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HIV-1 phenotypic resistance detection vector and construction method thereof

An HIV-1, 1. HIV-1 technology, applied in the field of HIV-1 phenotype drug resistance detection vector and its construction, can solve the problems of long detection time, failure, substitution of drug resistance fragments in RT region, etc. Detection time and the effect of improving detection efficiency

Active Publication Date: 2013-04-24
盛世菁华(北京)生物科技有限公司
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AI Technical Summary

Problems solved by technology

However, the emergence of HIV drug resistance has greatly limited the effectiveness of antiretroviral therapy
[0003] HIV drug resistance not only reduces the effect of antiviral treatment in patients, but even fails completely
The emergence and prevalence of HIV drug-resistant strains will bring serious public health problems: 1) The failure of treatment caused by drug resistance will increase the number of AIDS deaths; 2) The replacement of "second-line drugs" will increase the cost of treatment; spread
However, the pseudovirus backbone carrier molecule is very large, and it is difficult for the traditional phenotypic drug resistance detection system to replace the drug-resistant fragment in the RT region in a timely and effective manner, resulting in a long detection time, which is not conducive to the timely formulation of a reasonable clinical treatment plan

Method used

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  • HIV-1 phenotypic resistance detection vector and construction method thereof
  • HIV-1 phenotypic resistance detection vector and construction method thereof
  • HIV-1 phenotypic resistance detection vector and construction method thereof

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Embodiment Construction

[0025] The novel HIV-1 phenotypic drug resistance detection carrier of the present invention is constructed by the following method, the steps are:

[0026] (1) pcDNA6.2gw_miR vector reconstruction based on Invitrogen's gateway recombination system

[0027] 1. Construction of pcDNA6.2-gag-1 vector

[0028] 1.1 PCR amplification of the gag-1 fragment, the sequence of which is shown in SEQ ID No.1.

[0029] Primer: gag-S-BamH1: tgactagcggatcctagaaggaga

[0030] gag-ASE-NheI-BgII:GAGAGATCTGAGGGAAGCTAGCGGATACAG

[0031] The template is pNL4-3 plasmid (pNL4-3 is HIV-1 wild strain pseudovirus backbone plasmid, GenBank number: M19921.2) PCR conditions: pre-denaturation at 94°C for 2min, denaturation at 94°C for 30s, annealing at 50°C for 30s, extension at 72°C Cycle 30 times in 1.5 min, and finally extend at 72°C for 10 min.

[0032] 1.2 The gag-1 fragment was digested with BamhI and BglII to recover the gag-1 fragment, and inserted into the pcdna6.2gw_miR vector (Invitrogen, wit...

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Abstract

The invention relates to a HIV-1 phenotypic resistance detection vector. The vector is characterized in that: a structure of the vector is represented by a figure 6. According to the present invention, a novel general system for cloning or subcloning DNA sequence is provided. A powerful in vitro technology of the system adopts site-specific recombination without using restriction enzyme and ligase, such that time and labor are saved, and correct direction and correct reading frame of a target gene are ensured. . An entry vector cloned by a construction method provided by the present inventioncan be used repeatedly, and can transfer a target gene into various expression vectors modified by Gateway. Therefore, according to the novel phenotypic resistance detection vector, phenotypic resistance detection time is substantially reduced and phenotypic resistance detection efficiency is improved; a foundation for further researching HIV-1 phenotypic resistance is established.

Description

technical field [0001] The invention fully utilizes the molecular cloning technique and the site recombination principle to construct an entry clone and an objective expression clone. Constitute a new phenotypic drug resistance detection system. Background technique [0002] The widespread application of AIDS antiretroviral therapy has significantly prolonged the life expectancy of patients, improved the quality of life of patients, and greatly reduced HIV / AIDS-related morbidity and mortality. However, the emergence of HIV drug resistance has greatly limited the effect of antiviral therapy. [0003] HIV drug resistance not only reduces the effect of antiretroviral therapy in patients, but even fails completely. The emergence and prevalence of HIV drug-resistant strains will bring serious public health problems: 1) The failure of treatment caused by drug resistance will increase the number of AIDS deaths; 2) The replacement of "second-line drugs" will increase the cost of t...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/63C12N15/66C12Q1/68
Inventor 陈德喜乔录新魏飞力张玉林丁渭李宁
Owner 盛世菁华(北京)生物科技有限公司
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