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Flavivirus human monoclonal antibody and application

A technology of antibodies and antigens, applied in the field of medicine, can solve the problems of unknown affinity, poor specificity, limited antibody use and sensitivity, etc., and achieve the effect of huge clinical detection and basic research application value

Active Publication Date: 2017-04-26
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Studies have found that there are some broad-spectrum antibodies against flaviviruses, such as: 2A10G6, 4G2, these antibodies are all antibodies against FL produced by the fusion of mouse hybridomas, and the affinity between 2A10G6 and E protein is K D =2.7nM, the affinity between 4G2 and E protein is unknown, and has not been reported in the literature. Therefore, in basic research and clinical testing, there may be problems of low affinity, poor specificity, and high background, which limit the use of antibodies to a certain extent and sensitivity

Method used

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  • Flavivirus human monoclonal antibody and application
  • Flavivirus human monoclonal antibody and application
  • Flavivirus human monoclonal antibody and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1: Expression and purification of Zika E protein

[0050] The DNA fragment of the extracellular region of ZIKVE (the amino acid sequence is shown in SEQ ID NO:39, and the nucleotide sequence is shown in SEQ ID NO:40) was digested with NdeI and XhoI, and then connected to the pET21a vector. The 3' end of the ZIKV 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, culture at 37°C until OD600=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 by refolding the inclusion bodies in the dilution method. The refolding solution was concentrated and replaced with 20mM Tris, 150mM NaCl, pH8.0 buffer. The concentrated prote...

Embodiment 2

[0051] Example 2: Isolation of ZIKV-E protein-specific memory B cells

[0052] With the patient's informed consent, 15 mL of blood was collected and PBMCs were isolated. Separated PBMCs in 10 7 / mL density and final concentration of 100nM ZIKV-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.

[0053] Collect PE by FACSAria III sorting - Cy5 - APCs - APCs - Cy7 + Pacific Blue + FITC + PE + The cells were directly collected into a 96-well plate, 1 cell / well.

Embodiment 3

[0054] Example 3: Single B cell PCR and sequence analysis

[0055] The cells obtained in Example 2 were reverse-transcribed with Superscript III reverse transcriptase (Invitrogen), and the reverse transcription primers were listed in Table 1, and reacted at 55° C. for 60 min. 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 primers are listed in Tables 2 and 3, and the reaction conditions are 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 for 7min. Use this as a template for another round of PCR (PCRb), the conditions are as follows: 95°C for 5min; 95°C for 30s, 58°C (heavy chain) / 60°C (κ chain) / 64°C (λ chain) for 30s, 72°C for 90s, 35 cycles, 72°C for 7min.

[0056] 1.2% agarose gel electrophoresis to separate the PCR products. The bands with a band size of 400-500 bp were recover...

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Abstract

The invention discloses a flavivirus human monoclonal antibody and application, which belong to the technical field of medicine. According to the flavivirus human monoclonal antibody and the application provided by the invention, three antibodies capable of protein-binding with ZIKV-E are acquired, and binding sites of the three antibodies are confirmed. The three antibodies are completely different from a reported ZIKV antibody sequence, and are three new-found antibodies. The binding constants of the three antibodies and the ZIKV-E are 39.9pM (Z5), 44.7pM (Z6) and 200pM (Z7), which show that the three human monoclonal antibodies have a higher ZIKV-E protein-binding capacity. Through competiton experiments, the binding sites of the three antibodies have competitive relationships with 2A10G6, and the phenomenon shows that the binding sites of the three antibodies are near FL, and E protein of flaviviridae is highly conserved at the FL part. The antibody provided by the invention effectively detects the infection of common flaviviridae viruses: ZIKV, dengue 1-4 type and yellow fever viruses, and has huge application values on clinic detection and fundamental research.

Description

technical field [0001] The invention relates to a flavivirus human monoclonal antibody and its application, belonging to the technical field of medicine. Background technique [0002] The Zika outbreak in Central and South America in 2015 has infected millions of people in 69 countries, and several imported cases have also appeared in my country. With the flow of population and climate change, there is a high risk of Zika virus epidemic in my country. However, due to Zika virus infection, 80% of patients have no obvious symptoms, and 20% of patients have mild symptoms, which will not cause patients to pay attention. Zika virus (ZIKV) belongs to the Flaviviridae family and is mainly transmitted by mosquito bites. At the same time, Zika virus can pass through the mother-fetal barrier and cause microcephaly in newborns. In addition, Zika virus can be transmitted sexually. Clinical data show that Zika virus can be transmitted to women through male patients, and can also be t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/10C12N15/13C12N15/63A61K39/12A61P31/14
CPCA61K2039/505C07K16/10Y02A50/30
Inventor 王奇慧严景华高福
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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