Broad-spectrum neutralizing novel coronavirus SARS-CoV-2 nano antibody as well as preparation method and application thereof

Abstract

The invention discloses a nano antibody for broad-spectrum neutralization of novel coronavirus SARS-CoV-2 as well as a preparation method and application of the nano antibody. The invention also discloses nucleic acid or a gene for coding the nano antibody, an expression cassette, a recombinant vector, a recombinant cell or a recombinant bacterium and application of the nano antibody in preparation of drugs or vaccines for treating diseases infected by the novel coronavirus SARS-CoV-2 or the variant thereof. The amino acid sequence of the nano antibody has the characteristics of small molecular weight, high stability and high affinity, and the nano antibody can be combined with various mutants of S protein RBD to neutralize novel Spike pseudovirus of coronavirus. The invention also relates to the nano antibody and the related product thereof, and the nano antibody and the related product thereof have remarkable binding capacity and broad-spectrum effect in preventing and / or treating related diseases infected by the novel coronavirus and the variant thereof.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to broad-spectrum nanoantibodies that neutralize the novel coronavirus SARS-CoV-2, and a preparation method and application thereof. Background Art

[0002] The development of vaccines and antibody drugs will provide people with effective protection and treatment measures. The new coronavirus SARS-CoV-2 binds to their common receptor angiotensin-converting enzyme 2 (ACE2) through the receptor binding domain (RBD) of the S protein, mediating the virus's entry into host cells. Neutralizing antibodies target the highly conserved RBD region on the coronavirus surface spike protein (Spike, S), inhibiting the membrane fusion of the virus envelope and the host cell membrane, thereby inhibiting the virus from entering the host cell and achieving the purpose of neutralizing the virus. Neutralizing antibodies are an effective means of preventing and treating new coronavirus infection. Neutralizing antibodies have a clear and immediate therapeutic effect in the emergency treatment of infected patients, which has been fully confirmed in the development of drugs for the Ebola virus epidemic.

[0003] Nanobodies are heavy chain antibodies that are naturally missing light chains. They are found in animals such as alpacas, dromedaries, and sharks. They are the smallest structural units that can accurately identify antigen molecules. At the same time, nanobodies have many advantages, including better heat resistance and stability, stronger affinity with antigens, stronger tissue penetration, higher water solubility, and high expression levels. Based on the above characteristics, nanobodies can be used for early diagnosis and targeted treatment of diseases, improving the diagnostic accuracy and treatment effects of diseases. The development of the nanoantibody market benefits from the continued growth of the antibody drug market, the development of nanoantibody technology and the biopharmaceutical field, and its huge potential as a new treatment method in high-incidence diseases such as cancer, cardiovascular disease, and infectious diseases.

[0004] Nanobodies have shown great potential in the prevention and control of the new coronavirus. Studies have shown that nanobodies can specifically block the binding of ACE2 / SARS-CoV-2S-RBD, thereby effectively neutralizing the virus and preventing it from infecting host cells. In addition, nanobodies can more easily penetrate tissue barriers, such as lung tissue, due to their small size and high stability, to achieve local treatment. Therefore, they are developed into aerosolized inhalable preparations, which are delivered directly to the lungs by inhalation to achieve the purpose of treating new coronavirus infection. Nanobodies can be used as vaccines to prevent new coronavirus infection and block the spread of the epidemic, as well as to develop rapid and accurate new coronavirus detection kits, providing strong support for epidemic prevention and control. Although the new coronavirus nanoantibodies have many advantages and application prospects, their research and development and application still face some challenges, such as production costs, stability, immunogenicity and other issues. Therefore, further exploration and optimization are needed in the future to give full play to the role of nanoantibodies in the prevention and control of the new coronavirus. Summary of the invention

[0005] Purpose of the invention: The technical problem to be solved by the present invention is to provide a broad-spectrum nanoantibody that neutralizes the new coronavirus SARS-CoV-2.

