyse the cells, and detect the overexpression level of CBX4 by western blot to determine the effect of the pcDNA3.1-CBX4-FLAG overexpression plasmid;

[0043] (5) Spread well-grown TZM-bl cells in a 24-well transparent plate, 1×105 cells per well...

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Abstract

The invention discloses application of CBX4 used as an HIV-1 latent infection activation target. It is found through researches that inhibition of CBX4 has good HIV-1 latent activation capability andknock-down of CBX4 can effectively promote transcription of HIV-1 LTR. Then, it is found in a J-lat 10.6 HIV-1 latent infection cell model that through knock-down of CBX4, CFP gene expression is up-regulated and HIV-1 can be effectively activated. Through overexpression of CBX4 protein in TZM-bl cell, transcriptional activity of LTR of HIV-1 can be effectively reduced. Through further detection, it is found that reduced CBX4 protein expression will reduce H3K9 trimethylation of HIV-1 LTR and enrichment of H3K27 trimethylation so as to activate HIV-1 latent infection. The CBX4, which is used asa brand new HIV-1 latent infection activation target, has important research and development value and significance of development in the aspect of HIV-1 latent infection activation.

Description

technical field [0001] The present invention relates to the technical field of disease-related functional targets, specifically, to the technical field of HIV-1 latent infection activation targets, and more specifically, to the application of CBX4 as an HIV-1 latent infection activation target. Background technique [0002] HIV-1 virus is a lentivirus that infects cells of the human immune system. It attacks human T lymphocytes and destroys the human immune system, eventually leading to the generation of acquired immunodeficiency syndrome "AIDS". The virus was first discovered in 1981, and researchers have been working on its control and elimination for more than 30 years since then, hoping to cure the disease. The current conventional treatment method is referred to as cART combined antiretroviral therapy, which can effectively inhibit the replication of the virus, thereby controlling the viral load in the patient's peripheral blood to a low level. However, the current gen...

Claims

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Application Information

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IPC IPC(8): A61K45/00A61P31/18
Inventor 张辉陈灿灿
Owner SUN YAT SEN UNIV
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