Nanobody specifically targeting the SARS-CoV-2 RBD

Abstract

The present invention provides a nanobody specifically targeting the SARS-CoV-2 RBD, which relates to the technical field of antibodies. The nanobody specifically targeting the SARS-CoV-2 RBD provided by the present invention is any one of those shown in SEQ ID NO.1-4. By screening the constructed antibody library, the nanobody of the present invention is obtained. This nanobody has good affinity with the SARS-CoV-2 RBD. When applied to the purification of RBD, it shows high specificity, and the purity of the purified protein is high, realizing "one-step" separation and purification.

Description

Technical Field

[0001] The present invention relates to the technical field of antibodies, and in particular to a nanobody specifically targeting the SARS-CoV-2 RBD. Background Art

[0002] In 2019, pneumonia caused by the novel coronavirus (SARS-CoV-2) spread around the world, posing a serious threat to public safety. At present, four types of vaccines have been approved for emergency use, including inactivated virus vaccines, recombinant adenovirus vector vaccines, DNA / mRNA vaccines and recombinant subunit protein vaccines. Among them, recombinant subunit vaccines can be used to prevent and control the spread of the epidemic due to their advantages of easy large-scale production and transportation, and high safety. The receptor-binding domain (RBD) on the S protein of SARS-CoV-2 contains most of the antibody neutralizing epitopes. Therefore, subunit protein vaccines against SARS-CoV-2 mainly use recombinant proteins containing the RBD region as immunogens to stimulate the body's immune response to the RBD domain and prevent the virus from binding to the host's ACE2 receptor.

[0003] There are already a variety of recombinant protein drugs in clinical research and production around the world. However, the separation and purification of recombinant proteins have always been the key links in drug process development and cost control. Since Professor Cuatrecasas proposed the concept of affinity chromatography in 1968, affinity chromatography has developed into a powerful tool in biomedical research and biotechnology. Currently, many recombinant protein drugs use the antibody Fc fragment as a tag, and can be purified using Protein A or Protein G chromatography columns. However, for some recombinant proteins that do not use the Fc tag, if 6×His is used as a tag, metal ions will remain in the protein after purification through a metal ion affinity chromatography column. Therefore, designing and screening specific ligands with low cost, high stability, and high specific binding to the target protein to prepare affinity chromatography columns is a key step in drug production.

[0004] Nanobodies are the antigen-binding regions in the heavy-chain antibodies (IgG2 and IgG3) in camelids, also known as VHH fragments. Due to their small molecular weight, high stability, and easy large-scale production in bacterial expression systems, they can serve as an important source of affinity ligands. Currently, multiple teams have used VHH antibodies against Fc to prepare affinity resins for purifying immunoglobulin G (IgG), and their purification efficiency is superior to that of classical Protein A purification. The Timothy Pabst team used camel VHH antibodies as immunoaffinity ligands for the "one-step" separation and purification of bioactive drugs such as recombinant immunotoxins and clotting factors, obtaining biomolecules with high purity and high activity. These applications demonstrate that nanobody-based affinity ligands can simplify the purification process of biomolecules while ensuring purity and activity.

[0005] Phage display technology is a molecular biology technique that displays various biomacromolecules, including proteins, polypeptides, antibodies, TCRs, etc., on the surface of phages. If a phage library displaying billions of biomolecule variants interacts with a target, monoclonal biomolecules that specifically bind to the target can be enriched after 3 to 5 rounds of biopanning. Based on different panning strategies, various targets such as antigen recombinant proteins, bacteria, viruses, cells, and small molecules can be selected to screen the phage library. For example, the Rafique A team screened VHHs against Fab fragments from alpacas immunized with Fab and performed affinity maturation, ultimately designing VHH affinity ligands that are alkali-resistant, easily eluted under acidic conditions, and have a high affinity for Fabs.

[0006] In view of this, the present invention is specifically proposed. Summary of the Invention

[0007] Through long-term unremitting efforts, the inventors of the present invention screened and obtained antibodies specifically targeting the SARS-CoV-2 RBD. Therefore, the present invention provides the following technical solutions.

[0008] In a first aspect, the present invention provides an antibody specifically targeting the SARS-CoV-2 RBD, which comprises 3 CDRs in SS1 shown in Table 1, 3 CDRs in SS3, 3 CDRs in SS4, or 3 CDRs in S12. Preferably, the antibody is a VHH antibody, a heavy-chain antibody, or a nanobody, and is characterized in that the amino acid sequence of the VHH antibody, heavy-chain antibody, or nanobody comprises any one of SEQ ID NOs. 1 to 4.

