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High affinity nanoantibodies of SARS-CoV-2 alpha, gamma, delta and o mutants camel source

A technology for sars-cov-2 and mutant strains, which is applied in the field of camel-derived high-affinity nanobodies for SARS-CoV-2 α, γ, δ, and ο mutant strains, and can solve the problem of difficulty in obtaining plasma and the small amount of plasma from patients who have recovered from serum therapy problem, to achieve the effect of high stability, high neutrality and ability, high sensitivity

Pending Publication Date: 2022-07-29
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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  • High affinity nanoantibodies of SARS-CoV-2 alpha, gamma, delta and o mutants camel source
  • High affinity nanoantibodies of SARS-CoV-2 alpha, gamma, delta and o mutants camel source
  • High affinity nanoantibodies of SARS-CoV-2 alpha, gamma, delta and o mutants camel source

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0085] Example 1. Construction of SARS-CoV-2 Nanobody Library

[0086] Take 200ug of SARS-CoV-2 virus Wild Type original strain S protein and RBD protein (Beijing Yiqiao Shenzhou Biological Co., Ltd.) mixed with an equal volume of complete Freund's adjuvant, fully emulsified and injected into camels, and then boosted immunization every two weeks One time, the mixture of incomplete Freund's adjuvant and immunogen was used in booster immunization, and multiple subcutaneous immunizations on the back of the neck were used for a total of 5 immunizations. From the third immunization, blood was collected from the jugular-clavicular vein one week after each immunization and serum titers were detected.

[0087] 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 below)...

Embodiment 2

[0113] Example 2. Screening of SARS-CoV-2 Nanobodies

[0114] The first well of a 96-well microtiter plate was coated with the SARS-CoV-2 virus S protein antigen at a concentration of 1ug / mL, overnight at 4°C; the next day, the coating solution was poured out and washed three times with PBST. Block the first and second wells of the ELISA plate with BSA and incubate at room temperature for 2 hours; pour out the blocking solution and wash with PBST for 3 times; add the phage nanobody library obtained in Example 1 to the first well and react for 2 hours; Discharge the liquid, pat dry on clean absorbent paper, and wash 5 times with PBST; add 100 μL of S1 protein of SARS-CoV-2 virus Wild type original strain to the first well, and react for 1 h; aspirate 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 amplification.

[0115...

Embodiment 3

[0117] Example 3. Expression of SARS-CoV-2 Nanobodies

[0118] 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-A8. After amino acid sequencing analysis, the amino acid sequences of the obtained nanobodies were as shown in SEQ IDNO: 1-8 shown.

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Abstract

The invention relates to a camel source high-affinity nano antibody of SARS-CoV-2 alpha, gamma, delta and o mutant strains, in particular to an antibody specifically bound with S protein of novel coronavirus (SARS-CoV-2) and an antigen binding fragment of the antibody, and more particularly relates to an antibody or an antigen binding fragment of the antibody capable of being bound with S protein on the surface of the SARS-CoV-2 alpha, gamma, delta and o mutant strains with high affinity, such as SARS-CoV-2 Alpha (B.1. 1.7), Gamma (P.1), Delta (B.1. 617.2) and Omicro (B.1. 1.529) mutant strains. The amino acid sequence of the gene comprises a CDR1 shown as any one of SEQ ID NO: 9 to 15, a CDR2 shown as any one of SEQ ID NO: 16 to 21, and a CDR3 shown as any one of SEQ ID NO: 22 to 29; the polypeptide can be used for preventing, detecting, diagnosing or treating infection caused by coronavirus, especially SARS-CoV-2 virus.

Description

technical field [0001] The invention belongs to the fields of biotechnology, immune detection and biomedicine, and specifically relates to specific nanobodies or antigen-binding fragments, antigen-recognition epitopes and their uses in the detection, diagnosis, prevention and treatment of coronaviruses such as SARS-CoV-2 , especially in the detection, diagnosis, prevention of SARS-CoV-2 Alpha (B.1.1.7), Gamma (P.1), Delta (B.1.617.2) and Omicron (B.1.1.529) mutants , Use in 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 viru...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/10C07K14/165G01N33/569A61K39/42A61P31/14
CPCC07K16/10C07K14/005G01N33/56983A61P31/14C07K2317/565C12N2770/20022G01N2333/165A61K2039/505
Inventor 杨鹏远王楷刘兰兰章新政曹端方范晓益
Owner INSITUTE OF BIOPHYSICS CHINESE ACADEMY OF SCIENCES
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