A method of screening an HIV-1 activity determinative factor based on high-throughput RNAi

A technology of HIV-1, determinant, applied in the field of molecular biology

Inactive Publication Date: 2016-10-19
SHANGHAI PUBLIC HEALTH CLINICAL CENT
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Problems solved by technology

[0003] The regulation of transcriptional activity of HIV-1 LTR involving kinases has become one of the focuses of research in this field. So far, there have been no reports on the functional silencing screening of human kinase genes and the search for host factors related to HIV replication. In view of the Therefore, the inventors of the present application intend to provide

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  • A method of screening an HIV-1 activity determinative factor based on high-throughput RNAi
  • A method of screening an HIV-1 activity determinative factor based on high-throughput RNAi
  • A method of screening an HIV-1 activity determinative factor based on high-throughput RNAi

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Embodiment 1

[0032] Example 1 Detection of HIV-1 LTR Regulated Transcriptional Activity Participated by Kinases

[0033] The HIV-1 LTR-hRluc / HSV TK-hLuc luciferase dual reporter plasmid was stably expressed in HEK293T by using shRNA library high-throughput RNAi screening technology for 474 kinases; Take the fluorescence intensity of hLuc (such as figure 1 shown in A);

[0034] Two positive controls, NFκB and AKT1, were used for screening as shRNA or specific inhibitors (eg figure 1 Shown in B); inhibitors of NFκB or AKT1 kinase activity inhibited the activity of HIV-1 LTR by more than 30%, indicating that the screening system was reliable; in addition, the initial screening results showed 155 positive samples against HIV-1 LTR inhibited by 136 genes activity over 20% (eg figure 1 Shown in C), then above-mentioned 155 samples enter the second screening system, obtained 15 positive genes, the results are as shown in table 1;

[0035] Table 1. qPCR. Primers

[0036]

[0037] The resul...

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Abstract

The invention relates to the technical field of molecular biology, and particularly relates to a method of screening an HIV-1 activity determinative factor based on high-throughput RNAi. Aiming at shRNA libraries of 474 kinases, and through a high-throughput RNAi screening technique, HIV-1 LTR-hRluc/HSVTK-hLuc luciferase double reporter plasmids are stably expressed in HEK293T, and kinase-involved HIV-1 LTR regulatory transcriptional activity is obtained by dividing HIV-1 LTR relative transcriptional activity of hRluc expression fluorescence intensity by hLuc fluorescence intensity. The method aims at human kinase genes, performs functional silence screening, searches HIV replication related host factors, and provides valuable reference basis for further understanding interactions between HIV and hosts and for searching HIV-infection resisting potential treatment targets.

Description

technical field [0001] The invention belongs to the technical field of molecular biology. A method for screening HIV-1 activity determinants, more specifically, a method for screening HIV-1 activity determinants based on high-throughput RNAi. Background technique [0002] The prior art discloses that the currently known human immunodeficiency virus (HIV-1) genome only encodes 15 proteins, and its life cycle from invasion, replication, assembly to budding release needs to be completed using host-encoded proteins. Therefore, HIV infection Host factors will be potential targets for the treatment of HIV. According to many research reports, kinases play an important regulatory role in the various stages of the above HIV life cycle. [0003] The regulation of transcriptional activity of HIV-1 LTR involving kinases has become one of the focuses of research in this field. So far, there have been no reports on the functional silencing screening of human kinase genes and the search ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/66C12Q1/02C12N15/867
Inventor 卢洪州蒋卫民
Owner SHANGHAI PUBLIC HEALTH CLINICAL CENT
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