Nano antibody for resisting RBD structural domain antigen of novel coronavirus mutant strain and application of nano antibody

Abstract

The invention discloses a nano antibody for resisting a novel coronavirus mutant strain RBD structural domain antigen. The amino acid full-length sequence of the nano antibody is shown as any one of SEQ ID NO.5-8. The nano antibody disclosed by the invention has good capability of blocking the combination of a novel coronavirus mutant strain RBD and a receptor thereof, and can effectively prevent cells from being infected by viruses through experimental verification.

Description

Technical Field

[0001] The present invention belongs to the field of biological products, and particularly relates to a nanobody against the RBD domain antigen of a novel coronavirus mutant strain and its application. Background Art

[0002] The infection of the novel coronavirus (SARS-CoV2) is a major public health problem faced globally. In the past two years (2019 - 2022), the number of deaths and infections has been increasing weekly. By the end of 2022, multiple variants of concern (VOC) have been discovered: B.1.1.7 (Alpha), B.1.351 (Beta), P.1 (Gamma), B.1.617.2 (Delta), and B.1.1.529, BA.2, BA.2.12.1, BQ.1, BQ.1.1, BA.5, BF.7 (Omicron), etc.

[0003] Current research has confirmed that the spike glycoprotein on the surface of the novel coronavirus is very similar to the SARS antigen in terms of the antigenicity of its three-dimensional conformation. Based on the structural research of the virus, it is found that the receptor binding domain protein (RBD) on the SARS-CoV2 virus can specifically recognize human angiotensin-converting enzyme (ACE2), which is also the structural basis and molecular mechanism for the Spike protein of the novel coronavirus to mediate cell invasion. Based on this research background, developing therapeutic antibodies that can highly specifically recognize this region is expected to prevent the novel coronavirus from binding to the human ACE2 protein through RBD, thereby preventing virus invasion. Therefore, developing therapeutic antibodies against the RBD domain antigen of the novel coronavirus mutant strain is expected to provide new strategies for the diagnosis and treatment of the novel coronavirus.

[0004] Most current antibody research and development adopt monoclonal antibody technology. Antibodies in mammals (including humans) consist of two heavy chains and two light chains. Among them, the Fc region of the heavy chain can ensure that the antibody has a longer half-life in the body, ensuring sufficient immune protection. However, traditional antibody drugs have some disadvantages: (1) They have a relatively large molecular weight and it is difficult to penetrate tissues, cells, and the blood-brain barrier, etc.; (2) Antibodies are prone to aggregation and precipitation during preparation, storage, and transportation; (3) They have strong immunogenicity and are prone to certain anti-antibody reactions; (4) It is difficult to screen high-affinity antibodies with biological activity.

[0005] Nanobodies were discovered by Belgian scientist Hamers-Casterman in camelid sera in 1993. This part of the antibody lacks the light chain and only contains a heavy chain variable region VHH and two heavy chain CH2 and CH3 regions. The VHH crystal is 2.5 nm in size, 4 nm in length, and has a molecular weight of only 15 kDa. It retains all of its antigen-binding ability and is the smallest fragment that retains the complete antigen-binding ability, and is also known as a single-domain antibody. Compared with traditional antibodies, nanobodies have four specific amino acids in FR2. The hydrophobic residues V37, G44, L45, and W47 in ordinary antibodies are quite conserved during evolution. In VHH, these four amino acids mutate into hydrophilic residues F37, E44, R45, and G47, increasing water solubility; in addition to the existing disulfide bonds in FR1 (Cys22) and FR3 (Cys92), there is a pair of disulfide bonds between CDR1 and CDR3 within the molecule, which can further stabilize the loop structure formed by CDR3; in addition, VHH has a longer CDR3 region, which is beneficial for it to recognize antigenic epitopes that cannot be recognized by traditional antibodies; and there are only about 10 amino acids on the surface of nanobodies that are different from human VH, with low immunogenicity. Nanobodies also have the advantages of high affinity, high stability, strong penetration, and can be expressed in a prokaryotic expression system with relatively low production costs, making them a powerful tool for immuno-diagnosis, immuno-therapy, and immuno-analysis. Their significant advantage is that they are superior to traditional antibodies in terms of protein stability, enabling them to be atomized and specifically delivered to lung tissues, and have great potential for developing nanobody-based vaccines. CN115124615A discloses a nanobody against the RBD domain antigen of the novel coronavirus. Since the antigen used to prepare this antibody is wild-type RBD, its nanobody only has a neutralizing effect on SARS-CoV-2 and has no neutralizing effect on the currently mainly prevalent SARS-CoV-2 mutant strains, and cannot prevent the infection of SARS-CoV-2 mutant strains. Summary of the Invention

[0006] To solve the above problems, the present invention provides a nanobody against a novel coronavirus mutant strain, and the full-length amino acid sequence of the nanobody is shown as any one of SEQ ID NO.5 to SEQ ID NO.8.

