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Specific Zika virus neutralizing antibodies and application thereof

An antibody and sequence technology, applied in the field of medicine, can solve the problem of lack of vaccines and treatment methods

Active Publication Date: 2019-07-30
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is still a lack of effective vaccines and treatments for the virus

Method used

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  • Specific Zika virus neutralizing antibodies and application thereof
  • Specific Zika virus neutralizing antibodies and application thereof
  • Specific Zika virus neutralizing antibodies and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1: Expression and purification of Zika E protein

[0037]The DNA fragment of the extracellular region of ZIKV E (the amino acid sequence is shown in SEQ ID NO: 21, and the nucleotide sequence is shown in SEQ ID NO: 22) 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 and refolded by dilution. The refolding solution was concentrated and replaced with 20mM Tris, 150mM NaCl, pH8.0 buffer. The concentrated protein solution was further purifi...

Embodiment 2

[0038] Example 2: Isolation of ZIKV-E protein-specific memory B cells

[0039] 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.

[0040] 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

[0041] Example 3: Single B cell PCR, sequence analysis and human antibody design

[0042] The B cells obtained in Example 2 were reverse-transcribed by Superscript III reverse transcriptase (Invitrogen). The reverse transcription primers are shown in Table 1 (sequences are shown in SED ID NO.23 to SED ID NO.30), and reacted at 55° C. for 60 min.

[0043] Table 1. Primers for reverse transcription reactions

[0044]

[0045] 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 another 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 for 90s , 35 ...

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Abstract

The invention discloses specific Zika virus neutralizing antibodies and application thereof, and belongs to the technical field of medicine. Zika E protein expressed by colibacillus serves as an antigen, through fluorescence-activated cell sorting, memory B cells capable of being specifically bound with the Zika E protein are screened in PBMCs of a Zika patient in the rehabilitation stage, the screened single B cells are subjected to a RT-PCR, the sequence and segments of the variable region of the antibody are obtained, and the variable region and the constant region are connected in an expression vector. After mammalian cell expression and purification, a series of function detection is conducted, such as the binding force of the ZIKE-E protein, the in-vitro neutralizing effect and the in-vivo protection capacity, and the three human monoclonal antibodies capable of completely protecting Zika virus infection are obtained.

Description

technical field [0001] The invention relates to a Zika virus-specific neutralizing antibody and application thereof, belonging to the technical field of medicine. Background technique [0002] More and more evidence shows that Zika virus (Zika virus, ZIKV) can cause neonatal microcephaly and neurodevelopmental abnormalities, such as "Guillain-Barr syndrome". ZIKV, which belongs to flavivirus, is mainly transmitted by mosquito bites, but blood transmission, sexual transmission and maternal-fetal transmission have also been reported. ZIKV was first isolated from macaques in the Ugandan jungle in 1947. The virus is mainly endemic in Africa and tropical regions of Asia. In 2015, the Zika epidemic broke out in Brazil and spread to other countries with the flow of people. The outbreak has now resulted in more than 80,000 cases of infection in more than 60 countries or regions. However, there is still a lack of effective vaccines and treatments for this virus. [0003] Neutral...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/10C12N15/13A61K39/395A61P31/14
CPCC07K16/1081C07K2317/52C07K2317/56A61P31/14A61K2039/505A61K39/395C07K16/10C12N5/10C12N15/85Y02A50/30
Inventor 高福严景华王奇慧仝舟
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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