Human anti-HIV gp 120 specific antibody Z166 and application method thereof

A Z166, specific technology, applied to human anti-HIV gp120 specific antibody Z166 and its application field

Pending Publication Date: 2019-11-22
GUIZHOU MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

No patent application related to the human anti-HI

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  • Human anti-HIV gp 120 specific antibody Z166 and application method thereof
  • Human anti-HIV gp 120 specific antibody Z166 and application method thereof
  • Human anti-HIV gp 120 specific antibody Z166 and application method thereof

Examples

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example 1

[0024] Example 1: Preparation of Human Anti-HIVgp120 Specific Antibody

[0025] 1. Antigen labeling.

[0026] A plasmid (pcDNA) containing the full-length envelope protein (Accession NO.EU123924) of HIV Sf162 isolate was expressed from 293F (Thermo Fisher Ltd, CAT#R79007) cells TM 3.1 + , Thermo Fisher Ltd, CAT#V79020), the gp140 trimer (SF162) in the 293F supernatant was purified by nickel column purification. Dilute the trimer protein to 0.5-2 mg / ml with PBS, then use Thermo Fisher's biotin (CAT#21335) to label the gpl40 protein according to its kit procedure (the ratio of the number of molecules is protein:biotin=1: 20~100), incubate at room temperature in the dark for 0.5~2h, then centrifuge 4~6 times at 8000g with a 10kD centrifugal semi-permeable column (MerckMillipore Ltd, CAT#UFC501096), supplement with sterile PBS, and remove excess biotin molecules , the labeled gp120 protein molecules will be used to screen antigen-specific antibodies.

[0027] 2. Antigen-specif...

example 2

[0037] Example 2: Neutralizing activity of monoclonal antibody against HIV-1 pseudovirus

[0038] Pseudovirus packaging and antibody neutralization activity detection system according to the relevant literature "Reconstitution and characterization of antibody repertoires of HIV-1-infected"eliteneutralizers"." Antiviral Res 118:1-9. The description proceeds. Briefly described as follows, 293T cells are used to co-transfect PNL4-3 plasmid (containing VSV virus backbone and containing luciferase gene) and HIV-1 envelope protein particles to package HIV-1 pseudovirus. After culturing for 72 h, the virus supernatant was collected and the virus titer was detected. After infecting TZM-bl cells in a 96-well plate with an appropriate amount of virus and antibody gradient dilution, the cells were lysed after 72 hours, and the luciferase activity in the lysed cells was detected by a luciferase luminescence detection system (Promega Ltd) to determine the antibody Inhibitory effect on ...

example 3

[0039] Example 3: Detection of antibody-dependent cell-mediated cytotoxicity (ADCC) activity of monoclonal antibodies

[0040] TF228 cells were cultured in DMEM medium (see references (Puri, Hug et al.1998) for cell sources and characteristics), the cell density was about 90%, the cells were digested with trypsin, washed twice with PBS and resuspended, followed by the PKH67 labeling kit (Sigma, PKH67GL-lKT) process to mark TF228 cells, and adjust the cell density to be 106 / ml spare. Dilute the antibody to be tested in gradient with DMEM medium, mix with 100μl labeled TF228 cells, and place at 37°C, CO 2 Incubate in a cell culture incubator with a concentration of 5% for 15 minutes; add 100 μl to a density of 2x10 7 / ml of freshly isolated healthy human peripheral blood mononuclear cells (PBMC), mixed and placed at 37 ° C, CO 2 The concentration was 5% and cultured in a cell incubator for 4 hours; 1 μl 7AAD (ThermoFisher Ltd, Cat# 302232) was added to each reaction, mixed an...

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Abstract

The present invention discloses to a human anti-HIV gp 120 specific antibody Z166, provides amino acid sequences of the antibody and an active fragment thereof, provides coding genes of the antibody or the active fragment thereof, and simultaneously provides application methods of the antibody and the active fragment thereof in preparing medicines for treating or preventing HIV infection diseasesand application methods in preparing HIV diagnosis or detection reagents. The antibody Z166 can effectively neutralize a plurality of HIV virus subtypes, is used for treating HIV infected patients, effectively mediates killing of effector cells to HIV infected cells, can also mediate the killing (antibody-dependent cell-medicated cytotoxicity, ADCC) of the effector cells to an HIV envelope proteinstable expression cell line TF 228, and keeps a killing percentage of 30% or above when antibody concentration is 0.2 [mu]g/ml or more. The provided antibody Z166 can be used for treating the HIV infected patients and is suitable for antibody medicines based on all treatments of diseases caused by HIV based on the Z166.

Description

technical field [0001] The present invention relates to antibodies from humans, in particular, to a human-derived anti-HIV specific broad-spectrum neutralizing antibody prepared by genetic engineering, which has virus neutralization ability and induces strong antibody-mediated cytotoxicity ( ADCC) capability. Background technique [0002] Human Immunodeficiency Virus (HIV) was discovered in 1983. However, the research and development of HIV vaccines has not yet achieved a breakthrough. The main reason is that the virus has an ultra-high mutation frequency. Highly Active Antiretroviral Therapy (HAART), also known as cocktail therapy, is currently an effective means of suppressing the HIV virus. However, patients have to take medicine for a long time to effectively suppress the virus, and the side effects of the drug make the patient's persistence poor. The discovery of "long-term non-progressors (LTNP)" or "elite controllers" provides the possibility to screen high-titer fun...

Claims

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Application Information

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IPC IPC(8): C07K16/10C07K16/00
CPCC07K16/005C07K16/1063C07K2317/24C07K2317/55C07K2317/622
Inventor 张科孙泽华罗语思徐瑞雪
Owner GUIZHOU MEDICAL UNIV
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