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A fully human monoclonal antibody with high neutralizing activity against chikungunya and its application

A technology of monoclonal antibody and chikungunya virus, which is applied in the field of microbiology and immunology, can solve the problems of non-specific therapeutic drugs, achieve low immunogenicity, good stability, and improve the effect of curative effect

Active Publication Date: 2021-07-30
ACADEMY OF MILITARY MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, the treatment of chikungunya fever is mainly symptomatic treatment such as fever reduction and analgesia, and there is no specific therapeutic drug in clinical practice.

Method used

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  • A fully human monoclonal antibody with high neutralizing activity against chikungunya and its application
  • A fully human monoclonal antibody with high neutralizing activity against chikungunya and its application
  • A fully human monoclonal antibody with high neutralizing activity against chikungunya and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1. Screening and Preparation of Human Anti-Chikungunya Virus Monoclonal Antibody

[0030] 1.1 Blood sample collection

[0031] After obtaining informed consent, Guangzhou Eighth People's Hospital provided peripheral blood from a chikungunya infected patient 15 days after recovery for subsequent experiments.

[0032] 1.2 Sorting single cells by flow cytometry

[0033] The collected blood samples were separated from PBMC by Ficoll density gradient centrifugation, and the process was as follows:

[0034] 1.2.1 Take fresh anticoagulated whole blood (EDTA anticoagulated) and dilute the whole blood with an equal volume of PBS.

[0035] 1.2.2 Add a certain volume of separation liquid into the centrifuge tube, spread the diluted blood sample above the liquid surface of the separation liquid, and keep the interface between the two liquid surfaces clear. The volume of separation medium, anticoagulated undiluted whole blood, and PBS (or normal saline) is 1:1:1.

[0036...

Embodiment 2

[0118] Example 2. Cell neutralization experiment

[0119] 2.1 The day before the experiment, the Vero cells were diluted to 1.5×10 with medium (MEM+10% FBS) 5 Cells / mL concentration, inoculated into 96-well cell culture plate, inoculation volume 200 μL / well, cultured in 37°C 5% CO2 cell incubator;

[0120] 2.2 On the day of the experiment, the purified monoclonal antibody was mixed with medium MEM+2% FBS from the initial concentration (8D1 monoclonal antibody initial concentration 100ug / ml, Ab2 control antibody 200ug / ml (refer to the 8B10F8 heavy chain and light chain variable Region sequence, chemically synthesized variable region gene and obtained full-length antibody gene by fusion PCR technology, cloned into the expression vector pMH vector, transfected CHO-S cells, obtained and preserved by Protein A affinity chromatography purification, named by the laboratory Ab2), 4-fold dilution, added to a 96-well culture plate with a volume of 120 μL / well; then 120 μL of chikunguny...

Embodiment 3

[0125] Example 3. Research on monoclonal antibody recognition epitope

[0126] 3.1 ELISA screening for specific clones: Coat anti-Chikungunya virus monoclonal antibody (100 μg / mL, 150 μL / well) on a 96-well enzyme-linked plate, overnight at 4°C; discard the coating liquid, add 5 mg / mL BSA to block Buffer (0.1mol / L NaHCO3, pH8.6) was blocked at 4°C for 2h; the liquid was poured off, washed 6 times with TBST (1mL / L Tween-20, TBS); 100μL of the phage loop 7 peptide library was added (the original library used TBST According to 1:10 dilution, containing about phage 2×10 11 pfu / 100μL), incubated at room temperature for 30min with gentle shaking; discarded liquid, washed 10 times with TBST to wash off unbound phage; added 100μL eluent (1mg / mL BSA, 0.2mol / L Glycine-HCl, pH2.2 ), shake gently at room temperature for 10 minutes, quickly suck out the liquid and add 15 μL of neutralizing solution (1mol / L Tris-HCl, pH9.1) to neutralize; take 1 μL of the eluted phage to measure the titer, ...

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Abstract

The invention discloses a fully human monoclonal antibody with high neutralizing activity against Chikungunya virus, which is obtained through flow sorting-single-cell PCR technology screening and has unique CDR partitions. The invention also discloses Application of the antibody in the preparation of chikungunya fever therapeutic medicine. The monoclonal antibody disclosed by the invention has high-efficiency and specific anti-chikungunya virus activity, and also has the characteristics of high expression, full human source and good stability, and is suitable for industrial production.

Description

technical field [0001] The invention discloses an antibody and belongs to the fields of microbiology and immunology. Background technique [0002] Chikungunya fever (chikungunya fever) is a mosquito-borne infectious disease caused by chikungunya virus (chikungunya virus). Americas. The main clinical signs of chikungunya are fever, headache, myalgia, rash, and joint pain. In the past ten years, the number of outbreaks of chikungunya fever has increased and the scope of the epidemic has continued to expand. It has occurred in more than 100 countries and regions around the world, causing about 1 million cases worldwide every year. On December 20, 2015, based on the lessons learned from the Ebola outbreak response, the World Health Organization identified eight dangerous pathogens (Ebola, Marburg virus, etc.) Kenya fever, thrombocytopenia syndrome with severe fever and Zika flavivirus), chikungunya virus is one of the three less dangerous pathogens. At the same time, Chikung...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/10C12N15/13A61P31/14
CPCA61P31/14C07K16/1081C07K2317/24C07K2317/52C07K2317/56C07K2317/565C07K2317/76Y02A50/30
Inventor 陈薇李建民迟象阳付玲于长明徐俊杰侯利华宋小红刘树玲董韵竹张金龙房婷张复春
Owner ACADEMY OF MILITARY MEDICAL SCI
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