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Anti-SARS-CoV-2 single-chain antibody, and preparation method and application thereof

A single-chain antibody, sars-cov-2 technology, applied in the field of biomedicine, can solve the problems of side effects, difficulty in large-scale application, high production cost of human neutralizing antibodies, etc., achieve high virus neutralization activity, not easy to dissociate, The effect of low manufacturing cost

Active Publication Date: 2021-11-26
厦门福宸百奥生物技术有限公司 +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] CN112442120A discloses a neutralizing antibody against severe acute respiratory syndrome type II coronavirus SARS-CoV-2, which is extracted from the blood of recovered COVID-19 patients Peripheral immune cells, and screen B cells that can bind to the new coronavirus antigen protein-spike protein, and then analyze the single B cells that have produced antibodies at the single cell level to obtain the variable region of the neutralizing antibody encoded in the B cells The gene sequences of the heavy and light chains, which are used to reconstruct and express neutralizing antibodies, are expected to be used in the treatment and prevention of pneumonia and other diseases caused by the new coronavirus, but are limited due to lack of sources and potential side effects, and Human neutralizing antibodies are difficult to apply on a large scale because of high production costs, strict transportation conditions, large immunization doses, and possible ADE side effects

Method used

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  • Anti-SARS-CoV-2 single-chain antibody, and preparation method and application thereof
  • Anti-SARS-CoV-2 single-chain antibody, and preparation method and application thereof
  • Anti-SARS-CoV-2 single-chain antibody, and preparation method and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0078] This embodiment constructs a single-chain antibody phage library, and the construction method of the single-chain antibody phage library includes the following steps:

[0079] (1) Dissolve 100 μg of SARS-CoV-2S1 protein (purchased from Beijing Yiqiao Shenzhou) in 250 μL of PBS, emulsify with an equal volume of aluminum adjuvant, and inject subcutaneously and intramuscularly at multiple points to immunize Bamboo shark. times, with an interval of 2 weeks each time;

[0080] (2) From the second immunization, 1 mL of blood was collected from the tail vein one week after each immunization, 0.5 mL of which was treated with anticoagulation to separate lymphocytes, lysed with trizol, and stored at -80°C for later use; another 0.5 mL was taken and left at room temperature for 1 hour , centrifuge serum to detect serum titer, tail vein blood collection after the last immunization to detect serum pseudovirus neutralization titer;

[0081] (3) Take the lymphocyte lysate after the 3...

Embodiment 2

[0090] In this embodiment, the screening and expression of SARS-CoV-2 single-chain antibody comprises the following steps:

[0091] (1) Take 100 μL of the single-chain antibody phage library prepared in Example 1, inoculate it in 50 mL of 2YT medium containing ampicillin antibiotics and 1% glucose, cultivate to the logarithmic phase at 37° C., add 20 times the number of helper phage M13KO7, After mixing, let stand at 37°C for 20 minutes, shake and culture for 30 minutes, centrifuge, discard the medium, add 50mL 2YT ampicillin kanamycin medium, shake and culture at 30°C overnight, centrifuge the next day, and collect the supernatant , add 1 / 4 volume of PEG / NaCl, precipitate the recombinant phage at 4°C for 2 hours, collect the phage precipitate by centrifugation, dissolve it with 5mL PBS, repeat the precipitation once, dissolve the phage with PBS, add glycerol with a final concentration of 15%, and store in aliquots Store at -80°C for later use, and at the same time take 10 μL ...

Embodiment 3

[0100] This embodiment detects the binding ability of the SARS-CoV-2 single-chain antibody prepared in Example 2, including the following steps:

[0101] (1) The RBD (receptor binding site domain in the spike protein, which is the 332nd to 527th amino acid) proteins (WH-Hu-1 strain, British strain B.1.1.7 (Alpha , including N501Y mutation site), South African strain B.1.351 (Bbeta, including three site mutations K417N, E484K, N501Y) and Indian strain B.1.617 (Kappa, including L452R, E484Q mutation sites), B.1.617. 2 (Delta, including L452R, T478K mutation sites)) diluted in CBS buffer, coated with ELISA plate, 50ng / 100μL / well, incubated at 4°C for 12 hours, discarded the antigen, added blocking solution (5% skimmed milk Dissolve in PBST), 200 μL / well, block at 37°C for 2 hours, wash the plate, 0.05% PBST, 200 μL / well, wash 4 times, 2 minutes each time, shake off the liquid, and pat dry;

[0102] (2) Dilute the 20G6 and 17F6 antibodies in PBST, starting from 100 μg / mL, make se...

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Abstract

The invention relates to an anti-SARS-CoV-2 single-chain antibody, and a preparation method and application thereof. The complementary determining region of the anti-SARS-CoV-2 single-chain antibody comprises a CDR1 and a CDR3, wherein the CDR1 comprises an amino acid sequence as shown in SEQ ID NO.1 or SEQ ID NO.2, and the CDR3 comprises an amino acid sequence as shown in SEQ ID NO.3 or SEQ ID NO.4. The anti-SARS-CoV-2 single-chain antibody has high affinity, can be efficiently combined with SARS-CoV-2, is unlikely to dissociate, has a high virus neutralizing activity, can be efficiently expressed in a prokaryotic system, has a standard and controllable expression process, can greatly reduce the production cost, is small in molecular weight, is stable in physicochemical properties and good in heat resistance, can greatly reduce the transportation cost as a prevention and treatment drug, and has an important application value in the field of SARS-CoV-2 prevention and treatment.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and relates to an anti-SARS-CoV-2 single-chain antibody and its preparation method and application. Background technique [0002] The new coronavirus SARS-CoV-2 is a new type of β coronavirus named by the World Health Organization. The virus has an envelope, the particles are round or oval, and the main structural proteins include spike protein (Spike, S ), messenger protein (Envelop, E), membrane protein (Membrane, M) and nucleocapsid protein (Nucleocapsid, N), some studies have found that SARS-CoV-2 can interact with angiotensin converting enzyme 2 (Angiotensin converting enzyme 2, ACE2) receptors bind to enter host cells, carry out virus infection and replication, cause lung injury, pneumonia, severe acute respiratory syndrome, septic shock, difficult to correct metabolic acidosis and coagulation dysfunction and Multiple organ failure, etc. [0003] At present, the prevention and treatme...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/10C12N15/13C12N15/70C12N1/21A61K39/42A61P31/14C12R1/19
CPCC07K16/10C12N15/70A61P31/14C07K2317/565C07K2317/567C07K2317/622C07K2317/92C07K2317/76A61K2039/505
Inventor 陈志龙陈凌冯波
Owner 厦门福宸百奥生物技术有限公司
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