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High-functional-activity yellow fever virus humanized monoclonal antibody and application thereof

An antibody and sequence technology, applied in the field of medicine, can solve problems such as shortage, frequent disease outbreaks, and vaccine shortage vaccination rate

Active Publication Date: 2019-10-18
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, an attenuated vaccine (YFV17D) is clinically available, but the shortage of vaccine and insufficient vaccination rate lead to frequent outbreaks of the disease, and non-immune individuals are still at risk
After YFV infection, there is no effective specific drug available clinically for the treatment of the disease

Method used

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  • High-functional-activity yellow fever virus humanized monoclonal antibody and application thereof
  • High-functional-activity yellow fever virus humanized monoclonal antibody and application thereof
  • High-functional-activity yellow fever virus humanized monoclonal antibody and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Embodiment 1: Expression and purification of yellow fever virus E protein

[0032] YFV CNYF01 / 2016 (YFV-China) strain membrane protein E protein (amino acid sequence shown in SEQ ID NO: 11, nucleotide sequence shown in SEQ ID NO: 12) extracellular region DNA fragment by NdeI and XhoI enzymes After cutting, it was connected to the pET21a vector. The 3' end of the YFV E protein coding region is connected with the coding sequence of 6 histidine tags (hexa-His-tag) and the translation stop codon. Then the ligation product was transformed into BL21 Escherichia coli competent cells. Single clones were inoculated into 40mL LB medium and cultured for 6-8 hours. Inoculate into 4L of LB medium and culture to OD at 37°C 600 =0.4-0.6, add IPTG to a final concentration of 1 mM, and continue culturing at 37°C for 4-6 hours. Inclusion bodies were harvested and refolded by dilution. The refolding solution was concentrated and replaced with 20mM Tris, 150mM NaCl, pH8.0 buffer solut...

Embodiment 2

[0033] Example 2: Isolation of specific memory B cells combined with YFV China-E protein

[0034] With the patient's informed consent, 20 mL of blood was collected and PBMCs were isolated. Separated PBMCs in 10 7 / mL density and final concentration of 400nM YFV-E protein was incubated on ice for half an hour, then washed twice with PBS, and then incubated with the following antibodies: anti-human CD3 / PE-Cy5, anti-human CD16 / PE- Cy5, anti-human CD235a / PE-Cy5, anti-human CD19 / APC-Cy7, anti-human CD27 / Pacific Blue, anti-human CD38 / APC, anti-human IgG / FITC, and anti-His / PE. After the antibody was incubated on ice for half an hour, it was washed twice with PBS. Collect PE-Cy5 by FACSAria III sorting - APCs - APC-Cy7 + Pacific Blue + FITC + PE + The cells were directly collected into a 96-well plate, 1 cell / well.

Embodiment 3

[0035] Example 3: Single B cell PCR, sequence analysis and human antibody design

[0036] The B cells obtained in Example 2 were reverse-transcribed with Superscript III reverse transcriptase (Invitrogen), and the reverse transcription primers are shown in Table 1 (sequences are shown in SED ID NO.13 to SED ID NO.20), and reacted at 55° C. for 60 min.

[0037] Table 1. Primers for reverse transcription reactions

[0038]

[0039] Using this reverse transcription product as a template, PCR was performed with HotStar Tap Plus enzyme (QIAgen) to amplify the antibody variable region sequence (PCRa). The corresponding primers were designed, and the reaction conditions were as follows: 95°C, 5min; 95°C for 30s, 55°C (heavy chain / κ chain) / 50°C (λ chain) for 30s, 72°C for 90s, 35 cycles; 72°C, 7min. Use this as a template for the second round of PCR (PCRb), the conditions are as follows: 95°C, 5min; 95°C for 30s, 58°C (heavy chain) / 60°C (κ chain) / 64°C (λ chain) for 30s, 72°C 90s, 3...

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Abstract

The present invention discloses a high-functional-activity yellow fever virus humanized monoclonal antibody and an application thereof, and belongs to the technical field of medicines. A yellow fevervirus E protein expressed by escherichia coli is used as an antigen, memory B cells specifically combined with the yellow fever virus E protein are screened from PBMCs of a case of a convalescent patient through fluorescence-activated cell sorting, then the screened single B cells are subjected to RT-PCR and PCR amplification to obtain an antibody variable region segment, and the antibody variableregion segment is further connected with a constant region into an expression vector. After mammalian cell expression and purification, a series of function detections are carried out to obtain the humanized monoclonal antibody with a function of protecting from yellow fever virus infection. The antibody has affinity with the antigen at 0.588 nM, has strong yellow fever virus neutralization activity, has IC50 at 3.7ng / ml, can completely protect mice from being attacked by yellow fever viruses with a lethal dose, and has application values of clinical treatment and prevention of the yellow fever viruses.

Description

technical field [0001] The invention relates to a yellow fever virus human monoclonal antibody with high functional activity and application thereof, belonging to the technical field of medicine. Background technique [0002] Yellow fever virus (yellow fever virus, YFV), a single-stranded positive-sense RNA virus, belongs to the Flaviviridae family and is a mosquito-borne pathogen that can cause human disease. The same family of viruses also includes Zika virus ( zikavirus, ZIKV), dengue virus (dengue virus, DENV), West Nile virus (west nile virus, WNV), etc. YFV is an important pathogen that causes yellow fever. In severe cases, it can cause hemorrhagic fever with multiple organ failure, especially the liver, spleen, lymph nodes, heart, and kidneys. [0003] In 1996, scientists estimated that yellow fever virus causes 200,000 infections and 300,000 deaths each year in Africa and South America. In the past two years, yellow fever outbreaks have occurred in places such as B...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/10C12N15/13G01N33/569A61K39/42A61P31/14
CPCA61K2039/505A61P31/14C07K16/1081C07K2317/24C07K2317/76C07K2317/92G01N33/56983G01N2333/185Y02A50/30
Inventor 严景华高福李燕马素芳仵丽丽
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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