Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Anti-H7N9 whole human monoclonal antibody 5G22, and preparation method and application thereof

A monoclonal antibody, fully human technology, applied in the field of immunology, can solve the problems of rimantadine drug resistance and no effective treatment methods, and achieve the goal of reducing cumbersome operations and costs, high affinity and specificity, and low production costs Effect

Active Publication Date: 2019-07-02
SHENZHEN INST OF ADVANCED TECH CHINESE ACAD OF SCI
View PDF4 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

H7N9 virus is a kind of influenza virus, which is resistant to the traditional antiviral drugs amantadine and rimantadine, and there is no effective treatment at present

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Anti-H7N9 whole human monoclonal antibody 5G22, and preparation method and application thereof
  • Anti-H7N9 whole human monoclonal antibody 5G22, and preparation method and application thereof
  • Anti-H7N9 whole human monoclonal antibody 5G22, and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] (1) Construction of NTH-3T3 cell line stably expressing CD40L

[0044] 3T3-CD40L feeder cells were established using lentivirus. The lentiviral expression vector pLVX-CD40L was constructed, transfected into 293T cells, and the virus supernatant was collected on the fourth day of transfection. NIH-3T3 cells were activated, cultured for 3 generations, infected with lentivirus, continued to be cultured and passed 3 times. Use a flow cytometer to sort the cells whose FITC fluorescence intensity is near the MFI, and add them back to the culture flask at 37°C, 5% CO 2 Cultivate and detect in the incubator, and the test results are as follows: figure 1 As shown, 3T3 cells expressing CD40L and 3T3 cells transfected with empty vector pLVX (with ZxGreen) were stained with anti-CD40L with APC, and then analyzed by flow cytometry. It was found that all 3T3-CD40L feeder cells expressed CD40L. When the cells grow to 80%-90%, digest and collect the cells at a concentration of 1×10...

Embodiment 2

[0066] Example 2 Cloning, recombination and expression of humanized monoclonal antibody 5G22 gene

[0067] The B cells obtained in Example 1 capable of secreting the antibody 5G22 binding to the H7N9 virus were lysed, and the lysate was taken for reverse transcription of RNA to obtain the PCR template cDNA of the human antibody gene. The heavy chain and light chain genes of the antibody were cloned using cDNA as a template, and recombinantly expressed and purified in eukaryotic 293F or HEK293 cells.

[0068] specifically:

[0069] (1) Transfer the B cell liquid to a 96-well plate (Eppendorf, 030133366).

[0070] (2) Reverse transcription system: 150ng random primer (invitrogen, 48190-011), 0.5ul 10mM dNTP (Invitrogen, 18427-088), 1μl 0.1M DTT (Invitrogen, 18080-044), 0.5% v / v Igepal CA -630 (Sigma, I3021-50ML), 4U RNAsin (Promega), 6U Prime RNAse Inhibitor

[0071] (Eppendorf) and 50U III reverse transcriptase (Invitrogen, 18080-044), add DEPC water to 14μl / well.

[0072...

Embodiment 3

[0093] Example 3 In vitro neutralization experiment of humanized monoclonal antibody 5G22

[0094] Using the virus-infected cell model (canine kidney cell MDCK), the inhibitory effect and effect of the antibody on the H7N9 influenza virus with PR-8 as the backbone were evaluated by microneutralization-ELISA experiment, and the anti-influenza virus activity of the antibody was detected. The specific operation is as follows:

[0095] 1 cell plating

[0096] Digest logarithmic growth phase cells with trypsin, collect by centrifugation after termination, blow evenly, and prepare single cell suspension;

[0097] Adjust the cell concentration to 5×104 cells / ml with cell culture medium, inoculate in 96-well cell culture plate, and store the cells at 37°C and 5% CO 2 Incubate overnight in the incubator.

[0098] 2 Antibody and virus pretreatment

[0099] 5G22 antibody set up 10 concentration gradients, followed by 10-10 10 Three-fold dilutions were performed, and three parallel w...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The present invention relates to an anti-H7N9 whole human monoclonal antibody 5G22, and a preparation method and an application thereof, the anti-H7N9 whole human monoclonal antibody 5G22 or a biologically active fragment derived from the monoclonal antibody and capable of specifically binding H7N9. The antibody has a heavy chain variable region and a light chain variable region. The heavy chain variable region and light chain variable region respectilvey have 3 complementarity determining regions (CDRs). The antibody 5G22 is capable of specifically binding, detecting and neutralizing H7N9 viruses, and more suitable and more promising as a macromolecular drug for treating influenza viruses.

Description

technical field [0001] The invention relates to an anti-H7N9 fully human monoclonal antibody and its preparation method and application, in particular to the anti-H7N9 fully human monoclonal antibody 5G22 and its preparation method and application, belonging to the technical field of immunology. Background technique [0002] Among the top ten best-selling drugs in the world in 2015, six are fully human or humanized monoclonal antibody drugs. Ranked first is AbbVie's anti-TNFa monoclonal antibody Humira for the treatment of arthritis. This is a fully human monoclonal antibody and has been the king of drugs with sales of more than 10 billion for three consecutive years. Since the first monoclonal antibody drug was launched in 1986, monoclonal antibody drugs have experienced mouse monoclonal antibody drugs (Orthoclone OKT3), chimeric monoclonal antibody drugs (Rituximab), humanized monoclonal antibody drugs (Herceptin) and fully human monoclonal antibody drugs. Monoclonal anti...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
IPC IPC(8): C07K16/10C12N15/13C12N15/85C12N5/10A61K39/42A61P31/16G01N33/577G01N33/569
CPCA61K2039/505C07K16/1018C07K2317/24C07K2317/565
Inventor 万晓春李俊鑫刘绿艳
Owner SHENZHEN INST OF ADVANCED TECH CHINESE ACAD OF SCI
Features
  • Generate Ideas
  • Intellectual Property
  • Life Sciences
  • Materials
  • Tech Scout
Why Patsnap Eureka
  • Unparalleled Data Quality
  • Higher Quality Content
  • 60% Fewer Hallucinations
Social media
Patsnap Eureka Blog
Learn More

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2025 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com