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Humanized anti-H7N9 avian influenza virus high-affinity antibody 10K and application thereof

An avian influenza virus, affinity technology, applied in antiviral agents, antiviral immunoglobulins, applications, etc., can solve the problems of HA7 affinity and subtype specificity need to be further deepened

Inactive Publication Date: 2017-08-18
深圳普兰达科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the existing antibodies have a good ability to neutralize H7N9 influenza virus, the research on their affinity for HA7 and subtype specificity needs to be further studied

Method used

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  • Humanized anti-H7N9 avian influenza virus high-affinity antibody 10K and application thereof
  • Humanized anti-H7N9 avian influenza virus high-affinity antibody 10K and application thereof
  • Humanized anti-H7N9 avian influenza virus high-affinity antibody 10K and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1 Human Anti-H7N9 Avian Influenza Virus High Affinity Antibody 10K and Its Preparation Method

[0039] 1. Antigen labeling

[0040] Purchase HA from Acrobiosystems (HA9-V5227) 7 protein, diluted to 0.5-2mg / ml with PBS, and then using Thermal company's biotin (EZ-Link TM Sulfo-NHS-LC-Biotin, 21335) according to its kit flow process will HA 7 Protein labeling (the ratio of the number of molecules is protein:biotin=1:20-100), incubated at room temperature in the dark for 0.5-2h, and then centrifuged 4-6 times at 8000g with a 10KD semi-permeable centrifugal column (MerckMillipore, UFC501096). Supplement with sterile PBS, remove excess biotin molecules, and mark HA 7 Protein molecules will be used to screen HA 7 specific memory B cells.

[0041] 2. Antigen-specific memory B cell sorting and reverse transcription

[0042] The convalescent peripheral blood mononuclear cells were isolated from patients with H7N9 infection, washed once with PBS, and then resuspende...

Embodiment 2

[0109] Example 2 Monoclonal antibody 10K is directed against HA 7 protein EC 50 determination

[0110] Dilute HA with ELISA coating solution 7 The protein was adjusted to 1 μg / ml, and then 50 μl per well was coated with an ELISA plate (Corning, 3690) at 4° C. overnight. Plates were washed with PBST and blocked with PBS containing 5% skimmed milk for more than 2 hours. Starting from the initial concentration of 50 μg / ml, the monoclonal antibody was diluted 4-fold with PBS containing 5% skimmed milk, and then added to the blocked ELISA plate. A total of 10 gradients were set up, with two repetitions. The wells without antibodies were used as negative controls, and IgG9114L (ie CR9114, Dreyfus C, Laursen NS, Kwaks T, Zuijdgeest D, Khayat R, Ekiert DC, et al. protective epitopes on influenza B viruses. Science 2012 Sep 14; 337(6100): 1343-8) is an antibody control. Goat anti-human IgG Fc-HRP (1:10000, Jackson immunolab, 109-036-098) as the secondary antibody, 100μl TMB for co...

Embodiment 3

[0111] Example 3 Determination of neutralizing activity of monoclonal antibody 10K against H7N9 (A / Shenzhen / SP17 / 2014 (H7N9))

[0112] Inoculate MDCK cells in a 96-well cell culture plate, culture until the cell density is about 70%-90%, wash twice with PBS and set aside; dilute the monoclonal antibody to be detected 3 times on a 96-well microtiter plate (starting at 100μg / ml), each antibody to be detected is repeated in 4 wells, 8 gradients; according to the TCID of the virus 50 Titer dilute the virus so that the diluted virus titer is 200TCID 50 / 100μl; mix 60μl of the diluted virus solution with 60μl of the diluted monoclonal antibody sample, and incubate in a 37°C incubator for 2 hours to fully act on the antigen and antibody; then take 100μl of the virus plasma mixture and put it into the washed 96-well MDCK cells, Infect in an incubator at ℃ for 1 h, replace the virus liquid with 150 μl of MEM medium supplemented with TPCK trypsin, and store at 37 °C CO 2 Cultivate in ...

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Abstract

The invention discloses a humanized anti-H7N9 avian influenza virus high-affinity antibody 10K filtered and obtained based on a single cell separation technology, the amino acid sequences of light chain and heavy chain variable regions of the antibody are shown as SEQ ID No. 2 and SEQ ID No. 5 respectively. The high-affinity specificity of the antibody is combined with H7N9 avian influenza virus 7 type hemagglutinin protein, and can mediate the kill and wound (ADCC) of effector cells using NK cells as main parts for H7N9 influenza virus infected cells. The antibody 10K can be used for therapeutic development of highly pathogenic avian influenza infection, and also can be used for development of H7N9 influenza virus antigen dectection reagents.

Description

technical field [0001] The invention relates to the fields of genetic engineering, single cell sorting technology and antibody library display technology, in particular to a human-sourced anti-H7N9 avian influenza virus high-affinity antibody 10K and its application. Background technique [0002] Since February 2013, cases of human infection caused by H7N9 avian influenza virus have been found in southeast China, gradually spreading from Shanghai and Anhui to other provinces and cities across the country. As of February 2017, a total of 1079 cases of H7N9 influenza infection have been reported nationwide, with 417 deaths and a death rate as high as 38.6%. At present, there is no specific drug for the treatment of highly pathogenic avian influenza infection, and the curative effect of the existing non-specific neuraminidase inhibitor-oseltamivir is limited to the early stage of infection. Since the early symptoms of highly pathogenic H7N9 infection are not significantly diff...

Claims

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Application Information

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IPC IPC(8): C07K16/10C12N15/13A61K39/42A61P31/16G01N33/569
CPCC07K16/1018C07K2317/24C07K2317/565C07K2317/732C07K2317/92
Inventor 杨争
Owner 深圳普兰达科技有限公司
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