A single-chain antibody against the S1 protein on the surface of SARS-CoV-2 and its application

Abstract

This invention discloses a single-chain antibody against the S1 protein on the surface of SARS-CoV-2, comprising a heavy chain sequence and a light chain sequence. The invention also discloses a gene encoding the above sequence, a recombinant vector containing the above gene, a host cell containing the above vector, a biological agent containing the above antibody, and the application of the above antibody against SARS-CoV-2. This effectively solves the problem of a lack of highly potent antibodies against SARS-CoV-2.

Description

Technical Field

[0001] This invention relates to the field of single-chain antibody technology, specifically to a single-chain antibody against the S1 protein on the surface of SARS-CoV-2 and its application. Background Technology

[0002] The global spread of the novel coronavirus has drawn widespread attention to the antiviral drug industry. Greater focus is now placed on vaccines and antiviral drugs, and the development of antibody drugs is poised to become a medium- to long-term trend.

[0003] Neutralizing antibodies are a type of antibody produced by human B lymphocytes. When pathogens such as viruses and bacteria invade human cells, these antibodies can bind to antigens on the surface of the pathogens and neutralize them. Scientific research shows that the "weapon" used by the novel coronavirus (Severe acute respiratory syndrome coronavirus-2, SARS-CoV-2) to invade human cells is its spike protein (S protein). There are two types of S protein: S1 and S2. After this protein binds to the cell surface receptor—angiotensin-converting enzyme 2—the novel coronavirus can invade human cells. The receptor domain of the S protein is an important receptor binding site on the surface of coronaviruses, making it an important target for developing antiviral neutralizing antibodies.

[0004] Currently, there are no antibodies against the novel coronavirus that can be officially used in clinical practice. It is difficult to predict the effectiveness of antibodies in actual application when they are in the experimental stage. There is still an extreme shortage of different types of highly effective antibodies against the novel coronavirus. Summary of the Invention

[0005] To address the aforementioned shortcomings in the existing technology, this invention provides a single-chain antibody against the S1 protein on the surface of SARS-CoV-2 and its application, which effectively solves the problem of a lack of highly potent antibodies against the novel coronavirus.

[0006] To achieve the above objectives, the technical solution adopted by the present invention to solve its technical problem is as follows:

[0007] A single-chain antibody against the surface S1 protein of SARS-CoV-2 is provided. The single-chain antibody includes a heavy chain sequence and a light chain sequence. The heavy chain sequence includes CDR1, CDR2 and CDR3 sequences as shown in SEQ ID NO. 1~3. The light chain sequence includes CDR1, CDR2 and CDR3 sequences as shown in SEQ ID NO. 4~6.

[0008] Furthermore, the heavy chain sequence comprises an amino acid sequence having at least 70% homology with SEQ ID NO.7; the light chain sequence comprises an amino acid sequence having at least 70% homology with SEQ ID NO.8.

[0009] Furthermore, the single-chain antibody binds to the RBD region of the S1 protein.

[0010] Furthermore, the heavy chain sequence and the light chain sequence are linked by a linker peptide, which consists of a repeating GGGGS amino acid sequence or a variant thereof. For example, a linker having the amino acid sequence (GGGGS)4 can be used, but variants thereof can also be used.

[0011] A coding gene that encodes the heavy chain and light chain sequences of the single-chain antibody against the SARS-CoV-2 surface S1 protein described above.

[0012] A recombinant vector containing the aforementioned gene.

[0013] A host cell containing the aforementioned recombinant vector.

[0014] A biological agent comprising the above-mentioned single-chain antibody.

[0015] The application of the above-mentioned single-chain antibody against the S1 protein on the surface of SARS-CoV-2 in the preparation of reagents against SARS-CoV-2.

[0016] A drug for the prevention and / or treatment of COVID-19, comprising the aforementioned single-chain antibody against the SARS-CoV-2 surface S1 protein.

[0017] The application of the above-mentioned single-chain antibodies in the preparation of COVID-19 diagnostic kits.

[0018] According to the above technical solution, the present invention has the following advantages:

[0019] 1. Single-chain antibodies have low molecular weight, small size, and strong permeability, enabling them to bind better to antigens and block antigen damage to cells;

[0020] 2. This single-chain antibody has a strong specific binding ability to the S1 protein on the surface of SARS-CoV-2, and can significantly block the binding of the S1 protein on the surface of SARS-CoV-2 to human ACE2 at the protein and cellular levels, and can prevent the SARS-CoV-2 pseudovirus from infecting target cells.

