T lymphocyte for screening and activating dormant infection HIV-1 compound and preparation thereof

A latent infection, lymphocyte technology, applied in the field of genetic engineering and cell engineering, can solve problems such as unsatisfactory results

Inactive Publication Date: 2009-02-25
BEIJING BIOHEALTHCARE BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although there are several plans to eliminate the virus storage pool, satisfactory clinical results have not yet been obtained. Therefore, the dev

Method used

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  • T lymphocyte for screening and activating dormant infection HIV-1 compound and preparation thereof
  • T lymphocyte for screening and activating dormant infection HIV-1 compound and preparation thereof
  • T lymphocyte for screening and activating dormant infection HIV-1 compound and preparation thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1 Preparation method of lentiviral vector

[0032] The lentiviral vector FUGW was digested with PacI+BamhI, the 8691BP fragment was recovered, ligated with T4 ligase, and the transformed bacterial clone was extracted for DNA sequence analysis, and finally the FGW plasmid with the deletion of the ubiquitin regulatory region was obtained.

Embodiment 2

[0033] Example 2 Preparation of lentiviral vector

[0034]The FGW vector was mixed with the packaging construct plasmid CMVΔ8.9 and the envelope plasmid (VSVG) at a mass ratio of 2:1:1. Use liposome LipofectamineTM to transfect on 293T cells, observe under a fluorescent microscope after 24-48 hours, collect the virus supernatant after a large amount of fluorescence appears, and collect the collected virus supernatant after concentration for equipment or use immediately. Recombinant lentivirus activity During the measurement, the concentrated virus stock solution was diluted in different proportions, and the fluorescence count was performed under a fluorescence microscope after infecting the cells for 48 hours to determine the titer. FGW vector served as a control. The results showed that the site-specific integration lentivirus titer was 6.8X10 -7 TU / ML.

Embodiment 3

[0035] Example 3 Analysis of cells infected by lentivirus system

[0036] The above 1 ul lentiviral system FGW- (packaged by plasmid CMVΔ8.9) was infected with the Jurkat cell line on a 6-well plate. After 72 hours, observation under a fluorescent microscope showed that Jurkat cells showed green fluorescence; the infection efficiency was 82.1%. Non-fluorescing cells were harvested and subjected to single cell clonal expansion in 96-well plates.

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Abstract

The invention belongs to the fields of genetic engineering and cell engineering, pertaining to a human T-lymphocytic series model for screening and reactivating latent infection HIV-1 compound and a preparation method thereof. The invention discloses a T lymphocyte used for screening and activating latent infection HIV-1 drug, and the T lymphocyte is a Jurkat stable strain that is infected by but not expresses HIV slow virus carrying reporter gene. The preservation number of the T lymphocyte is CCTCC NO.C200821. The invention also provides a preparation method for the T lymphocyte, including infecting human T-lymphocytic series Jurkat with the HIV-1 slow virus carrying EGFO reporter gene, and carrying out cell sorting and HIV integration test so as to obtain the clone with HIV integration while not expressing EGFP. The invention is the cell model for the virus preservation library to control drug screening.

Description

technical field [0001] The invention belongs to the field of genetic engineering and cell engineering, and relates to a human T lymphocyte line model used for screening and reactivating latently infected HIV-1 compounds and a preparation method thereof. Background technique [0002] Acquired Immunodeficiency Syndrome (AIDS) is an infectious disease caused by HIV infection that seriously endangers people's life and health. According to WHO statistics, there are more than 40 million AIDS patients in the world, with 5 million new patients and 3 million deaths each year. At present, the clinical treatment of AIDS is mainly Highly active antiretroviral therapy (HAART), because it can effectively reduce the viral load (virus load) and protect the immune function, thus prolonging the life expectancy of HIV-infected patients accordingly. survival time. However, this therapy can only inhibit virus replication, and cannot completely eliminate HIV infection. Most patients will experi...

Claims

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Application Information

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IPC IPC(8): C12Q1/02C12Q1/68C12N5/10C12N15/867
Inventor 朱焕章辛清婷刘绍辉唐丽莎余龙
Owner BEIJING BIOHEALTHCARE BIOTECH
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