[0006] The technical problem that the present invention also aims to solve is to provide a nucleic acid or gene encoding the nanobody.

[0007] The technical problem that the present invention also solves is to provide a method for preparing the broad-spectrum nanoantibody that neutralizes the new coronavirus SARS-CoV-2.

[0008] The final technical problem to be solved by the present invention is to provide the use of the broad-spectrum nanoantibody that neutralizes the new coronavirus SARS-CoV-2, the nucleic acid or gene, the expression cassette, the recombinant vector, the recombinant cell or the recombinant bacteria in the preparation of a drug or vaccine for treating infection with the new coronavirus SARS-CoV-2 or its variants.

[0009] Technical solution: In order to solve the above technical problems, the present invention provides a broad-spectrum neutralizing novel coronavirus SARS-CoV-2 nanoantibody, the amino acid sequence of which is shown in SEQ ID NO: 1.

[0010] The present invention also includes a nucleic acid or gene encoding the nanobody, whose nucleotide sequence is shown in SEQ ID NO: 2.

[0011] The present invention also includes expression cassettes, recombinant vectors, recombinant cells or recombinant bacteria, which contain the nucleic acid or gene.

[0012] Wherein, the recombinant vector is obtained by introducing the nucleic acid or gene into an expression vector.

[0013] Wherein, the expression vector includes but is not limited to a eukaryotic expression vector.

[0014] Wherein, the recombinant bacteria are obtained by introducing the recombinant vector into a host bacteria.

[0015] The host bacteria include but are not limited to Pichia pastoris.

[0016] The present invention also includes a method for preparing the broad-spectrum nanoantibody that neutralizes the novel coronavirus SARS-CoV-2, comprising the following steps:

[0017] 1) Obtaining a nucleic acid or gene of a broad-spectrum nanoantibody that neutralizes the novel coronavirus SARS-CoV-2, wherein the sequence of the nucleic acid or gene is shown in SEQ ID NO: 2;

[0018] 2) connecting the nucleic acid or gene to an expression vector and introducing it into a host bacterium to obtain a recombinant bacterium;

[0019] 3) Inducing the expression of the recombinant bacteria to obtain the recombinant protein and further purifying it to obtain the nanobody.

[0020] The present invention also includes the use of the broad-spectrum nanoantibody that neutralizes the new coronavirus SARS-CoV-2, the nucleic acid or gene, the expression cassette, the recombinant vector, the recombinant cell or the recombinant bacteria in the preparation of a drug or vaccine for treating diseases infected by the new coronavirus SARS-CoV-2 or its variants.

[0021] Among them, the mutants include Delta mutants, Omicron mutants, Omicron subtype mutants XBB.1 or other mutants of the new coronavirus SARS-CoV-2.

[0022] Beneficial effects: Compared with the prior art, the outstanding effect of the present invention is that: the present invention provides a broad-spectrum, specific nano-antibody targeting the novel coronavirus S protein receptor binding region (RBD) and the nucleic acid sequence and amino acid sequence of the encoding gene, the amino acid sequence has the characteristics of small molecular weight, high stability and high affinity, and can bind to multiple mutants of the S protein RBD to neutralize the novel coronavirus Spike pseudovirus. The present invention also relates to nano-antibodies and related products that have significant binding ability and broad-spectrum effects in the prevention and / or treatment of diseases related to novel coronavirus and its variants. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 A is a graph showing the agarose gel electrophoresis results of total RNA extracted from peripheral lymphatic blood of alpacas, which can show the quality and purity of total RNA; Figure 1 B is the agarose gel electrophoresis of the first round of PCR amplification library using oligo dT Primer and random 6-mers cDNA templates for amplification, showing clear bands and no spurious bands; Figure 1 C is agarose gel electrophoresis of the second-round PCR amplified library using different amounts of cDNA template; Figure 1 D is the agarose gel electrophoresis result of the PCR amplification product of the positive clone colony. 46 out of the 48 selected single clones were positive clones, and the positive rate was 95.83%.