[0021] 3. By adding linker peptides to the heavy chain and light chain sequences, steric hindrance between the heavy chain and light chain sequences can be prevented, ensuring that the single-chain antibody against the SARS-CoV-2 surface S1 protein forms the correct spatial conformation and increasing the binding ability of the single-chain antibody to the antigen. Attached Figure Description

[0022] Figure 1 Results of monoclonal ELISA identification;

[0023] Figure 2 To detect the binding of single-chain antibody ABF-013 to antigen by flow cytometry;

[0024] Figure 3 The results of in vitro neutralization identification of different concentrations of single-chain antibodies against different strains of SARS-CoV-2;

[0025] Figure 4 IC50 for single-chain antibodies against different strains of SARS-CoV-2 50 value. Detailed Implementation

[0026] The present invention will be further described in detail below with reference to specific embodiments, but the scope of protection of the present invention is not limited to the content described herein. Unless otherwise specified, the raw materials mentioned in the embodiments are all commercially available products. The experimental methods mentioned in the embodiments are not specifically described, and are performed according to conventional biological experimental methods.

[0027] Example 1: Single-chain antibody ABF-018 against SARS-CoV-2 surface S1 protein

[0028] I. Construction of Phage Single-Chain Antibody Display Library

[0029] 1. Peripheral blood was collected from four COVID-19 convalescent patients, and B cells were isolated from the peripheral blood.

[0030] The experiment was conducted on October 17, 2020. With informed consent, 20 mL of peripheral blood was collected from each of four recovered COVID-19 patients. PBMCs were isolated using density gradient centrifugation.

[0031] 2. RNA extraction and cDNA synthesis from PBMCs

[0032] RNA was extracted from PBMC cells and then reverse transcribed into cDNA using a synthesis kit.

[0033] 3. PCR amplification of VK, VL and VH

[0034] (1) The VK&VL system was expanded, as shown in Table 1.

[0035] Table 1. Amplification of VK & VL systems

[0036]

[0037] (2) The heavy chain VH segment system was amplified, as shown in Table 2.

[0038] Table 2. Amplified Heavy Chain VH Segment System

[0039]

[0040] (3) The reaction procedure is shown in Table 3.

[0041] Table 3 Reaction Procedure

[0042]

[0043] The PCR products were subjected to 2% agarose gel electrophoresis, and fragments of approximately 750 bp were recovered.

[0044] 4. Light chain cloning (cloning VK and VL into the pComb3H vector)

[0045] VK and VL were digested with XbaI and SacI, then ligated into the pComb3H vector, which was also digested with XbaI and SacI. The ligation products were recovered, and then electroporated into XL1-Blue competent cells. The electroporated bacterial culture was plated on 15cm Petri dishes, and the cells were scraped off the next day to extract the plasmids, which were the light chain libraries. At this point, the recombinant plasmids were pComb3H-VK and pComb3H-VL.

[0046] 5. Heavy chain cloning (cloning the VH gene into the pComb3H-VK and pComb3H-VL light chain libraries)

[0047] The light chain library pComb3-L and Fd fragments were double-digested with XhoI and SpeI, respectively, and then ligated with pComb3H-VK and pComb3H-VL, which were also double-digested with XhoI and SpeI. Electroporation was then performed to obtain the phage single-chain antibody display library.

[0048] II. Phage Display Library Screening

[0049] Library screening was performed using the SARS-CoV-2 S1 protein to obtain positive clones, which were then identified and sequenced. The specific process is as follows:

[0050] 1. Take an appropriate amount of single-chain antibody library into 500mL of 2xTY medium and adjust OD. 600 =0.1, place in a shaker at 37℃, 250 rpm for about 2 hours, until OD 600 Take it out when =0.5.

[0051] 2. Add an excess of helper phage KM13, then incubate in a 37°C water bath for 1 hour. Centrifuge, discard the supernatant, resuspend the precipitate in 500 mL of 2xTY medium, and incubate overnight at 25°C and 250 rpm.