[0009] In a second aspect, the present invention provides biomaterials related to the antibody described in the first aspect, and the biomaterials are any one of the following:

[0010] (a) A nucleic acid molecule encoding the antibody described in the first aspect;

[0011] (b) An expression cassette containing the nucleic acid molecule in (a);

[0012] (c) A recombinant vector containing the nucleic acid molecule in (a) or the expression cassette in (b);

[0013] (d) A recombinant eukaryotic cell containing the nucleic acid molecule in (a), the expression cassette in (b) or the recombinant vector in (c);

[0014] (e) A recombinant prokaryotic cell containing the nucleic acid molecule in (a), the expression cassette in (b) or the recombinant vector in (c).

[0015] Preferably, the recombinant vector in (c) is a plasmid, such as pET-25b.

[0016] More preferably, the prokaryotic cell in (e) is Escherichia coli, such as Escherichia coli Rosetta2.

[0017] The third aspect of the present invention provides a pharmaceutical composition, which contains the antibody described in the first aspect.

[0018] The fourth aspect of the present invention provides a method for preparing the antibody described in the first aspect, which is characterized in that a nucleic acid molecule encoding the antibody described in the first aspect is introduced into a recipient cell to obtain a transgenic cell, and the transgenic cell is cultured to obtain the antibody.

[0019] Preferably, the recipient cell is a microbial cell or a mammalian cell.

[0020] The fifth aspect of the present invention provides the use of the antibody described in the first aspect or the biological material described in the second aspect in the preparation of a product for detecting SARS-CoV-2.

[0021] The sixth aspect of the present invention provides the use of the antibody described in the first aspect or the biological material described in the second aspect in the affinity chromatography purification of SARS-CoV-2 RBD.

[0022] The seventh aspect of the present invention provides the use of the antibody described in the first aspect or the biological material described in the second aspect in the preparation of a drug for treating COVID-19.

[0023] More specifically, one of the purposes of the present invention is to provide a nanobody specifically targeting SARS-CoV-2 RBD to at least solve one of the technical problems existing in the prior art.

[0024] The second purpose of the present invention is to provide biological materials related to the above-mentioned nanobody.

[0025] The third purpose of the present invention is to provide a heavy chain antibody containing the above-mentioned nanobody.

[0026] A fourth object of the present invention is to provide a method for preparing the above-mentioned nanobody.

[0027] A fifth object of the present invention is to provide the use of the above-mentioned nanobody or biomaterial.

[0028] In order to achieve the above objects of the present invention, the following technical solutions are specifically adopted:

[0029] A nanobody specifically targeting the SARS-CoV-2 RBD, wherein the amino acid sequence of the nanobody is any one of those shown in SEQ ID NO.1-4.

[0030] A biomaterial related to the above-mentioned nanobody, wherein the biomaterial is any one of the following:

[0031] (a) A nucleic acid molecule encoding the above-mentioned nanobody;

[0032] (b) An expression cassette containing the nucleic acid molecule in (a);

[0033] (c) A recombinant vector containing the nucleic acid molecule in (a) or the expression cassette in (b);

[0034] (d) A recombinant eukaryotic cell containing the nucleic acid molecule in (a), the expression cassette in (b) or the recombinant vector in (c);

[0035] (e) A recombinant prokaryotic cell containing the nucleic acid molecule in (a), the expression cassette in (b) or the recombinant vector in (c).

[0036] Further, the backbone vector in (c) is a pET-25b plasmid.

[0037] Further, the prokaryotic cell in (e) is Escherichia coli Rosetta 2.

[0038] A heavy chain antibody containing the above-mentioned nanobody.

[0039] The method for preparing the above-mentioned nanobody, wherein a nucleic acid molecule encoding the above-mentioned nanobody is introduced into a recipient cell to obtain a transgenic cell, and the transgenic cell is cultured to obtain the nanobody.

[0040] Further, the recipient cell is a microbial cell or a mammalian cell.

[0041] The use of the above-mentioned nanobody or biomaterial in the preparation of a product for detecting SARS-CoV-2.

[0042] The use of the above-mentioned nanobody or biomaterial in the affinity chromatography purification of SARS-CoV-2 RBD.

[0043] Use of the above-mentioned nanobody or biomaterial in the preparation of a drug for treating COVID-19.