[0024] Figure 2 A is the result of the ELISA test of the binding of the nanoantibody to the S protein RBD of the original strain, Delta mutant strain, and Omicron mutant strain of the new coronavirus; Figure 2B is an inverted fluorescence electron micrograph of 293T cells infected with Omicron pseudovirus after incubation with nanoantibodies, showing that nanoantibodies have a good effect in preventing pseudoviruses from invading cells.

[0025] Figure 3 A is the result of testing the stability of nanoantibodies at different temperatures. The antibodies were treated at different temperatures for 10 minutes, and the antigens were coated on the surface of the solid phase carrier. The results of the enzyme-linked immunosorbent assay showed that the antibodies still maintained good activity after being treated at various temperatures. Figure 3 B is to detect the stability of nano antibodies at an extreme temperature of 95°C. The antibodies were treated at 95°C for different time periods and the antigens were coated on the surface of the solid phase carrier. The results of the enzyme-linked immunosorbent assay showed that the antibody could still maintain good activity after being treated at 95°C for 2 hours, and the antibody activity decreased significantly after being treated for 6 hours, and the antibody was completely inactivated after being treated for 12 hours. Figure 3 C detected the stability of the nanobody at 37°C to evaluate the stability of the subsequent nanobody during transportation or storage as a drug. The antibody was treated at 37°C for different time periods and the antigen was coated on the surface of the solid phase carrier. The results of the enzyme-linked immunosorbent assay showed that the antibody still maintained good activity after being treated at 37°C for 10 days.

[0026] Figure 4 To detect the affinity of nanoantibodies to the Omicron subtype variant XBB.1 by BLI, an antibody concentration gradient was set to analyze the molecular interaction kinetics and determine the antigen-antibody affinity KD value of 5.974×10 -7 ±3.877×10 -8 , has a strong affinity, among which K D : dissociation constant; Kon: association rate constant; Koff: dissociation rate constant; R 2 : Coefficient of determination. DETAILED DESCRIPTION

[0027] The embodiments of the present invention will be described in detail below in conjunction with the examples, and the following examples are only used to illustrate the present invention. If the specific conditions are not specified in the examples, they are carried out according to the conventional conditions or the conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be obtained commercially.

[0028] Example 1 Establishment of Nanobody Library and Acquisition of Nanobodies

[0029] Alpacas (purchased from Shenzhen Kangti Life Science Co., Ltd., 2.5 years old, weighing 55 kg) were immunized with the original strain of SARS-CoV-2 spike-RBD protein (purchased from Baiying Biotechnology, catalog number B232003, 400 μg per immunization). After 6 weeks of immunization, peripheral lymphatic blood was collected from the alpacas, and 1×10 7Peripheral blood lymphocytes were collected and transferred to a 1.5 mL centrifuge tube, and 1 / 5 volume of chloroform was added to mix; after standing at room temperature for 5 minutes, centrifuge at 4 degrees and 12000g for 15 minutes; carefully transfer the supernatant after centrifugation to a new centrifuge tube; add 0.5-1 times the volume of isopropanol to the new centrifuge tube; after standing at room temperature for 10 minutes, centrifuge at 4 degrees and 12000g for 10 minutes; use 75% ethanol equal to the volume of peripheral blood lymphocytes preserved by Trizol to wash the precipitate, centrifuge at 4 degrees and 7500g for 5 minutes, dissolve in an appropriate amount of RNase-free water, and combine all samples to obtain the total RNA extracted. The total RNA obtained was reverse transcribed using the Takara reverse transcription kit to obtain total cDNA. The first round of PCR reaction was carried out using cDNA as a template.