[0052] Centrifuge at 3-4°C for 10 minutes, and filter the supernatant through a 0.45-micron filter membrane. Add an appropriate amount of PEG solution to the filtrate at a ratio of 100 mL PEG solution / 400 mL filtrate, place on ice for 1 hour, then centrifuge at 4°C for 30 minutes, discard the supernatant, and resuspend the precipitate in 1 mL PBS.

[0053] 4. Add the above phage solution to a blank 96-well plate and incubate at room temperature for 1 hour for pre-blocking.

[0054] 5. Remove the 96-well plate coated with S1 protein from the previous day (6 wells per protein, 12 wells in total). Wash three times with PBS, then add 5% skim milk and block for about 1 hour.

[0055] 6. Add the pre-blocked phage solution to each well coated with the S1 protein and incubate at room temperature with shaking for 2 hours. Then add elution buffer to wash off the positive phages from each well and add them to the TG1 bacterial culture in the logarithmic growth phase. Infect at 37°C for 1 hour.

[0056] 7. Centrifuge the above bacterial suspension at room temperature for 10 minutes, and resuspend the precipitate in 2xTY solution. Finally, spread the mixture onto several 15cm 2xTY Agar plates. Incubate overnight at 30°C.

[0057] 8. On the second day, collect the clones and transfer them to a 2xTY solution.

[0058] 9. Repeat the above screening process twice. Take the 192 single clones obtained from the third round of screening for subsequent ELISA identification. Select 42 clones with an OD value greater than 1.8 for sequencing, obtaining 14 different anti-S1 scFv sequences, such as... Figure 1 As shown.

[0059] III. Flow cytometry detection of the binding of single-chain antibody ABF-018 to antigen

[0060] 1. Transiently transfect the expression plasmid containing ABF-018 into 293T cells using PEI transfection reagent;

[0061] 2. 24 h after transfection, add biotinylated S1 protein to each well and incubate at room temperature for 1 hour. Collect the cells and wash them 3 times with PBS.

[0062] 3. Add APC-Streptavidin to each well and incubate at room temperature for 30 minutes, then wash 3 times with PBS;

[0063] 4. FACS analysis.

[0064] like Figure 2As shown, Ctrl is the control group that was not transfected. Both S1 and Neg Ctrl S2 groups were transfected with single-chain antibodies. Then, biotinylated S1 protein or S2 protein was added to ABF-018 as a negative control. The results showed that ABF-018 could specifically bind to S1 protein, but did not bind to the negative control S2 protein.

[0065] Example 2: Identification of the in vitro neutralizing activity of the single-chain antibody ABF-018

[0066] ABF-018 was then cloned into a secretory expression vector containing Fc, and the supernatant was used to further verify its blocking effect on the infection of target cells by the pseudovirus of SARS-CoV-2. The specific steps are as follows:

[0067] 1. Take a 96-well plate and add 100 μL / well of DMEM complete medium (containing 1% antibiotic, 25 mM HEPES, 10% FBS) to the cell control wells; add 100 μL / well of DMEM complete medium to the virus control wells; and add 100 μL / well of DMEM complete medium to the experimental wells to dilute the test antibody to various concentration gradients. The antibody concentrations used in the experiment are 10... -2 nM, 10 -1 nM, 10 0 nM, 10 1 nM, 10 2 nM, 10 3 nM, 10 4 nM and 10 5 nM.

[0068] 2. Dilute the SARS-CoV-2 pseudovirus to approximately 1.0 × 10⁻⁶ using DMEM complete medium. 4 / mL, and then add 50μL / well of SARS-CoV-2 pseudovirus to the virus control well and experimental well.

[0069] 3. Place the 96-well plate in a cell culture incubator (37℃, 5% CO2) and incubate for 1 hour.

[0070] 4. After incubation, add 50 μL of 0.02 M HuH-7 cells to the cell control wells, virus control wells, and experimental wells, and place the 96-well plate in a cell culture incubator (37℃, 5% CO2) for 48 hours.

[0071] 5. Remove the 96-well plate from the cell culture incubator, aspirate the supernatant from each well, add 50 μL of lysis buffer, and incubate at room temperature for 5 min.