[0044] Compared with the prior art, the organic effect of the present invention is as follows:

[0045] The nanobody specifically targeting SARS-CoV-2 RBD provided by the present invention is any one of those shown in SEQ ID NO.1-4. By screening the constructed antibody library, the nanobody of the present invention is obtained. This nanobody has good affinity with SARS-CoV-2 RBD. When it is applied to the purification of RBD, it shows high specificity, and the purity of the purified protein is high, achieving "one-step" separation and purification. Description of the Drawings

[0046] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required for use in the description of the specific embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0047] Figure 1 For the four groups with differences in the CDRH3 region obtained by NGS sequencing provided in Example 4 of the present invention;

[0048] Figure 2 For the SDS-PAGE (12%) electrophoresis pattern of the Anti-RBD nanobody provided in Example 5 of the present invention;

[0049] Figure 3 For the affinity detection of the nanobody VHHs and the antigen (100 ng rRBD, human FC tag) provided in Example 6 of the present invention;

[0050] Figure 4 For the Octet RED384 detection of the competitive binding of antibodies (SS1, SS4, S12) and rRBD provided in Example 7 of the present invention;

[0051] Figure 5 For the Octet RED384 detection of the non-competitive binding of antibodies (SS3, SS4) and rRBD provided in Example 7 of the present invention;

[0052] Figure 6This is the result diagram of purifying rRBD with SS4-Sepharose provided in Example 8 of the present invention. Among them, Lane 1: RBD positive control; Lane 2: After SS4-Sepharose 4FF binds to rRBD, 20 μL of the packing material is taken and mixed with 20 μL of 6× Protein Loading Buffer, and treated at 98 °C for 20 min; Lane 3: Elution;

[0053] Figure 7 This is the result diagram of purifying rRBD with SS3-Sepharose 4FF provided in Example 8 of the present invention. Among them, Lane 1: rRBD eluted from the packing material; Lane 2: The packing material after elution;

[0054] Figure 8 This is the affinity detection of the rRBD recovered after purifying rRBD with SS3-Sepharose 4FF provided in Example 8 of the present invention. Detailed implementation manners

[0055] Next, the technical solutions of the present invention will be clearly and completely described in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts fall within the protection scope of the present invention.

[0056] The inventor immunized alpacas with S1 recombinant protein, constructed a VHH phage library, and obtained nanobody clones that bind to RBD through biopanning. Specifically, they are four nanobodies shown in SEQ ID NO.1-4.

[0057] The present invention also provides the biological materials related to the above-mentioned nanobodies, which can be nucleic acid molecules, expression cassettes, recombinant vectors, recombinant eukaryotic cells, and recombinant prokaryotic cells.

[0058] The "nucleotide molecule" can be DNA, such as cDNA or recombinant DNA, or RNA, such as mRNA or hnRNA, etc.

[0059] The "expression cassette" contains a polynucleotide sequence. The polynucleotide sequence encodes a polypeptide to be expressed (single-domain antibody) and sequences that control its expression, such as a promoter and optionally an enhancer sequence, including any combination of cis-acting transcriptional control units. Sequences that control gene expression (i.e., its transcription and translation of the transcription product) are often referred to as regulatory units. Most parts of the regulatory unit are located upstream of the coding sequence of the gene and are effectively linked to it. The expression cassette can also contain a downstream 3' untranslated region containing a polyadenylation site.

[0060] The vector of the "recombinant vector" can be a plasmid, phage, or virus.

[0061] The host cell of "recombinant eukaryotic cell" can be yeast, mammalian cell, etc.

[0062] The host cell of "recombinant prokaryotic cell" can be bacterium, alga, etc.

[0063] In a preferred embodiment, the vector as the nucleic acid molecule encoding the nanobody of the present invention in the recombinant vector is a backbone vector, for example, it can be pET-25b plasmid. The host cell in the recombinant prokaryotic cell can be, for example, Escherichia coli Rosetta 2.

[0064] The present invention also protects the heavy chain antibody containing the nanobody of the present invention.

[0065] A method for preparing the nanobody of the present invention, comprising introducing the nucleic acid molecule encoding the nanobody into a recipient cell to obtain a transgenic cell, and culturing the transgenic cell to obtain the nanobody. Among them, the recipient cell can be a microbial cell or a mammalian cell, preferably Escherichia coli Rosetta 2.

