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Method for detecting ADCC (antibody-dependent cell-mediated cytotoxicity) activity of anti-HIV (human immunodeficiency virus) antibody

A carrier and packaging carrier technology, applied in the fields of molecular virology and genetic engineering, can solve the problem that the detection method is difficult to meet the evaluation of large-scale clinical trial samples

Active Publication Date: 2017-06-06
NAT INST FOR FOOD & DRUG CONTROL
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0010] To sum up, the currently existing anti-HIV antibody ADCC activity detection schemes all rely on live HIV virus culture, and it is difficult to break away from the BSL-3 laboratory, which is limited by the laboratory safety level and the number of virus strains. Existing detection methods It is difficult to meet the evaluation of large-scale clinical trial samples, and it is urgent to develop a simple, rapid and high-throughput method for evaluating the ADCC activity of anti-HIV antibodies

Method used

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  • Method for detecting ADCC (antibody-dependent cell-mediated cytotoxicity) activity of anti-HIV (human immunodeficiency virus) antibody
  • Method for detecting ADCC (antibody-dependent cell-mediated cytotoxicity) activity of anti-HIV (human immunodeficiency virus) antibody
  • Method for detecting ADCC (antibody-dependent cell-mediated cytotoxicity) activity of anti-HIV (human immunodeficiency virus) antibody

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Experimental program
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Effect test

Embodiment 1

[0084] Construction and identification of embodiment 1.GX-SF expression plasmid

[0085] CX-SF recombinant envelope protein comes from the transformation of pcDNA3.1-SF162 and pcDNA3.1-GX88. pcDNA3.1-SF162 comes from NIH AIDS Reagent in the United States; pcDNA3.1-GX88 comes from the isolates from patient serum in our laboratory. The isolated strain is obtained by extracting HIV RNA from plasma with the Qiagen Viral RNA Extraction Kit, reverse-transcribing it into cDNA, amplifying the membrane region fragment by PCR, connecting the T vector, picking a single clone, sequencing, and finally obtaining a monoclonal isolate The membrane protein GX88. The GX-SF envelope protein transformation process is as follows: figure 1 As shown, the specific steps include:

[0086] 1) Using pcDNA3.1-SF162 as a template, design primers:

[0087] SF-F: 5'-GTGGAGATATGAGGGACAAT-3'

[0088] SF-R: 5'-TTATAGCAAAGCCCCTTTCAAAGCC-3'

[0089] The sequence was amplified by 1386-2544bp to obtain the f...

Embodiment 2

[0100] The preparation of embodiment 2.HIV pseudovirus

[0101] 2.1 HIV pseudovirus packaging system

[0102] Pseudoviruses were prepared by co-transfecting eukaryotic cells with envelope protein expression plasmids and backbone plasmids. Among them, the HIV backbone plasmid used and the specific conditions for co-transfecting eukaryotic cells are described in detail in "Chinese Patent Application CN104830908A", which is incorporated herein by reference in its entirety. In this example, an exemplary envelope protein expression plasmid (pcDNA3.1) and a backbone plasmid (pSG3.Aenv.cmvFluc, referred to as pSG3-Fluc) were used to prepare HIV pseudoviruses.

[0103] 2.2 Expression of envelope protein expression plasmid and backbone plasmid in eukaryotic cells

[0104] 293FT cells were digested with trypsin, counted, and 6×10 6 Cells / 15ml inoculated on 75cm 2 Cell culture flasks, 37 °C, 5% CO 2 cultured in an incubator. When the confluence rate of the cells reaches about 50%, ...

Embodiment 3

[0113] Embodiment 3.GX-SF pseudovirus ADCC detection method

[0114] Use CEM-NKr cells as target cells. After counting the cells, dilute the number of cells to 1E6 / mL, and take 1 mL of cells. According to the calculation results, dilute the virus of the desired strain to MOI=1.0, put it into a T25 culture bottle, and make up the medium volume to 5 mL. Add 5 μL of 15mg / mL DEAE and mix well. Centrifuge at 1200g room temperature for 120min. 5% CO at 37°C 2 Incubate for 2 hours. The prepared target cells were washed 3 times with PBS. Follow 1 x 10 per well 4 cells / 50 μL, the cells were seeded in a U-shaped 96-well plate, and the wells around the plate were left as controls. Control wells were set as follows: Ee wells had only effector cells (NK92-6DF5 cells), Te and Tm had only target cells (CEM-NKr cells), P and V had only 150 μL medium, and empty wells were filled with 150 μL PBS. Dilute NK92-6DF5 cells to 1 x 10 5Cells / 50 μL were seeded in 96-well plates. Gently tap th...

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Abstract

The invention relates to the fields of molecular virology and genetic engineering. In particular, the invention relates to an HIV (human immunodeficiency virus) envelope protein, a coding sequence thereof, and an HIV pseudovirus comprising the HIV envelope protein; the HIV pseudovirus can be used to detect ADCC (antibody-dependent cell-mediated cytotoxicity) activity of an anti-HIV antibody. The invention also relates to a kit for detecting the ADCC activity of the anti-HIV antibody. The invention further relates to a method for detecting the ADCC activity of the anti-HIV antibody by using the HIV pseudovirus, the method can achieve rapid, simple and high-throughput detection of the ADCC activity of the anti-HIV antibody, and thereby the method is of great significance for protection effect on analysis of vaccine immune response, and development and quality control of a new vaccine.

Description

technical field [0001] The invention relates to the fields of molecular virology and genetic engineering. Specifically, the present invention relates to an HIV envelope protein, its coding sequence, and an HIV pseudovirus containing it, and the HIV pseudovirus can be used to detect the ADCC activity of an anti-HIV antibody. The invention also relates to a kit for detecting the ADCC activity of the anti-HIV antibody. The invention also relates to a method for detecting the ADCC activity of the anti-HIV antibody by using the HIV pseudovirus. Background technique [0002] Human immunodeficiency virus (Human Immunodeficiency Virus, HIV) belongs to the Retroviridae family and is a double-stranded positive-sense RNA enveloped virus. In the early days when HIV was discovered, it was also called LAV (Lymphadepathy Associated Virus) or HTLV-III (Human T Lymphotropic Virus III). Two types of HIV have been identified, HIV-1 and HIV-2. Among them, HIV-1 has a wide range of epidemics...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/12C12N7/01G01N33/68G01N33/577C12Q1/02
CPCC07K14/16C07K2319/00C12N7/00C12N2740/16021G01N33/5008G01N33/577G01N33/6854
Inventor 王佑春王萌聂建辉黄维金刘强
Owner NAT INST FOR FOOD & DRUG CONTROL
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