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Application of recombinant lentivirus to HIV (human immunodeficiency virus) phenotypic drug resistance detection

A recombinant lentivirus and phenotype technology, applied in the field of medical molecular biology, can solve the problems of biosafety and high cost, and achieve the effect of strong versatility, safety, high efficiency and toxic side effects

Active Publication Date: 2016-06-08
广州佰芮慷生物科技有限公司
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  • Abstract
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  • Claims
  • Application Information

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Problems solved by technology

Although these assays have great application prospects for phenotypic drug resistance analysis, due to the high-throughput quantitative firefly luciferase expression system, the cost is still very high, and there may be biosafety issues

Method used

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  • Application of recombinant lentivirus to HIV (human immunodeficiency virus) phenotypic drug resistance detection
  • Application of recombinant lentivirus to HIV (human immunodeficiency virus) phenotypic drug resistance detection
  • Application of recombinant lentivirus to HIV (human immunodeficiency virus) phenotypic drug resistance detection

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Embodiment Construction

[0039] The present invention will be described in detail below in conjunction with the accompanying drawings and embodiments.

[0040] A recombinant lentiviral system of the present invention is constructed by the following method, the steps are:

[0041] (1) Construction of transfer plasmid

[0042] The transfer plasmid of the present invention has double reporter gene luciferase Luciferase and ZsGreen, and its specific structure is as follows figure 1 shown. Luciferase and ZsGreen are independently expressed proteins, not fusion proteins.

[0043] Such as figure 2 As shown, the construction method of the transfer plasmid is as follows:

[0044] (1.1) Use BamHI and KpnI to double digest the pMD2.G plasmid, recover the digested product, and collect the 119bp CMV fragment;

[0045] (1.2) The pHAGE-EF1α-IRES-ZsGreen plasmid was double digested with BamHI and KpnI, and the 4736bppHAGE-IRES-ZsGreen fragment was recovered;

[0046] (1.3) Ligate the recovered CMV fragment and...

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Abstract

The invention discloses application of recombinant lentivirus to HIV (human immunodeficiency virus) phenotypic drug resistance detection. The recombinant lentivirus is a package body obtained through cotransfection on a 293FT cell or 293T cell by a packaging plasmid, an envelope plasmid and a transferred plasmid, wherein the envelope plasmid is a psPAX2m-pol plasmid; the transferred plasmid is a pHAGE-CMV-Luc-IRES-ZsGreen plasmid. The recombinant lentivirus is safe and efficient; the toxic or side effect is small; the cost is low; when the recombinant lentivirus is applied to HIV phenotypic drug resistance detection, important reference basis can be provided for reasonably and effectively selecting anti-HIV medicine in clinics. The reporter genes Luciferase and ZsGreen of the transferred plasmid are positioned on pHAGE-CMV-Luc-IRES-ZsGreen; the expression of the reporter genes Luciferase and ZsGreen is controlled by a CMV (cytomegalovirus) promoter, so that the antiviral effect of an HIV-1 (human immunodeficiency virus-1) inhibitor can be simply obtained through the observation under an inverted fluorescence microscope or the determination of luciferase activity; after a pol region is imported, various cells can realize drug resistance detection by the recombinant lentivirus with the transferred plasmid; the universality is high.

Description

technical field [0001] The invention relates to the field of medical molecular biology, in particular to the application of a recombinant lentivirus in the detection of HIV phenotype drug resistance. Background technique [0002] Over the past few decades, a variety of HIV-1 inhibitory drugs have been approved for the treatment of HIV-infected patients. Highly active antiretroviral therapy (HAART), based on the combination of two or more inhibitors of HIV-1 reverse transcriptase and (or) protease, is recognized as one of the most effective methods for treating HIV-infected patients. It can not only improve the quality of life of HIV-infected patients but also reduce the risk of HIV infection. Although HAART inhibits viral replication and reduces the risk of HIV infection, it does not clear the virus from HIV-1 infected individuals. As a result, antiretroviral therapy is compromised by emerging drug-resistant strains under drug-selective pressure. Therefore, it is necessar...

Claims

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Application Information

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IPC IPC(8): C12Q1/66C12Q1/02C12N7/01C12N15/867
CPCC12N7/00C12N15/86C12N2740/15021C12N2740/15043C12Q1/66G01N33/502G01N33/5044
Inventor 黎诚耀翁云层张玲李金峰
Owner 广州佰芮慷生物科技有限公司
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