[0066] The nanobody provided by the present invention and the related biological materials can be used for preparing products for detecting SARS-CoV-2, such as immunochromatographic test strips, ELISA test kits, etc.; can also be used for affinity chromatography purification of SARS-CoV-2 RBD; can also be used for preparing drugs for treating COVID-19.

[0067] The present invention will be further illustrated by the following examples. Unless otherwise specified, the materials in the examples are prepared according to the existing methods or directly purchased from the market.

[0068] Example 1: Antigen preparation and alpaca immunization

[0069] Alpaca immunization: Subcutaneously and intramuscularly inject the SARS-COV-2 Spike S1 recombinant protein into multiple points on the back of the alpaca's neck to form multiple lumps, and track and observe the absorption of the subcutaneous injection lumps to confirm correct immunization. For the first immunization, 0.5 mg of antigen is mixed with Freund's complete adjuvant at a ratio of 1:1, emulsified and then injected, with a volume of 1 mL per alpaca; for the second immunization: 3 weeks after the first immunization, 0.25 mg of antigen is mixed with Freund's incomplete adjuvant at a ratio of 1:1, emulsified and then injected, with an injection volume of 1 mL per alpaca; for the third immunization: 3 weeks after the second immunization, 0.25 mg of antigen is mixed with Freund's incomplete adjuvant at a ratio of 1:1, emulsified and then injected, with an injection volume of 1 mL per alpaca; for the fourth immunization: 3 weeks after the third immunization, 0.25 mg of antigen is mixed with Freund's incomplete adjuvant at a ratio of 1:1, emulsified and then injected, with an injection volume of 1 mL per alpaca.

[0070] Serum treatment and titer detection: One week after the fourth immunization, 50 mL of peripheral blood was collected, and serum and lymphocytes were separated. The RBD-his antigen was coated on an ELISA 96-well plate, and the antibody titer in the serum was determined by ELISA. The ELISA results showed that the titer of the alpaca's quadruple-immunized serum > 1:32000, meeting the library construction standard.

[0071] Example 2: Construction and screening of a phage display immune antibody library

[0072] Since the titer of the fourth serum > 1:32000, it indicates that there are high-affinity antibodies against rRBD in the serum. Further construct a phage display immune antibody library and obtain positive monoclonal nanobodies against human rRBD through biological screening.

[0073] Collect the blood of the alpaca after the fourth immunization and isolate lymphocytes PBMC; take 2×10 7 of PBMC, and extract total RNA using an RNA extraction kit; take an appropriate amount of RNA (such as 3 - 5 μg) and obtain cDNA through an RT-PCR reverse transcription kit.

[0074] Obtain the IgG2 and IgG3 heavy chain variable region sequences (the heavy chain variable region VHH of nanobodies) step by step through nested PCR. The experimental steps are as follows:

[0075] 1) Design two pairs of specific primers to amplify the alpaca heavy chain antibody gene fragments. Among them, the outer nested primers are located in the highly conserved regions of the antibody heavy chain signal peptide region and the CH2 domain. This pair of primers is used to amplify VH-CH1-CH2 fragments of about 900 bp and VH-CH2 fragments of about 700 bp; the inner nested primers are used to amplify a heavy chain antibody variable region VHH fragment of about 400 bp from the 700 bp VH-CH2 fragment.

[0076] 2) Using cDNA as a template, perform the first round of PCR amplification with the outer nested primers. Separate the products by DNA gel electrophoresis, and cut and recover the 700 bp PCR product. 3) Use the inner nested primers to perform PCR amplification on the 700 bp product obtained in the first round to obtain the target gene VHH fragment, and use a PCR product purification kit for purification and recovery.

[0077] The heavy chain variable region sequence was inserted into the digested linearized phagemid vector VHH-libTemplate by homologous recombination or enzymatic digestion and ligation to obtain a recombinant vector; after purification and recovery, it was transformed into super-competent SS320 cells (containing helper phage M13K07); the transformed bacterial solution was resuspended and activated in SOC medium for 1 hour; a small amount of the bacterial solution was taken for 10-fold serial dilution, an appropriate dilution titer was selected, and it was plated on LB / tet10 and LB / Carb50 plates and placed in a 37°C biochemical incubator overnight for calculating the library capacity the next day; the remaining bacterial solution was transferred into a large volume of 2YT / Carb50 / Kan25 liquid medium, placed on a 37°C shaker, and cultured overnight, and the supernatant was harvested the next day. 1 / 4 volume of PEG / NaCl solution was added, and after precipitating the phage, an appropriate amount of PBT solution was taken to resuspend and dilute to the required concentration to obtain a phage display immune antibody library (stored at -80°C for later use).

