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Camel-source high-affinity nano antibody for SARS-CoV-2alpha mutant strain and SARS-CoV-2beta mutant strain

A sars-cov-2, antibody technology, applied in the direction of antibodies, antiviral agents, chemical instruments and methods, etc., can solve the problems of difficult to obtain plasma of patients who have recovered from serum therapy, and the amount is small, and achieves simple pretreatment process, high neutralization Ability, high sensitivity effect

Pending Publication Date: 2022-05-27
INSITUTE OF BIOPHYSICS CHINESE ACADEMY OF SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the limitation of patient serum therapy is that it is difficult to obtain the plasma of recovered patients, and the quantity is small, which cannot meet the needs of a large patient population. Therefore, alternative engineered antibodies are needed for treatment.

Method used

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  • Camel-source high-affinity nano antibody for SARS-CoV-2alpha mutant strain and SARS-CoV-2beta mutant strain
  • Camel-source high-affinity nano antibody for SARS-CoV-2alpha mutant strain and SARS-CoV-2beta mutant strain
  • Camel-source high-affinity nano antibody for SARS-CoV-2alpha mutant strain and SARS-CoV-2beta mutant strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0075] Example 1. Construction of SARS-CoV-2 Nanobody Library

[0076] Take 200ug of SARS-CoV-2 virus original strain S protein and RBD protein (Beijing Yiqiao Shenzhou Biological Co., Ltd.) and mix it with an equal volume of complete Freund's adjuvant, fully emulsify and inject it into camels, and then boost the immunization every two weeks. Among them, the mixture of incomplete Freund's adjuvant and immunogen was used in booster immunization, and subcutaneous immunization on the back of the neck was performed at multiple points for a total of 5 times. From the third immunization, blood was collected from the jugular-clavicular vein one week after each immunization and serum titers were detected.

[0077] Leukocytes were isolated from the peripheral blood after the fifth immunization, total RNA was extracted, and the VHH gene was cloned by reverse transcription PCR and nested PCR (wherein, the systems and parameters of reverse transcription PCR and nested PCR are described be...

Embodiment 2

[0103] Example 2. Screening of SARS-CoV-2 Nanobodies

[0104] The first well of a 96-well microtiter plate was coated with the S protein antigen of the original strain of SARS-CoV-2 at a concentration of 1ug / mL, overnight at 4°C; the next day, the coating solution was poured out and washed with PBST for 3 The first and second wells of the ELISA plate were blocked with BSA and incubated at room temperature for 2 hours; the blocking solution was poured out and washed 3 times with PBST; the phage nanobody library obtained in Example 1 was added to the first well and reacted for 2 hours ; Pour out the liquid, pat dry on clean absorbent paper, and wash 5 times with PBST; add 100 μL of SARS-CoV-2 virus original strain S1 protein to the first well, and react for 1 h; aspirate out the first well The liquid was added to the second well, reacted for 1 h, and the phage bound to BSA was removed; the eluate was collected, and 5 μL was used for titer determination, and the rest was used for...

Embodiment 3

[0107] Example 3. Expression of SARS-CoV-2 Nanobodies

[0108] The positive monoclonal plasmid was extracted, transformed into E. coli TOP10F' competent cells (purchased from ThermoFishier), and then spread on solid medium for overnight culture after recovery. The next day, a single clone was picked and cultured in SB-carboxybenzyl medium, and IPTG was added to induce overnight expression; the next day, the cells were lysed with a high-pressure homogenizer, filtered through a membrane, and purified with a nickel column, that is, using a histidine tag to bind with the cells. The nanobodies were separated and purified by affinity chromatography of nickel chloride in the nickel column to obtain high-purity anti-SARS-CoV-2 nanobodies, namely antibodies A1-A6. After amino acid sequencing analysis, the amino acid sequences of the obtained nanobodies were as shown in SEQ IDNO: 1-6.

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Abstract

The invention relates to a camel source high-affinity nano antibody of SARS-CoV-2 alpha mutant strain and beta mutant strain, in particular to an antibody specifically bound with S protein of coronavirus (such as SARS-CoV-2) and an antigen binding fragment of the antibody. More specifically, the present invention relates to camel-derived nanoantibodies or antigen-binding fragments thereof capable of binding with high affinity to surface S proteins of coronaviruses such as SARS-CoV-2 wild-type original strains and mutants thereof (especially B.1. 1.7 mutant strains and B.1.351 mutant strains), which can be used for the prevention, detection, diagnosis or treatment of infections caused by coronaviruses, especially SARS-CoV-2 viruses.

Description

technical field [0001] The invention belongs to the fields of biotechnology, immune detection and biomedicine, and in particular relates to broad-spectrum high-affinity antibodies or antigen-binding fragments and their uses in the detection, diagnosis, prevention and treatment of coronaviruses, in particular to SARS-CoV-2 Wild-type original strain, B.1.1.7 mutant strain (ie α mutant strain) and / or B.1.351 mutant strain (ie β mutant strain) for detection, diagnosis, prevention and treatment. Background technique [0002] The novel coronavirus SARS-CoV-2 is a betacoronavirus RNA virus. The virus has the characteristics of strong transmission, high lethality and rapid mutation rate. SARS-CoV-2 can cause respiratory infections, leading to viral pneumonia and acute respiratory distress syndrome (ARDS) in some patients. At the same time, it can also trigger a cytokine storm, causing multiple organ damage. Since the original strain of the new coronavirus was isolated, new mutant...

Claims

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Application Information

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IPC IPC(8): C07K16/10A61K39/42A61P31/14G01N33/569
CPCC07K16/10A61P31/14G01N33/56983C07K2317/565C07K2317/569C07K2317/92C07K2317/22C07K2317/76C07K2317/94G01N2333/165G01N2469/10
Inventor 杨鹏远王楷刘兰兰
Owner INSITUTE OF BIOPHYSICS CHINESE ACADEMY OF SCIENCES
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