[0030] (1) The PCR reaction system is as follows:

[0031]

[0032] (2) Carry out PCR reaction according to the reaction procedure in the following table:

[0033]

[0034] 1μl, 2μl, 3μl, 4μl, and 5μl of oligo dT Primer (Takara, item number 6110A) and random6-mer (Takara, item number 3801) cDNA were used as templates to configure the PCR reaction system, and the PCR products were subjected to 1% agarose gel electrophoresis, and the gel was cut to recover the target fragment band of about 600bp. The first round of PCR amplification products were used as templates for the second round of PCR reaction, and 0.1μl, 0.2μl, 0.3μl, 0.4μl, and 0.5μl were used as templates to configure the PCR reaction system. After the reaction, 10μl of PCR products were taken for 1% agarose gel electrophoresis. Finally, the template amount with a single target band and a fragment size of 600bp in the electrophoresis results was selected as the optimal template amount. The recovered products were measured for concentration and then combined and collected in a centrifuge tube, which was the second round of PCR amplification products. Use pComb3XSS (Addgene, Catalog No. 63890) as a phage plasmid vector to construct a phage library. Use SpeI (NEB, Catalog No. R3133) and SacI (NEB, Catalog No. R3156) to double-digest 14μg pComb3XSS vector and 5μg second-round PCR amplification product, incubate at 37℃ for 4h; use a DNA recovery and purification kit to purify the pCpmb3XSS vector and the second-round PCR amplification product and store at 4℃. Subsequently, connect the vector and the fragment, and prepare the connection reaction system according to the following table:

[0035]

[0036] The ligation reaction was incubated at 4°C overnight (about 16h); the ligation reaction solution was purified using a universal DNA purification recovery kit (Tiangen, catalog number DP214), the concentration of the recovered product was detected, and it was stored at 4°C. TG1 competent cells (Beijing Huayueyang Biology, catalog number WG1221) were taken for electrotransformation; after waiting for the completion of electrotransformation, SOC culture medium (Sigma, catalog number S1797) was immediately added to shake the bacteria for 1h; 100μl was taken from 1ml of the revived bacterial solution for 10-fold gradient dilution and plated, and the number of transformed colonies that can be obtained in each reaction was calculated according to the dilution multiple and the number of single colonies, which is the transformation efficiency of the ligation product; at the same time, 48 monoclonal bacteria were randomly selected for colony PCR, and the monoclonal positive rate was estimated. After library screening and ELISA verification, positive clones were selected and inoculated to 2 certificates, and medium (Sigma, catalog number Y2377) was selected for large-scale amplification and expression, and further purification was performed using an AKTA protein purifier.

[0037] Depend on Figure 1 It can be seen that the agarose gel electrophoresis results showed that the total RNA was of high quality and purity; the agarose gel electrophoresis of the first and second rounds of PCR amplification of the library showed that the amplified bands were clear and free of mixed bands; the colony PCR agarose gel electrophoresis results showed that 46 of the 48 selected monoclonal clones were positive clones, with a positive rate of 95.83%, and the positive clone rate of the bacterial library met the requirements; the eukaryotic expression vector of the positive clone was electrotransformed into the competent Pichia pastoris X-33 (Shanghai Weidi Biotechnology Co., Ltd.), and the monoclonal yeast expression strain was picked and inoculated into 2 ml of the culture system (expansion medium containing 10×YNB, 50% glycerol, 500× biotin, 100ug / ml bleomycin), 30℃, 250rpm overnight culture, transfer the seed bacteria to 200ml culture system (expansion culture medium, containing 10×YNB, 50% glycerol, 500× biotin, without bleomycin), 30℃, 250rpm, 24 hours of culture, centrifugation, discard the supernatant, add 200ml induction culture solution (10×YNB, 1% methanol, 500× biotin, 1000× chloramphenicol) to induce expression, add methanol with a final concentration of 1% every 24 hours, collect samples after 72 hours of induction expression, centrifuge and filter the supernatant, and then purify it with a nickel column. The antibody has a high purity after purification, with a purity of >95%. It can be used in subsequent experiments. The amino acid sequence of the nanobody is: VQLQESGGGLVQAGGSLRLSCAASGGTFSSYAMGWFRQAPGKERYVAAISSGRTTFYADSVKGRFTISRDNAKNTVDLQMNSLEPEDTAVYYCAADRSPNYGRYSASFMASRYDYWGQGTQVTVSS.