[0007] Further, the full-length amino acid sequence of the nanobody is shown as SEQ ID NO.5 or SEQ ID NO.7.

[0008] The present invention also provides a nucleic acid encoding the aforementioned nanobody.

[0009] Further, the sequence of the nucleic acid is shown as SEQ ID NO.1 to SEQ ID NO.4, preferably as SEQ ID NO.1 or SEQ ID NO.3.

[0010] The present invention also provides a recombinant nucleic acid vector, which is a vector ligated with a nucleic acid sequence as shown in any one of SEQ ID NOs. 1 to 4.

[0011] Further, it is a vector ligated with a nucleic acid sequence as shown in SEQ ID NO. 1 or SEQ ID NO. 3.

[0012] Still further, the vector is a prokaryotic expression vector.

[0013] The present invention also provides a recombinant cell, which contains the aforementioned vector.

[0014] The present invention also provides the use of the aforementioned nanobody in the preparation of a drug against a mutant strain of novel coronavirus.

[0015] Finally, the present invention provides a drug against a mutant strain of novel coronavirus, and the drug uses the aforementioned nanobody as an active ingredient.

[0016] The "mutant strain of novel coronavirus" referred to in the present invention means SARS-CoV-2 Lambda mutant strain, SARS-CoV-2 omicron mutant strain, and SARS-CoV-2 Delta mutant strain.

[0017] The nanobody of the present invention has a strong affinity for the RBD of the novel coronavirus mutant strains Lambda, omicron, and Delta. In particular, the full-length amino acid sequences of L2 and L25 nanobodies as shown in SEQ ID NOs. 5 and 7 have the ability to well block the binding of the RBD of the novel coronavirus mutant strain to its receptor, and it has been experimentally verified that they can effectively prevent the virus from infecting cells and have practical application value for popularization.

[0018] Obviously, based on the above content of the present invention, according to the common general technical knowledge and conventional means in the art, without departing from the above basic technical idea of the present invention, various other forms of modification, substitution, or change can also be made.

[0019] The following is a further detailed description of the above content of the present invention through specific embodiments in the form of examples. However, this should not be construed as limiting the scope of the above subject matter of the present invention to the following examples. All technologies implemented based on the above content of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 : SDS-PAGE diagram of the RBD domain of the SPIKE protein on the surface of the SARS-CoV-2 Lambda mutant strain.

[0021] Figure 2: Agarose gel electrophoresis pattern of the VHH target gene amplified by PCR from the camel immunized with the RBD antigen.

[0022] Figure 3 : Alignment of antibody sequences obtained by panning.

[0023] Figure 4 : Alignment of antibody complementary determining region sequences.

[0024] Figure 5 : Purification pattern of antibodies L2, L40, L23, and L25.

[0025] Figure 6 : SDS-PAGE pattern of the RBD domain of the SPIKE protein on the surface of the SARS-CoV-2 Omicron mutant strain.

[0026] Figure 7 : Purification pattern of the antibody and Omicron RBD complex.

[0027] Figure 8 : ELISA binding verification results of the antibody and the full-length S protein of the Omicron mutant strain.

[0028] Figure 9 : Results of microscale thermophoresis for determining the dissociation equilibrium constant of the antibody and Omicron RBD. Detailed implementation methods

[0029] Example 1 Preparation of nanobody against the RBD domain antigen of the novel coronavirus

[0030] I. Preparation of the RBD antigen

[0031] In this experiment, the immunizing antigen used for camels was the RBD domain of the SPIKE protein on the surface of the SARS-CoV-2 Lambda mutant strain virus. The insect expression system was used. Since it is a secreted protein, the culture medium supernatant was collected after centrifugation and separated and purified using a nickel column. The purified protein was quantitatively analyzed using a 12.5% SDS-PAGE gel (see Figure 1 ). Then, the purified protein was aliquoted, freeze-dried, and stored at -80 °C. (Avoid repeated freeze-thaw cycles that may affect protein activity).

