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Strain kit for screening and evaluating AIDS (acquired immune deficiency syndrome) therapeutic drugs

A technology of virus strains and kits, applied in the field of virus strain kits, can solve the problems of high toxicity and side effects, few varieties, and inability to meet clinical needs

Inactive Publication Date: 2015-07-15
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

[0006] At present, there are about 9 kinds of drugs included in the anti-HIV free treatment list in my country (Kalitra is the only free protease inhibitor), most of which are generic drugs with expired patents, few varieties, and large toxic and side effects, which cannot meet clinical needs

Method used

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  • Strain kit for screening and evaluating AIDS (acquired immune deficiency syndrome) therapeutic drugs
  • Strain kit for screening and evaluating AIDS (acquired immune deficiency syndrome) therapeutic drugs
  • Strain kit for screening and evaluating AIDS (acquired immune deficiency syndrome) therapeutic drugs

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Embodiment 1, construction of recombinant plasmid

[0047] 1. Using the pNL4.3 plasmid as a template, the primer pair consisting of OUT-1315 and OUT-3525 was used for PCR amplification to obtain PCR amplification products.

[0048] 2. Using the PCR amplification product obtained in step 1 as a template, a primer pair consisting of IN-Sph I and IN-Age I is used for PCR amplification to obtain a PCR amplification product. After sequencing, the PCR amplification product is shown in the 1434th to 3514th nucleotides of Sequence 1 in the sequence listing.

[0049] 3. Ligate the PCR amplification product obtained in step 2 with the pMD18-T vector to obtain a recombinant plasmid.

[0050]4. Using the recombinant plasmid obtained in step 3 as a template, perform PCR amplification with a primer pair consisting of F(M46I) and R(M46I), and obtain recombinant plasmid M46I under the action of T4 DNA ligase. Because circular DNA is amplified, F(M46I) and R(M46I) amplify the sense st...

Embodiment 2

[0070] Embodiment 2, construct HIV mutant strain

[0071] 1. Suspend 293T cells in DMEM medium containing 10% fetal bovine serum (FBS) by volume to obtain 4×10 5 cells / ml of cell suspension.

[0072] 2. Add the cell suspension obtained in step 1 to a 6-well culture plate, 2ml per well, 37°C, 5% CO 2 Incubate for 24 hours.

[0073] 3. With the help 2000 (operated according to the instructions), transfect the cells of step 2 with the recombinant plasmid pNL4.3-M46I (transfection of 2.5 micrograms of recombinant plasmid per well), 37 ° C, 5% CO 2 After culturing for 36 hours, collect the culture supernatant, which is the HIV-M46I strain virus culture supernatant, and store at -80°C after aliquoting.

[0074] The above steps were carried out by using the recombinant plasmid pNL4.3-I54V instead of the recombinant plasmid pNL4.3-M46I to obtain the HIV-I54V strain virus culture supernatant.

[0075] The above steps were carried out by using the recombinant plasmid pNL4.3-V82A i...

Embodiment 3

[0081] Embodiment 3, the virus infectivity identification of mutant virus strain

[0082] The HIV-M46I strain virus culture supernatant prepared in Example 2, the HIV-I54V strain virus culture supernatant, the HIV-V82A strain virus culture supernatant, the HIV-M46I / N88S strain virus culture supernatant, the HIV-G48V / I54V strain Virus culture supernatant, HIV-M46I / V82T / I84V strain virus culture supernatant, HIV-G48V / I54V / V82A strain virus culture supernatant and HIV-1B subtype pNL4.3 wild strain virus culture supernatant were used as the virus culture supernatant to be tested. Clear, carry out the following identification respectively:

[0083] 1. Suspend TZM-bl cells in DMEM medium containing 10% FBS by volume to obtain 1.1×10 4 cells / ml of TZM-bl cell fluid.

[0084] 2. Add the cell suspension obtained in step 1 to a white 384-well plate, and add 90 μl to each well.

[0085] 3. Dilute the culture supernatant of the virus to be tested in a 3-fold gradient with DMEM culture ...

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Abstract

The invention discloses a strain kit for screening and evaluating AIDS (acquired immune deficiency syndrome) therapeutic drugs. The invention provides a kit which comprises 7 recombinant HIVs (human immunodeficiency viruses): HIV-M46I virus strain, HIV-I54V virus strain, HIV-V82A virus strain, HIV-M46I / N88S virus strain, HIV-G48V / I54V virus strain, HIV-M46I / V82T / I84V virus strain and HIV-G48V / I54V / V82A virus strain. Site-directed mutagenesis is utilized to establish a set of HIV-1 proteinase inhibitor drug-resistant mutant strains. The earlier stage test proves that the HIV-1 proteinase inhibitor drug-resistant mutant strains have certain drug resistance to the proteinase inhibitors in the market at present. The new drugs can only have potential marketing value when having favorable antivirus effects for the strains. Therefore, the kit is applicable to preclinical pharmacodynamic evaluation of AIDS therapeutic drugs.

Description

technical field [0001] The invention relates to a virus strain reagent kit for screening and evaluating AIDS treatment drugs. Background technique [0002] Human immunodeficiency virus (Human immunodeficiency virus, HIV) is the pathogen that causes human acquired immunodeficiency syndrome (Acquired immunodeficiency syndrome, AIDS). AIDS has spread rapidly around the world since it was first diagnosed in the United States in 1981. According to the latest UNAIDS report in July 2014, the number of HIV infections worldwide has reached 35 million. In 2013, there were 63,498 newly diagnosed HIV infections in China, and it is estimated that there are currently 780,000 surviving HIV infections in China. [0003] HIV-1 protease is an aspartic protease consisting of two homodimers each 99 amino acids in length. HIV-1 protease mainly acts on the translation of the virus, cutting the gag and gag-pol protein polymers to produce structural proteins and some enzymes. The hydrophobic sub...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/02C12R1/93
CPCC12Q1/025
Inventor 常帅李林庄道民刘思扬李敬云李天一张文福
Owner MICROBE EPIDEMIC DISEASE INST OF PLA MILITARY MEDICAL ACAD OF SCI
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