[0038] Example 2 Broad-spectrum binding of nanoantibodies to the original strain and mutant strains of the new coronavirus

[0039] 2 μg of the S protein RBD of the original strain of the new coronavirus, the Delta mutant strain, and the Omicron mutant strain (purchased from Baiying Biotechnology, item number B232003, Sino Biological, item number 40592-V08H88, and Sino Biological, item number 40592-V08H144, respectively) were coated on the surface of a 96-well plate at 4°C overnight, and a 100mM NaHCO3 solution was used as a control group (CBS); 100 μL of milk blocking solution was added to each well for incubation for 1 hour, and then washed 5 times with 300 μL PBS; 8 μg of the nanoantibody prepared in Example 1 was added for incubation for 1 hour, and washed 10 times with 300 μL PBS; color development was performed subsequently, and the values ​​were read using an enzyme reader. Results refer to Figure 2 A, from Figure 2 A. Nanoantibodies can bind to the S protein RBD of the original strain, Delta mutant strain, and Omicron mutant strain of the new coronavirus. The nanoantibody has a broad spectrum of binding to the new coronavirus strains.

[0040] In the antibody neutralization experiment, 293T cells were first plated in a 96-well cell culture plate with a cell confluence of about 40%. 1 μL of the novel coronavirus Omicron mutant pseudovirus (Yisheng Bio, Cat. No. 12024ES50) was taken and incubated with 1×PBS (pH7.4) and nanoantibodies with a final concentration of 50 μM, 100 μM, and 200 μM at 37°C for 1 hour. After the incubation, it was added to the 293T cells and cultured for 24 hours. The cell nuclei were stained with Hoechst dye, and the fluorescence signals of each group of cells were observed under an inverted fluorescence microscope. Figure 2 B shows that the nanoantibody has a good effect in preventing pseudoviruses from invading cells.

[0041] Example 3 Temperature sensitivity experiment of nanobodies

[0042] 100 μl (1 μg / mL) of the original strain of the new coronavirus spike-RBD protein was coated on the surface of a 96-well plate at 4°C overnight, and a 100mM NaHCO3 solution was used as the control group (CBS). After taking it out the next day, 100 μL of milk blocking solution was added to each well and incubated for 1 hour, and then washed 5 times with 300 μL 1×PBS (pH7.4). The nanoantibodies with a final concentration of 100 μM were divided and placed in a water bath at 4°C, 40°C, 60°C, 80°C, 90°C, and 95°C for 10 minutes. After treatment, they were added to the surface of the 96-well plate and incubated at room temperature for 1 hour, washed 5 times with 300 μL PBS, and horseradish peroxidase colorimetric solution was added for color development, and the values ​​of each well were read with an enzyme reader. The results are as follows Figure 3A. Nanobodies have good temperature stability. After being treated at different temperatures for 10 minutes, their activity is basically consistent with that of untreated nanobodies. Even after being treated at a high temperature of 95°C for 10 minutes, the nanobodies can still maintain about 80% of their activity.

[0043] In order to evaluate the stability of nanobodies under high temperature conditions, the activity of nanobodies was detected by enzyme-linked immunosorbent assay after treatment at 95°C for 0 min, 10 min, 30 min, 60 min, 2 h, 6 h, 12 h, 18 h, and 24 h. Figure 3 B. Treating the nanoantibodies at extreme temperature (95°C) showed that the antibodies still maintained good activity after being treated at 95°C for 2 hours. The antibody activity decreased significantly after 6 hours of treatment, and the antibody was completely inactivated after 12 hours of treatment. In order to evaluate the temperature stability of the nanoantibodies during subsequent transportation or storage as drugs, the nanoantibodies were placed at 37°C for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, and 10 days, and then subjected to enzyme-linked immunosorbent assay. Figure 3 C shows that the nanoantibody has good stability when treated at 37°C, and the results of the enzyme-linked immunosorbent assay show that the antibody still maintains good activity after being treated at 37°C for 10 days.