[0078] The number of clones on the LB / Carb50 plate was counted, and the library capacity was calculated to be 7.8×10 9 . 10 monoclonal colonies were randomly picked from the plate for sequencing, and the insertion efficiency was 90%.

[0079] Example 3: Screening of the antibody library

[0080] 5 μg / mL of RBD-his was added to a 96-well plate (100 μL / well) and coated overnight at 4°C; NEB5αF' Escherichia coli was streaked on a 2YT / Tet10 plate and cultured overnight in a 37°C incubator; the next day, NEB5αF' monoclonal colonies were picked from the overnight 2YT / Tet10 plate and added to 3 mL of 2YT / Tet10 liquid medium, and cultured with shaking at 37°C until OD600 = 0.8; meanwhile, the antigen supernatant in the 96-well plate was removed, and 200 μL of 1% BSA was added to each well for blocking. At the same time, 200 μL of 1% BSA was added to the blank well as a negative control well, and it was placed on a 3D rotary shaker at room temperature for 2 hours; then, the supernatants of the protein wells and the control wells were removed, washed with 200 μL of PT, and 100 μL of the phage antibody library was added to each well, and it was placed on a 3D rotary shaker at room temperature for 2 h; the supernatants of the protein wells and the control wells were removed, and washed with 200 μL of PT; 100 μL of 100 mM HCl was added to the wells and placed at room temperature for 5 minutes; the supernatant was aspirated and added to a 1.5 mL centrifuge tube, and neutralized with 1 M Tris-HCl. The above mixture was added to a centrifuge tube containing 1 mL of NEB5αF' bacteria, and cultured on a shaker at 37°C for 1 h; 20 μL of the culture solution in the centrifuge tube was taken for appropriate dilution and plated on an LB / Carb50 plate, and placed in a 37°C biochemical incubator overnight for calculating the titer and enrichment degree the next day; 1 L of helper phage M13K07 (final concentration of 1010 (at a concentration of 1×10⁹ CFU / mL), incubate in a shaker at 37 °C for 1 h; transfer the above culture medium into 35 mL of 2YT / Carb50 / Kan25 culture medium, place it in a shaker, and incubate overnight at 37 °C to collect the antibody library formed by phages in each round.

[0081] Repeat the above operation 3 - 5 rounds until phage enrichment occurs. If the number of colonies in the antigen-binding wells on the LB / Carb50 culture plate is more than 10 times that of the negative control wells, it is considered successful enrichment. In this experiment, after the second round of screening, the number of colonies in the antigen-binding wells was 1000 times that of the negative control wells, indicating successful enrichment, and then enter Phage ELISA to select high-affinity positive clones.

[0082] Example 4: High-throughput sequencing

[0083] Design primers in the constant regions on both sides of VHH, amplify the variable region of the phage library, perform E-gel detection on the PCR products, and then use the kit to purify and recover the PCR library fragments for NGS sequencing. The obtained data is statistically analyzed using SPSS 2.0 and Microsoft Excel 2019, and the high-throughput sequencing data is statistically analyzed and plotted using Geneious and Clustal Omega. According to the CDRH3 differences of the sequences, the sequences with the top 200 DNA frequencies are divided into 4 groups, and the VHHs (SS1, SS3, SS4, S12) with the highest DNA frequencies are selected from each group for the next experiment (such as Figure 1 ). The amino acid sequences of SS1, SS3, SS4, and S12 are shown in Table 1 below.

[0084] Table 1 Amino acid sequences of anti-RBD nanobody clones

[0085]

[0086] The italicized and underlined sequences are the CDR1, CDR2, and CDR3 sequences in turn.

[0087] Example 5: Prokaryotic expression of nanobodies

[0088] We performed prokaryotic expression on the sequences in Table 1 of Example 4, namely sequences SS1, SS3, SS4, and S12. The experimental steps are as follows:

[0089] 1) The VHH fragment amplified by PCR was cloned into the pET-25b plasmid through the restriction enzyme sites BamH I and Xho I, electrotransformed into Escherichia coli Rosetta 2, and after screening with ampicillin, the correct recombinant plasmid pET-25b-VHH-His was obtained by monoclonal sequencing;

[0090] 2) Select monoclonal colonies containing the recombinant plasmid, shake culture at 37 °C until OD600 = 0.6 - 0.8, add 0.5 mM IPTG, and shake culture overnight at 25 °C; collect the bacteria the next day, lyse the bacteria by sonication, collect the supernatant, and purify the VHH-His fusion protein by nickel affinity chromatography. The prokaryotic expression information is shown in Table 2 below, and the SDS-PAGE (12%) electrophoresis results are as Figure 2 shown.