[0032] II. Collection of serum after immunizing camels with the RBD antigen and detection of antiserum titer

[0033] The alpaca was immunized 6 times in total using RBD as the antigen. The injection site of the antigen was at the cervical lymph nodes of the camel. Half of the sample was injected into each of the left and right cervical lymph nodes of the camel. During the operation, special attention should be paid to storing the protein and adjuvant at low temperature throughout the process to avoid protein degradation caused by repeated freezing and thawing. On the first day, the camel was primed with an emulsified mixture of 1 mg Lambda-RBD protein and an equal volume of complete Freund's adjuvant. On the 15th, 29th, 43rd, 57th, and 71st days, the camel was boost-immunized 5 times with 0.5 mg RBD antigen and an equal volume of incomplete Freund's adjuvant.

[0034] On the seventh day after the fifth and sixth immunizations, blood was collected to detect the antiserum titer for calculating the antibody titer against the mutant strain RBD protein. The serum before immunization was used as a control.

[0035] The specific steps are as follows:

[0036] (1) Coating antigen, 200 ng of recombinant Lambda mutant strain RBD protein per well, diluted with PBS buffer, overnight at 4 °C;

[0037] (2) Wash the plate 2 times with PBS, add 300 μL of 2.5% skim milk powder per well, and block at 26 °C for 1 h;

[0038] (3) Wash 3 times with PBS. The alpaca serum before and after immunization was first diluted 1000 times and then serially diluted 2-fold. Pipette 100 μL and add it to the wells coated with RBD protein, and incubate at 26 °C for 1 h;

[0039] (4) After washing 4 times with PBS, add 100 μL / well of the polyclonal antibody of HRP conjugated Goat anti-Camel IgG diluted 1:3000, and incubate at 26 °C for 1 h;

[0040] (5) After washing 5 times with PBS, add 100 μL / well of TMB (3,3',5,5'-tetramethylbenzidine) chromogenic solution and react for 8 min;

[0041] (6) Add 50 μL of 1 M phosphoric acid termination solution to each well and read OD 450nm .

[0042] After the sixth immunization, the titer of the specific antibody of the camel serum against the Lambda mutant strain RBD recombinant protein was ≥1:128000. It can be used for the construction of the next phage display library.

[0043] III. Construction and panning of the VHH phage library

[0044] 100 ml of peripheral blood was collected from camels immunized with RBD antigen. Note that fresh blood samples should be stored at 18°C-20°C. Lymphocytes should be separated as soon as possible within 2-6 hours after blood collection. PBMCs of alpacas were separated using lymphocyte separation medium (GEFicoll-Paque Plus). Total RNA was extracted using Trizol and reverse transcribed into cDNA using oligo dT. VHH fragments were obtained through two rounds of amplification using molecular biology techniques. The VHH gene was cloned into the pCOmb3 phagemid vector and transformed into TG1 competent cells to construct a library with a capacity of 2.7×10 8 cfu Lambda-RBD phage antibody library. In order to further identify whether the RBD-VHH phage library was successfully constructed, the corresponding primers were designed, and the VHH target gene produced after the alpaca was immunized with RBD antigen was identified through bacterial liquid. It can be seen that the target band is about 400bp, which is consistent with the expected size ( Figure 2 ), which further indicates that the RBD-VHH phage antibody library contains the VHH target gene, and the insertion rate is 95.83%. 24 colonies were selected for amplification and sequencing, and the DNA sequence was translated into an amino acid sequence, and the sequence identity was 61.53%.

[0045] Subsequently, the M13KO7-assisted phage-phagemid system was used to package the library. First, the phage antibody library was revived with bacteria transformed with VHH-pComb3, and the phage coat protein was precipitated with PEG / NaCl. The phage antibody library was enriched by three rounds of immune selection by ELISA-phage. The enriched phage was eluted, transformed, plated, and a single clone was picked to induce expression for phage and Lambda mutant RBD protein ELISA binding identification. The single clones with a binding read value greater than 4 to the Blank control were sequenced, and the sequencing results were translated into amino acid sequences. The sequence alignment results are shown in Figure 3 shown.

