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HIV-1B subtype drug-resistant mutant and construction method and application thereof

A technology for HIV-1B, drug-resistant mutation, applied in the fields of biochemical equipment and methods, introduction of foreign genetic material using vectors, determination/inspection of microorganisms, etc.

Inactive Publication Date: 2012-03-28
MICROBE EPIDEMIC DISEASE INST OF PLA MILITARY MEDICAL ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The long duration of antiviral treatment and the characteristics of the above antiviral mode suggest that there may be new drug resistance mutations that have not yet been recognized in HIV-1B subtype strains

Method used

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  • HIV-1B subtype drug-resistant mutant and construction method and application thereof
  • HIV-1B subtype drug-resistant mutant and construction method and application thereof
  • HIV-1B subtype drug-resistant mutant and construction method and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Embodiment 1, discovery of drug resistance mutation site

[0026] Comparing the full-length gene sequence of the protein between patients who failed antiviral therapy and those who were not treated, there were 7 mutations at 6 sites in the 560 codons in the reverse transcriptase region in the two groups of people, one of which was T369V.

[0027] The amino acid sequence of the reverse transcriptase of HIV-1B subtype is shown in sequence 1 of the sequence listing, and its coding sequence is shown in sequence 2 of the sequence listing.

[0028] From the protein level, the T369V mutation is about to mutate the reverse transcriptase (shown in sequence 1 of the sequence listing) of the HIV-1B subtype wild strain from threonine (T) to valine from the 369th amino acid residue at the N-terminal (V). From the genetic level, the T369V mutation can be a mutation of the reverse transcriptase coding gene (shown in sequence 2 of the sequence listing) of the HIV-1B subtype wild strai...

Embodiment 2

[0029] Embodiment 2, the construction of HIV-1B subtype drug-resistant mutant strain (HIV-1B subtype T369V mutant strain)

[0030]pNL4.3 plasmid: The public can obtain it from the Institute of Microbial Epidemiology, Academy of Military Medical Sciences of the Chinese People's Liberation Army; References: Wang C, Mitsuya Y, Gharizadeh B, Ronaghi M, Shafer RW.Characterization of mutation spectrum with ultra-deep pyrosequencing: application to HIV-1 drug resistance. Genome Res. 2007; 17(8): 1195-201.; Hoffmann C, Minkah N, Leipzig J, Wang G, Arens MQ, Tebas P, Bushman FD. NA bar coding and pyrosequencing to identify rare HIV drug resistance mutations.Nucleic Acids Res.2007;35(13):e91.;pNL4.3 plasmid contains the whole genome sequence of HIV-1B subtype wild strain, transfected cells and expressed can get HIV-1B subtype wild strain strain.

[0031] 293T cells: purchased from the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences.

[0032] MT-2 cells: The pu...

Embodiment 3

[0055] Example 3, Identification of drug resistance of HIV-1B subtype drug-resistant mutants

[0056] 1. Add 10 microliters of drug solution to the cell culture plate (the drug solution is composed of drug and water; three drugs, AZT, EFV and NVP, are used respectively; 6 gradient concentrations are set for each drug: AZT is 2000nM~2.74nM, dilute degree is 3 0 ~3 -6 ; EFV is 100nM ~ 0.14nM, the dilution is 3 0 ~3 -6 ; NVP is 6000nM ~ 8.23nM, the dilution is 3 0 ~3 -6 ; each concentration of each drug was set up in triplicate wells).

[0057] 2. Adjust the concentration of the HIV-1B subtype T369V mutant strain virus liquid (or the HIV-1B subtype wild strain virus liquid obtained by the step 3 of embodiment 2) obtained in step 2 of embodiment 2 with PBS buffer to obtain virus Diluent.

[0058] 3. Add 20 μl of the virus dilution in step 2 to each well of the cell culture plate in step 1, and then add 70 μl of MT-2 cell suspension to each well (the cell concentration in th...

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Abstract

The present invention discloses an HIV-1B subtype drug-resistant mutant and a construction method and application thereof. In accordance with the method provided by the present invention, the reverse transcriptase of an HIV-1B subtype wild strain is mutated from threonine to valine from the 369th amino acid residue at the N tail end, so as to obtain an HIV-1B subtype drug-resistant mutant. The present invention is significant for the sifting of HIV-resistant drugs, thereby having far-reaching influence on the cure of AIDS.

Description

technical field [0001] The invention relates to an HIV-1B subtype drug-resistant mutant strain and its construction method and application. Background technique [0002] Human immunodeficiency virus (HIV) is the pathogen that causes human acquired immunodeficiency syndrome (Acquired immunodeficiency syndrome, AIDS). Since AIDS was first diagnosed in the United States in 1981, AIDS has spread rapidly around the world. AIDS has been prevalent in China for 25 years. As of the end of October 2010, China has reported more than 370,000 HIV-infected and AIDS patients, including more than 130,000 AIDS patients, and more than 68,000 deaths. With the support of the national "Four Frees and One Care" policy, my country has gradually implemented free AIDS antiretroviral treatment nationwide since 2003, and by the end of 2010, the number of people treated had exceeded 70,000. Clinical observations show that antiviral therapy has a good effect on alleviating the disease, improving the qu...

Claims

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Application Information

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IPC IPC(8): C12N15/63C12N7/00C12Q1/70C12Q1/02
Inventor 李韩平李敬云耿庆茂郭伟鲍作义李林刘永健庄道民刘思扬李天一王晓林
Owner MICROBE EPIDEMIC DISEASE INST OF PLA MILITARY MEDICAL ACAD OF SCI
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