[0091] Table 2. Prokaryotic expression of anti-RBD nanobody clones

[0092]

[0093] Example 6: Evaluation of the affinity of VHH by ELISA

[0094] Coat the RBD-his antigen (2 μg / mL) on a 96-well plate and incubate overnight at 4 °C. The next day, remove the antigen supernatant from the 96-well plate, add 200 μL of 1% PVA to each well for blocking, and at the same time add 200 μL of 1% PVA to the blank well as a negative control well, and place it on a 3D rotary shaker at room temperature for 2 h; then, remove the supernatant from the protein wells and the control wells, wash 3 times with 200 μL of PT, pat dry, add 100 μL of serially diluted primary antibodies (SS1, SS3, SS4, S12), incubate at room temperature for 2 h, remove the supernatant from the protein wells and the control wells, and wash with 200 μL of PT; detect the bound nanobodies with HRP-conjugated anti-His secondary antibody, detect at room temperature for 1 h, after washing clean, add 100 μL of TMB for color development, and measure the OD value at 450 nm. The analysis results show that SS1, SS3, SS4, and S12 all show good antigen-binding activity (as Figure 3 ).

[0095] Example 7: Detection of epitope differences recognized by VHHs by Epitope Binning method

[0096] First, dilute the antigen with PBST solution to a final concentration of 5 μg / mL; antibody preparation: dilute the antibodies with PBST solution, the final concentrations of SS1, SS4, and S12 are 1 μM, and the final concentration of SS3 is 60 μM; then, detect the epitope differences of VHHs with Octet. Take Protein A Sensor to immobilize the antigen (5 μg / mL), and the immobilization height is 3.0 nm; as a preliminary experiment, immobilize the antigen on the protein A sensor through the Fc region, and detect the short-time binding signals of the nanobodies SS1, SS3, SS4, and S12 to the antigen. The antibodies SS1, SS3, SS4, and S12 bind well to the antigen;

[0097] Then, the antibody SS4 was immobilized on the protein A sensor and saturated with rRBD, and then the complex was incubated with each of the designated antibodies (SS1, SS3, S12) separately; when the antibody on the sensor competes with the antibody in the solution for the same antigen epitope, no additional binding to the antigen was observed. After SS4 bound to the antigen, no obvious binding reaction was shown by SS1 and S12 (as Figure 4 , at the position indicated by the arrow), indicating that the antigen epitopes bound by SS1 and S12 highly overlap with the antigen epitope bound by SS4. In contrast, after the antigen was saturated by the binding of SS4, SS3 could bind to the antigen again (as Figure 5 , at the position indicated by the arrow), indicating that the antigen epitope bound by SS3 is different from the antigen epitope bound by SS4.

[0098] Example 8: Purification of rRBD by SS4-Sepharose 4FF and SS3-Sepharose 4FF

[0099] ① Resuspend the CNBr-4FF medium with 5 column volumes of buffer A (0.001 M HCl, 0.5 M NaCl, pH 3.0), and after 5 min, drain all the solvent, and repeat this process 5 times. ② Select nanobodies (SS4, SS3) that bind different epitopes according to Example 7 as affinity ligands and conjugate them with the affinity filler Sepharose 4FF: Replace the conjugation sample into a buffer of 0.2 M NaHCO3, 0.5 M NaCl, pH 8.3, and concentrate the sample with an ultrafiltration tube. Mix the washed medium and the concentrated sample in equal volumes, gently mix at room temperature for 3 h, then draw out the sample solution and measure the concentration of the sample extract after conjugation. ③ Resuspend the conjugated medium with 5 volumes of buffer B (0.02 M Tris-HCl solution, pH 8.3), completely drain the solvent, and then repeat this step 3 times. Then resuspend with 5 volumes of buffer B, gently mix at room temperature for 2 h, and then drain the solution. ④ Resuspend the blocked medium with 5 volumes of buffer C (0.05 M Tris-HCl, 0.5 M NaCl, pH 8.0), drain the solvent after 5 min; then resuspend the medium with 5 volumes of buffer D (0.05 M Glycine, 0.5 M NaCl, pH 3.5), drain the solution after 5 min. Repeat this process 3 times and store with 20% ethanol. Determination of the conjugation amount: Measure the antibody content (280 nm) before and after the reaction by Nanodrop, and calculate the conjugation amount according to formula (2-1):

