H7N9 virus-resistant monoclonal antibody

A monoclonal antibody and antibody technology, applied in the fields of molecular biology and cellular immunology, can solve problems such as unfavorable development prospects, uncontrollable results, and complicated humanization process

Active Publication Date: 2017-08-04
JIANGSU PROVINCIAL CENT FOR DISEASE PREVENTION & CONTROL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the research on monoclonal neutralizing antibodies against H7N9 influenza at home and abroad is mostly mouse-derived monoclonal antibodies. The clinical application of this mouse-derived monoclonal antibody is limited due to the immune reaction of mouse proteins.
Although the humanized antibody that uses genetic engineering technology to humanize the mouse-derived monoclonal antibody is currently the dominant monoclonal antibody product market, it still retains the mouse-derived variable region sequence, coupled with human The source process is complicated and the result is uncontrollable, and its development prospects are not optimistic

Method used

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  • H7N9 virus-resistant monoclonal antibody
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  • H7N9 virus-resistant monoclonal antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0101] Example 1 Preparation of monoclonal antibody mAb6137 against H7N9 virus

[0102] 1. Memory B cell sorting

[0103] 1.1 Isolation of PBMCs

[0104] Three samples of peripheral anticoagulant blood from H7N9 convalescent patients were collected, and PBMCs from peripheral blood were separated by Ficoll density gradient centrifugation and stored in liquid nitrogen.

[0105] 1.2 Acquisition of memory B cells

[0106] The purified HA protein was labeled with PE and APC fluorescent dyes, and then mixed with Anti-CD3-Cy7APC, Anti-CD14-FITC, Anti-CD20-Cy7PE, Anti-CD19-PE-CF594, Anti-IgG-BV421, Anti-IgM -Cy55 fluorescently labeled antibodies were mixed in corresponding proportions. Take out PBMC cells from liquid nitrogen, wash with PBS after thawing, first stain the dead cells with AQUA staining solution, and then incubate with the above-mentioned mixed antibody on ice to bind. Unbound antibodies were washed by centrifugation at 800g, washed with PBS and passed through a cell...

Embodiment 2

[0123] Example 2 Detection of mAb6137 Antibody Binding Specificity

[0124] Detection of binding of purified antibody to H7N9 virus HA protein by immunoblotting

[0125] 1. Steps:

[0126] (1) The HA protein of the purified H7N9 virus was subjected to SDS-PAGE, and the HA protein of the purified H3N2 virus was used as a control at the same time. After the electrophoresis was completed, the protein in the gel was transferred to a PVDF membrane, and the PVDF membrane was 5% skimmed milk 4 Block overnight at ℃, then add 50μg of purified antibody, incubate at 37℃ for 2h, and then elute with PBST;

[0127] (2) Add anti-human IgG secondary antibody at 1:2000, incubate at 37°C for 1 hour, and then elute with PBST;

[0128] (3) PVDF membrane plus chemiluminescent liquid is exposed in a dark room.

[0129] 2. Results

[0130] The result is as image 3 As shown, the purified mAb6137 antibody binds to the H7N9 virus HA protein. image 3 "1" represents the lane where the HA protein ...

Embodiment 3

[0131] Example 3 Hemagglutination inhibition experiment of mAb6137 antibody

[0132] 1. Treatment of serum and antibody

[0133] A positive control serum is the serum of a patient in the recovery period of H7N9 virus infection, and a negative control serum is the serum of a healthy adult. Add 3 times the volume of RDE to the serum and antibody, incubate at 37°C for 16-18 hours, inactivate at 56°C for 30 minutes before use, add 6 times the volume of PBS to the serum, and the final concentration is 1:10, without adding the antibody, the final concentration is 1:1: 4.

[0134] 2. Preparation of 1% turkey red blood cells

[0135] Inhale 2ml of Alfred's solution in the syringe in advance, and take 2ml of venous blood from the root of the turkey wing to form a 4ml whole blood mixture. Add 4ml of blood to a 50ml centrifuge tube, add cold PBS solution to the centrifuge tube to 50ml, mix gently and centrifuge at 2000rpm for 5 minutes to remove the supernatant suspension, then wash p...

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Abstract

The invention discloses an H7N9 virus-resistant monoclonal antibody and in particular discloses an antibody capable of identifying and combining an N7N9 virus HA protein, a coding gene thereof, an expression vector and an application. The monoclonal antibody disclosed by the invention can prevent H7N9 virus from infecting susceptible cells and is fully derived from a human body. Compared with other animal-derived H7N9 virus-resistant molecules, the immunogenicity is greatly reduced, the monoclonal antibody is good in affinity, and not only is good in treatment effect, but also is low in side effect.

Description

technical field [0001] The invention belongs to the fields of cellular immunology and molecular biology, and relates to a fully human monoclonal antibody, in particular to a fully human monoclonal neutralizing antibody against H7N9. In addition, the present invention also relates to the preparation method and application of the neutralizing antibody. Background technique [0002] The H7N9 virus belongs to the Orthomyxoviridae family. It is a segmented RNA virus with a single negative strand and an envelope. The genome is divided into 8 segments. The virus is spherical, about 100nm in size, and consists of a capsid and an envelope. Eight viral RNA segments are covered by NP to form a helically symmetrical viral capsid. The envelope comes from the host cell membrane, and HA and NA protein spikes are embedded on the surface. HA is chimerized on the surface of the envelope in the form of a trimer. It is the main antigenic component of influenza virus, can agglutinate red blood...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/10C12N15/13C12N15/85A61K39/42A61P31/14G01N33/569
CPCA61K39/00C07K16/1018C07K2317/24C07K2317/54C07K2317/55C07K2317/56C07K2317/565C07K2317/76G01N33/56983
Inventor 朱凤才张黎李靖欣唐蓉孟繁岳胡月梅
Owner JIANGSU PROVINCIAL CENT FOR DISEASE PREVENTION & CONTROL
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