Screening system of HIV infected cell and applications thereof

Abstract

The invention provides a screening system of HIV infected cell. A reporter protein gene is divided into a reporter gene N-end and a reporter gene C-end, wherein the reporter gene N-end and dnae-N which has the protein splicing function and is the part of DnaE intein N-end gene are blended to form an expression vector I; and the reporter gene C-end and dnae-C which has the protein splicing functionand is the part of DnaE intein C-end gene are blended to form an expression vector II. The two expression vectors respectively transfect an HEK293 cell or a CHO cell containing chemotactic factors and CD4 protein or containing envelope protein Env of HIV, and establish stable expression cell stains. When the two strains of stable cells are cultured mixedly, the cells are blended, thus promoting DnaE-C and DnaE-N to mutually contact and act; while the combined action of DnaE-N and DnaE-C connects the reporter protein N-end and the reporter protein C-end into one complete reporter protein, andfinally, the blending degree can be learned by the protein activity of a test report.

Description

(1) Technical field [0001] The invention relates to a cell-cell fusion drug screening system simulating HIV-infected cells and its application in screening chemokine CCR5 and CXCR4 antagonists. (2) Background technology [0002] The envelope glycoprotein gene env of HIV, after being encoded into mRNA, is transported from the infected cell nucleus to the cytoplasm under the action of the Rev protein, and then synthesizes the precursor protein gp160 in the rough endoplasmic reticulum; gp160 is in its internal gp41 Under the guidance of the signal peptide on the domain, it immediately enters the lumen of the endoplasmic reticulum, and the formation of intramolecular disulfide bonds, oligomerization, and high glycosylation modification of the gp120 domain part; in the process of transferring from the endoplasmic reticulum to the Golgi apparatus In the process, the gp160 precursor protein is cleaved by the host protease (furinora furin-like enzyme) to form a complex of mature env...

AI Technical Summary

Problems solved by technology

Using different cell lines and fusion detection methods, there are many test methods, including: 1. Observation of cell-cell fusion through cell membrane and nucleus staining. The lengthy operation steps of this method are not suitable for large-scale drug screening test

2. Detection of cell fusion through the expression of transcriptional activation signal genes after cell fusion. This method has been used in large-scale screening experiments, but because it takes a certain amount of time to activate the transcriptional activation of signal proteins, the screening time needs to be improved

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