[0100]

[0101] Add PBS buffer with a volume 5 times that of the column, let the solution flow out, and the packing material will settle to the bottom. Add the sample to be purified (rRBD), and let it flow out slowly at a flow rate of generally 1 mL / min. Collect the effluent. If there are special requirements, the sample can be loaded repeatedly or in a cycle. Wash with PBS at a flow rate of approximately 1 mL / min, and monitor the protein concentration using a UV detector until the A280 value stabilizes. After washing, add conventional elution buffers, including 1) 0.1 M glycine-HCl, pH 2.5; 2) 0.1 M citric acid, 0.5 M NaCl, pH 2.5; 3) 0.1 M glycine-NaOH, pH 10; 4) 45 mM citric acid, 70 mM arginine, pH 3.5; 5) 20 mM Tris, 2 M MgCl2, pH 7.4; 6) 50 mM Tris, 0.2% SDS, 0.1% Tween-20, pH 8.0. The eluent flow rate is about 1 mL / min and is monitored by a UV detector until the A280 value stabilizes. Collect the eluate to calculate the protein content and protein recovery rate. Add 20% ethanol to the chromatography column and store it at 4°C. Immediately after elution, replace the protein into the storage solution of 10 mM sodium acetate, 270 mM sucrose, 0.01% Tween-20, pH 6.8, and store it at -80°C.

[0102] The coupling amounts of SS3 and SS4 with CNBr activation are 4.6 mg / mL and 8.3 mg / mL respectively. SS3 and SS4 show good coupling performance in the primary amino group immobilization method. Wash the packing material with 0.05 M Tris-HCl, 0.5 M NaCl buffer (pH 8.0) and 0.05 M glycine, 0.5 M NaCl buffer (pH 3.5), and almost no nanobody is lost, indicating that SS3 and SS4 can be stably coupled to the chromatography column. Compared with the positive control, there are almost no contaminating proteins around the rRBD band adsorbed by the affinity chromatography column, indicating that SS3 and SS4 have high specificity. When purifying rRBD protein with this affinity chromatography column and adding the loading buffer to rinse, the contaminating proteins that are not adsorbed by the immunoaffinity column flow out of the column. Compared with the positive control, there are almost no contaminating proteins around the rRBD protein band adsorbed by the affinity chromatography column, indicating that SS3 and SS4 have high specificity for rRBD. Add the conventional eluent and react for 10 min, and none of them can elute the rRBD adsorbed by SS4-Sepharose 4FF ( Figure 6 ). On the contrary, for the rRBD adsorbed by SS3-Sepharose 4FF, it is eluted under the condition of 50 mM Tris, 0.2% SDS, 0.1% Tween-20 (pH 8.0) buffer, with a protein recovery rate of 50% and a purity of 90% ( Figure 7 ), and the EC50 is 0.1 nM (Figure 8 )。