[0046] 4. Prokaryotic expression and purification of nanobodies

[0047] The sequencing results were analyzed, and representative sequences were selected and cloned into the E. coli expression vector pET20b, and transformed into the BL21 (DE3) strain to express and produce nanobodies. The base sequences of the nanobodies are shown below, and annotated according to the Kabat numbering system. The sequences of the three complementary determining regions (CDRs) are compared as follows: Figure 4 As shown:

[0048] L2 (SEQ ID NO.1)

[0049] CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTCGGTGCAGGCTGGAGGGTCTCTGA

[0050] GACTCACCTGTGCAGCCTCTGGATCCATCGACAGTAACTACTACATGGGCTGGTTCC

[0051] GCCAGGCTCCAGGAAAGGAGCGCGAGGGACTCGCAGGTATTGAAGCTGATGGAAC

[0052] CACGTCCTACGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAAAGACAACGCCC

[0053] AGAACACCGTGTATCTACAAATGAACAGCCTCAAACCTGAGGACTCTGGCATGTACT

[0054] ACTGTGCGGCAGACGAGGCGACGTGGGTCTCGATACGGACTTTGACCTGGAACTAC

[0055] TGGGGCCAGGGGACCCAGGTCACCGTCTCCTCG

[0056] L23(SEQ ID NO.2)

[0057] CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTCGGTGCAGGTTGGAGGCTCTCTGA

[0058] GACTCACCTGTGCATCCTCTGGGGCCACATCGTCGACTGTCTGCATGGCCTGGTTCC

[0059] GCCAGGCTCCAGGAAAGCAACGCGAGGGACTCGCTTCTATTAATCGTTGGCGTATAA

[0060] CAAAACACGCGTCTTTCCTGGAGGGTCGATTCACCATCTCCAAAGACAACGCCAAG

[0061] AATATTCTATATTTGCAAATGGACAGCCTGAAACCTGAGGACACTGCCATGTACTACT

[0062] GTGCGGCAGCAACGTCCCCGACAGGTTGTTCTGGTGGTATCTCCATCTCTGACTCTT

[0063] TCTTTAACTTCTGGGGACAGGGGACCCAGGTCACCGTCTCCTCGL25(SEQ ID NO.3)

[0064] CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTCGGTGCAGGCTGGAGGGTCTCTGA

[0065] GGCTCTCCTGTTTAGTCTCCGGAGACACTTCCAGCCGCTACTGCATGGGCTGGTTCC

[0066] GCCAGGCTCCGTGGAAGGAGCGCGAAGCGGTCGCGACTATTGATAAGAATGGTTTA

[0067] ACAACGTACACAGAATCCGTGAAGGGCCGATTCACCATCTCCAAAGACAACGCCAA

[0068] GAACACTCTCAATCTACAAATGAACAGCCTGAAACCCGAGGACAGTGCCATGTACT

[0069] ACTGTGCGGCAGATCCACCCAGTATAGAGACTATGACGTGTGATGTCTTGGCGTTTT

[0070] GGGTTAATCACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCGL40(SEQ ID NO.4)

[0071] CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTCGGTGCAGGCTGGAGGGTCTCTGA

[0072] GACTCTCCTGTGCAGCCTCTGGATACAGCTTTGGTGCATACTGCATGAACTGGTTCC

[0073] GCCAGACTCCAGGGAAGGAGCGCGAGGCGGTCGCGGCTATTGATAGTGATGGTAAC

[0074] ACAAGGTACGCAGAGTCCGTGAAGGGCCGATTCACCATCTCCAAAGACAACGCCAA

[0075] GAACACTCTGTATCTGCAAATGGACAGCCTGAAACCTGAAGACACTGCCACGTACT

[0076] ACTGTGCGGCCGCCCGCGGTAGTGGTGGTGCCTGCTACGGGTACGACTTTAATTACT

[0077] ACGGCATGGACTACTGGGGCAAAGGAACCCAGGTCACCGTCTCCTCG

[0078] The purification step is divided into two steps: nickel column affinity chromatography and molecular sieve (Superdex 75 10 / 300GL) purification. The purification results are shown in Figure 5 .