[0072] 6. Use a pipette to transfer the liquid from each well to the corresponding 96-well opaque chemiluminescence detection plate. Add 50 μL of detection buffer and react at room temperature in the dark for 5 min. Read the luminescence value using a Promega GloMax luminescence detector. Wherein, inhibition rate = 1 - (reading value) 样品 -reading 阴性对照 ) / (reading 空白对照 -reading 阴性对照 )

[0073] The results are as follows Figure 3 and Figure 4 As shown, the single-chain antibody 13 against the S1 protein of SARS-CoV-2 exhibits an IC50 neutralizing activity against the original SARS-CoV-2 strain, the Beta mutant strain (B.1.351 South African mutant), the Delta mutant strain (B.1.617.2 Indian mutant), and the Omicron mutant strain (B.1.1.529 mutant) S1 protein. 50 The concentrations were 4.6 nM, 24.8 nM, 41.9 nM, and 118.1 nM, respectively. This indicates that the single-chain antibody with the heavy chain sequence of SEQ ID NO.7 and the light chain sequence of SEQ ID NO.8 has extremely high efficacy against the S1 protein on the surface of SARS-CoV-2.

[0074] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention. sequence list <110> Chen Xiang <120> A single-chain antibody against the S1 protein on the surface of SARS-CoV-2 and its application <160> 8 <170> SIPOSequenceListing 1.0 <210> 1 <211> 5 <212> PRT <213> Artificial Sequence <400> 1 Ser Tyr Ala Ile Ser 1 5 <210> 2 <211> 17 <212> PRT <213> Artificial Sequence <400> 2 Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe Gln 1 5 10 15 Gly <210> 3 <211> 10 <212> PRT <213> Artificial Sequence <400> 3 Asp Arg Gly Tyr Tyr Tyr Tyr Gly Met Asp Val 1 5 10 <210> 4 <211> 13 <212> PRT <213> Artificial Sequence <400> 4 Ser Gly Ser Ser Ser Ser Asn Ile Gly Asn Asn Ala Val Asn 1 5 10 <210> 5 <211> 7 <212> PRT <213> Artificial Sequence <400> 5 Tyr Asp Asp Leu Leu Pro Ser 1 5 <210> 6 <211> 11 <212> PRT <213> Artificial Sequence <400> 6 Ala Ala Trp Asp Asp Ser Leu Asn Gly Trp Val 1 5 10 <210> 7 <211> 136 <212> PRT <213> Artificial Sequence <400> 7 Val Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 1 5 10 15 Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly 20 25 30 Ser Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser 35 40 45 Tyr Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp 50 55 60 Met Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys 65 70 75 80 Phe Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala 85 90 95 Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr 100 105 110 Cys Ala Arg Asp Arg Gly Tyr Tyr Tyr Gly Met Asp Val Trp Gly Gln 115 120 125 Gly Thr Thr Val Thr Val Ser Ser 130 135 <210> 8 <211> 108 <212> PRT <213> Artificial Sequence <400> 8 Leu Pro Val Leu Thr Gln Pro Pro Ser Val Ser Glu Ala Pro Arg Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Asn Asn 20 25 30 Ala Val Asn Trp Tyr Gln Gln Leu Pro Gly Lys Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Tyr Asp Asp Leu Leu Pro Ser Gly Val Ser Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Gln 65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu 85 90 95 Asn Gly Trp Val Phe Gly Gly Gly Thr Lys Leu Thr 100 105

Claims

1. A single-chain antibody against SARS-CoV-2 surface S1 protein, characterized in that, The single-chain antibody comprises a heavy chain sequence and a light chain sequence; the heavy chain sequence comprises CDR1, CDR2 and CDR3 sequences as shown in SEQ ID NO. 1~3; and the light chain sequence comprises CDR1, CDR2 and CDR3 sequences as shown in SEQ ID NO. 4~6.

2. The single-chain antibody against SARS-CoV-2 surface S1 protein according to claim 1, characterized in that, The single-chain antibody is combined with the RBD region of S1 protein.

3. A gene encoding a gene, characterized in that, The coding gene encodes the heavy chain sequence and the light chain sequence of the single-chain antibody against SARS-CoV-2 surface S1 protein according to claim 1.

4. A recombinant vector, characterized in that, The recombinant vector comprises the gene according to claim 3.

5. A host cell, characterized in that, The host cell comprises the recombinant vector according to claim 4.

6. A biological agent, characterized in that, The biological preparation comprises the single-chain antibody according to claim 1.

7. Use of the single-chain antibody according to claim 1 in the preparation of a new coronavirus diagnostic kit.

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