[0103] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention. Sequence Listing <110> Chengdu Shengshi Junlian Biotechnology Co., Ltd. <120> Nanobody Specifically Targeting SARS-CoV-2 RBD <160> 4 <170> PatentIn version 3.5 <210> 1 <211> 111 <212> PRT <213> Artificial Sequence <220> <223> SS1 Antibody <400> 1 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Val Gly Gly 1 5 10 15 Ser Leu Thr Leu Ala Cys Thr Ala Ser Gly Ser Gly Val Ser Ile Arg 20 25 30 Gly Met Gly Trp Tyr Arg Gln Ala Pro Gly Gln Gln Arg Glu Leu Val 35 40 45 Ala Arg Leu Thr Ala Val Leu Ala Thr Met Ile His Asn Ser Val Glu 50 55 60 Gly Arg Phe Thr Ile Ser Gly Asp Asn Ala Lys Asn Thr Ile Tyr Leu 65 70 75 80 Gln Met Asn Asn Leu Lys Pro Glu Asp Thr Gly Val Tyr Tyr Cys Asn 85 90 95 Ser Gly Gln Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 100 105 110 <210> 2 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> SS3 Antibody <400> 2 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Trp Val Gln Ala Gly Gly 1 5 10 15 Ser Val Arg Leu Ser Cys Val Ala Ser Gly Ser Ile Arg Asn Met Arg 20 25 30 Gly Ile Gly Trp Phe Arg Gln Val Pro Gly Lys Gln Arg Glu Leu Val 35 40 45 Ala Arg Phe Thr Ala Ala Gly Ala Thr Leu Ser Ser Asn Ala Val Glu 50 55 60 Gly Arg Phe Thr Leu Ser Ala Asp Arg Ala Lys Asn Thr Val Asp Leu 65 70 75 80 Val Leu Asn Asn Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn 85 90 95 His Arg Pro Ser Phe Gly Pro Glu Asn Glu Ser Trp Gly Gln Gly Thr 100 105 110 Gln Val Thr Val Ser Ser 115 <210> 3 <211> 111 <212> PRT <213> Artificial Sequence <220> <223> SS4 Antibody <400> 3 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Pro Ala Gly Gly 1 5 10 15 Ser Leu Thr Leu Ser Cys Thr Ala Ser Gly Asn Ile Arg Asn Leu Asn 20 25 30 Gly Val Gly Trp Tyr Arg Gln Thr Pro Gly Lys Gln Arg Asp Leu Val 35 40 45 Ala Gln Phe Thr Asn Val Gly Ala Thr Leu Ser Lys Asp Ser Leu Glu 50 55 60 Asp Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr Val Tyr Leu 65 70 75 80 Gln Met Asn Asn Leu Gln Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn 85 90 95 Leu Arg Ser Asn Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 100 105 110 <210> 4 <211> 116 <212> PRT <213> Artificial Sequence <220> <223> S12 antibody <400> 4 Gln Val Gln Leu Val Glu Ser Gly Gly Asp Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Thr Leu Ser Cys Ala Ala Ser Gly Ala Ile Ser Ser Phe Asp 20 25 30 Ala Val Thr Trp Tyr Arg Gln Val Pro Gly Asn Ala Arg Ala Phe Ile 35 40 45 Ala Ala Ile Ser Gly Gly Asp Val Arg Ser Tyr Ala Arg Ser Ala Lys 50 55 60 Asp Arg Phe Thr Ile Phe Arg Asp Asn Asp Lys Asn Thr Val Asn Leu 65 70 75 80 Glu Met Asn Lys Leu Thr Pro Glu Asp Thr Gly Thr Tyr Val Cys His 85 90 95 Trp Ala Ser His Ser Gly Gly Asp Tyr Trp Gly Gln Gly Thr Gln Val 100 105 110 Thr Val Ser Ser 115

Claims

1. An antibody specifically targeting the SARS-CoV-2 RBD, the variable region of whose heavy chain contains the following CDRs: CDR1: MRGIG; CDR2: RFTAAGATLSSNAVEGR; CDR3: RPSFGPENES, wherein the antibody is a VHH antibody, a heavy chain antibody or a nanobody.

2. The antibody according to claim 1, wherein The amino acid sequence of the VHH antibody, heavy chain antibody or nanobody contains the amino acid sequence shown in SEQ ID NO.

2.

3. A biological material related to the antibody according to claim 1 or 2, characterized in that, The biological material is any one of the following: (a) A nucleic acid molecule encoding the antibody according to claim 1 or 2; (b) An expression cassette containing the nucleic acid molecule in (a); (c) A recombinant vector containing the nucleic acid molecule in (a) or the expression cassette in (b); (d) A recombinant eukaryotic cell containing the nucleic acid molecule in (a), the expression cassette in (b) or the recombinant vector in (c); (e) A recombinant prokaryotic cell containing the nucleic acid molecule in (a), the expression cassette in (b) or the recombinant vector in (c).

4. The biomaterial according to claim 3, characterized in that, The recombinant vector in (c) is a plasmid.

5. The biomaterial according to claim 3, wherein The prokaryotic cell in (e) is Escherichia coli.

6. A pharmaceutical composition containing the antibody according to claim 1 or 2.

7. A method for preparing the antibody according to claim 1 or 2, characterized in that, The nucleic acid molecule encoding the antibody according to claim 1 or 2 is introduced into a recipient cell to obtain a transgenic cell, and the transgenic cell is cultured to obtain the antibody.

8. The preparation method according to claim 7, characterized in that, The recipient cell is a microbial cell or a mammalian cell.

9. Use of the antibody according to claim 1 or 2 or the biological material according to any one of claims 3-5 in the preparation of a product for detecting SARS-CoV-2.

10. Use of the antibody according to claim 1 or 2 or the biological material according to any one of claims 3-5 in the affinity chromatography purification of SARS-CoV-2 RBD.

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