[0044] Example 4 Affinity determination of nanobodies and Omicron subtype variant XBB.1

[0045] In order to determine the interaction between the nanobody and the Omicron subtype variant strain XBB.1 (Yisheng Bio, Cat. No.

[0046] 12024ES50), and the molecular interaction dynamics characteristics were analyzed by biomembrane interferometry (BLI). A preliminary experiment was carried out by diluting the nanoantibody to 1 μM with 0.1% BSA and the Omicron mutant RBD protein to 10 μg / mL to preliminarily determine the optimal antibody (original) concentration for sensor immobilization and the optimal concentration of the analyte to be tested. Antigen-antibody affinity determination was carried out; the antibody concentrations were set to 1 μM, 0.5 μM, 0.25 μM, 0.125 μM, 0.0625 μM, and 0 μM (2-fold dilution, and 0 concentration was set as a control), and then molecular interaction dynamics characteristics analysis (ForteBio) was performed to determine its affinity. By Figure 4 It can be seen that BLI detects the affinity of nanoantibodies to the Omicron subtype variant XBB.1, sets an antibody concentration gradient, performs molecular interaction kinetics analysis, and determines the antigen-antibody affinity K D Value 5.974×10 -7 ±3.877×10 -8, with strong affinity.

Claims

1. A broad-spectrum nanoantibody that neutralizes the novel coronavirus SARS-CoV-2, characterized in that: The amino acid sequence of the Nanobody is shown in SEQ ID NO:

1.

2. A nucleic acid or gene encoding the Nanobody according to claim 1, characterized in that Its nucleotide sequence is shown in SEQ ID NO:

2.

3. An expression cassette, a recombinant vector, a recombinant cell or a recombinant bacterium, characterized in that: It contains the nucleic acid or gene according to claim 2.

4. The recombinant vector according to claim 3, characterized in that The recombinant vector is obtained by introducing the nucleic acid or gene into an expression vector.

5. The recombinant vector according to claim 4, characterized in that The expression vector includes a eukaryotic expression vector.

6. The recombinant bacterium according to claim 3, characterized in that The recombinant bacteria are obtained by introducing the recombinant vector into a host bacteria.

7. The recombinant bacterium according to claim 6, characterized in that The host bacteria include Pichia pastoris.

8. The method for preparing a broad-spectrum nanoantibody that neutralizes the novel coronavirus SARS-CoV-2 according to claim 1, characterized in that: The following steps are involved: 1) Obtaining a nucleic acid or gene of a broad-spectrum nanoantibody that neutralizes the novel coronavirus SARS-CoV-2, wherein the sequence of the nucleic acid or gene is shown in SEQ ID NO: 2; 2) connecting the nucleic acid or gene to an expression vector and introducing it into a host bacterium to obtain a recombinant bacterium; 3) Inducing the expression of the recombinant bacteria to obtain the recombinant protein and further purifying it to obtain the nanobody.

9. Use of the broad-spectrum nanoantibody that neutralizes the new coronavirus SARS-CoV-2 according to claim 1, the nucleic acid or gene according to claim 2, the expression cassette, recombinant vector, recombinant cell or recombinant bacteria according to claim 3 in the preparation of a drug or vaccine for treating diseases infected by the new coronavirus SARS-CoV-2 or its variants.

10. The use according to claim 9, characterized in that: The variants include Delta mutants, Omicron mutants, Omicron subtype variants XBB.1 or other variants of the new coronavirus SARS-CoV-2.

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