[0079] The amino acid sequences of each nanobody are as follows:

[0080] L2: (SEQ ID NO.5)

[0081] QVQLQESGGGSVQAGGSLRLTCAASGSIDSNYYMGWFRQAPGKERE

[0082] GLAGIEADGTTSYADSVKGRFTISKDNAQNTVYLQMNSLKPEDSGMYYC

[0083] AADEATWVSIRTLTWNYWGQGTQVTVSS

[0084] L23: (SEQ ID NO.6)

[0085] QVQLQESGGGSVQVGGSLRLTCASSGATSSTVCMAWFRQAPGKQRE

[0086] GLASINRWRITKHASFLEGRFTISKDNAKNILYLQMDSLKPEDTAMYYCA

[0087] AATSPTGCSGGISISDSFFNFWGQGTQVTVSS

[0088] L25: (SEQ ID NO.7)

[0089] QVQLQESGGGSVQAGGSLRLSCLVSGDTSSRYCMGWFRQAPWKERE

[0090] AVATIDKNGLTTYTESVKGRFTISKDNAKNTLNLQMNSLKPEDSAMYYCA

[0091] ADPPSIETMTCDVLAFWVNHWGQGTQVTVSS

[0092] L40: (SEQ ID NO.8)

[0093] QVQLQESGGGSVQAGGSLRLSCAASGYSFGAYCMNWFRQTPGKERE

[0094] AVAAIDSDGNTRYAESVKGRFTISKDNAKNTLYLQMDSLKPEDTATYYCA

[0095] AARGSGGACYGYDFNYYGMDYWGKGTQVTVSS

[0096] V. Detection of the Binding Ability between Nanobody and RBD

[0097] 1. Assembly Experiment

[0098] The purified and homogeneous stable nanobody sample was incubated with the purified and homogeneous stable Omicron RBD domain sample at 4°C for 2 hours, followed by gel filtration chromatography to isolate the stable antigen-antibody complex. The purification result of the Omicron mutant RBD domain is as shown in Figure 6 and the assembly result is as shown in Figure 7 .

[0099] 2. Detection of the Antigen-Antibody Binding Ability

[0100] To further analyze the binding ability of the nanobody, ELISA was used to verify the binding ability between the nanobody and the full-length S protein of the Omicron mutant, and the result is as shown in Figure 8 . The binding ability between the aforementioned recombinantly expressed antibodies and the Omicron mutant RBD was verified using microscale thermophoresis (MST), and the result is as shown in Figure 9 . The EC50 and dissociation equilibrium constant (Kd) values are summarized in Table 1 below.

[0101] Table 1 Screening of Antibody Affinity

[0102]

[0103] It is concluded from the data in Table 1 that L2 and L25 have strong affinity for mutant strains. The affinity of all screened nanobodies is at the nM level.

[0104] In summary, the nanobody of the present invention has the ability to effectively block the binding of the receptor-binding domain (RBD) of the novel coronavirus mutant strain to its receptor, and has been experimentally verified to effectively prevent virus from infecting cells, thus having practical application value for popularization.

Claims

1. A nanobody against the mutant strain of novel coronavirus, characterized in that: The full-length amino acid sequence of the nanobody is shown as any one of SEQ ID NO.5 to 8.

2. The nanobody according to claim 1, wherein The full-length amino acid sequence of the nanobody is shown as SEQ ID NO.5 or SEQ ID NO.

7.

3. Nucleic acid encoding the nanobody according to claim 1 or 2.

4. The nucleic acid according to claim 3, wherein The sequence of the nucleic acid is shown as SEQ ID NO.1 to 4, preferably SEQ ID NO.1 or SEQ ID NO.

3.

5. A recombinant nucleic acid vector, characterized in that: It is a vector linked with a nucleic acid sequence shown as any one of SEQ ID NO.1 to 4.

6. The recombinant nucleic acid vector according to claim 5, wherein: It is a vector linked with a nucleic acid sequence shown as SEQ ID NO.1 or SEQ ID NO.

3.

7. The recombinant nucleic acid vector according to claim 5 or 6, wherein: The vector is a prokaryotic expression vector.

8. A recombinant cell, characterized in that: It contains the vector according to claim 5 or 6.

9. Use of the nanobody according to claim 1 or 2 in the preparation of a drug against a mutant strain of novel coronavirus.

10. A drug against novel coronavirus mutant strains, characterized in that: The drug uses the nanobody according to claim 1 or 2 as an active ingredient.

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