A nano-antibody with anti-sars-cov-2 neutralization activity and application

Abstract

The application discloses an anti-SARS-CoV-2 neutralization activity nanobody and application, and belongs to the technical field of biology.The anti-SARS-CoV-2 neutralization activity nanobody has an amino acid sequence as shown in SEQ ID NO.1; the nanobody can recognize a receptor binding domain (RBD) of SARS-CoV-2.A kind of anti-SARS-CoV-2 neutralization activity nanobody Nb1 is constructed by the method of molecular biology, and it is proved by experiment that Nb1 has very strong reactivity with the receptor binding region (RBD) of SARS-CoV-2, has good neutralization activity to SARS-CoV-2 of different strains, and has the potential to be applied to the prevention and treatment of SARS-CoV-2.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biotechnology, and particularly relates to an anti-SARS-CoV-2 neutralization activity nanobody and application. BACKGROUND

[0002] The continuous emergence of SARS-CoV-2 mutant strains makes the virus exhibit different characteristics in terms of infectivity, disease severity and vaccine resistance, thereby limiting the protective efficacy of the existing vaccines and antibodies.

[0003] Camelidae animals contain a kind of natural antibody called heavy chain antibody, which lacks light chains and the first constant region of heavy chains. The VHH fragment obtained by amplifying the variable region of the heavy chain antibody is called nanobody. Nanobody has better penetration, higher stability and lower immunogenicity, higher target affinity and is easy to modify due to its small size and stable structure, and is the smallest antigen-binding fragment known at present. Nanobody shows excellent biological activity in clinical imaging tracing, tumor targeted drugs, antiviral drug research and diagnosis. SUMMARY

[0004] The present application aims to overcome the shortcomings of the prior art and provide an anti-SARS-CoV-2 neutralization activity nanobody and application. The nanobody of the present application can efficiently and specifically bind to the RBD domain of SARS-CoV-2, and has good neutralization activity against multiple different strains of SARS-CoV-2 and its mutant strains. The nanobody has a good development prospect in the field of anti-SARS-CoV-2.

[0005] To achieve the above-mentioned purpose, the technical solution adopted by the present application is as follows: an anti-SARS-CoV-2 neutralization activity nanobody, wherein the nanobody has an amino acid sequence as shown in SEQ ID NO. 1; and the nanobody can recognize the receptor binding domain of SARS-CoV-2.

[0006] As a preferred embodiment of the anti-SARS-CoV-2 neutralization activity nanobody of the present application, the nanobody further includes an antibody with the same activity obtained by substituting, deleting and / or adding one or more amino acids from the amino acid sequence shown in SEQ ID NO. 1.

[0007] As a preferred embodiment of the anti-SARS-CoV-2 neutralization activity nanobody of the present application, the nanobody further includes an antibody obtained by humanization or long-acting modification of the amino acid sequence shown in SEQ ID NO. 1.

[0008] Preferably, in order to facilitate the purification of the anti-SARS-CoV-2 neutralization active nanobody, any of the following tags can be connected to the N-terminus or C-terminus of the nanobody: Poly-Arg (sequence: RRRRR), Poly-His (sequence: HHHHHH), FLAG (sequence: DYKDDDDK), Strep-tag II (sequence: WSHPQFEK), c-myc (sequence: EQKLISEEDL).

[0009] Preferably, in order to facilitate the secretion expression of the anti-SARS-CoV-2 neutralization active nanobody, a signal peptide sequence can be added to the amino acid sequence of the nanobody.

[0010] Preferably, in order to prolong the half-life of the anti-SARS-CoV-2 neutralization active nanobody, the nanobody can be fused with IgG Fc (amino acid sequence: SEQ ID NO. 3) or human serum albumin (HSA) (amino acid sequence: SEQ ID NO. 4) or modified with PEG.

[0011] The present application also provides a nucleic acid molecule encoding the nanobody.

[0012] As a preferred embodiment of the nucleic acid molecule of the present application, the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO. 2.

[0013] Preferably, the nucleic acid molecule further comprises a DNA molecule that hybridizes to the DNA sequence shown in SEQ ID NO. 2 under stringent conditions and encodes a protein having the same function.

[0014] Preferably, the nucleic acid molecule further comprises a DNA molecule that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology to the DNA sequence shown in SEQ ID NO. 2 and encodes a protein having the same function.

[0015] The present application also provides a biological material containing the nucleic acid molecule, which comprises a recombinant vector, a transgenic cell line, an expression cassette, or an engineered bacterium.

[0016] The present application also provides a recombinant antibody composed of the nanobody and IgG Fc.

[0017] The present application also provides the use of the nanobody, the nucleic acid molecule, the biological material, or the recombinant antibody in the preparation of a product against SARS-CoV-2.

[0018] The application also provides the application of the nanobody, the nucleic acid molecule, the biological material or the recombinant antibody in the preparation of a reagent or kit for detecting SARS-CoV-2.

[0019] The application also provides a reagent or kit for detecting SARS-CoV-2, comprising the nanobody, the nucleic acid molecule, the biological material or the recombinant antibody.

[0020] The application has the following beneficial effects: the application constructs an anti-SARS-CoV-2 neutralizing activity nanobody Nb1 by a molecular biology method, and experiments prove that Nb1 has strong reactivity with the receptor binding domain (RBD) of SARS-CoV-2, has good neutralizing activity to SARS-CoV-2 of different strains, and has the potential to be applied to the prevention and treatment of SARS-CoV-2. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 PCR identification map of recombinant expression vector PPICZ alpha A-Nb1.

[0022] Figure 2 Linearization identification map of recombinant expression vector PPICZ alpha A-Nb1.

[0023] Figure 3 SDS-PAGE identification map of small-scale induced expression of PPICZ alpha A-Nb1.

[0024] Figure 4 Western blot identification map of small-scale induced expression of PPICZ alpha A-Nb1.

[0025] Figure 5 SDS-PAGE identification map of purified nanobody Nb1.

[0026] Figure 6 SDS-PAGE identification map of concentrated nanobody Nb1.

[0027] Figure 7 Detection results of nanobody Nb1 and SARS-CoV-2 RBD reactivity.

[0028] Figure 8 Neutralization test results of nanobody Nb1 and SARS-CoV-2 pseudovirus.

[0029] Figure 9 Neutralization test results of nanobody Nb1 and SARS-CoV-2 live virus. DETAILED DESCRIPTION

[0030] For better illustrating the purposes, technical solutions and advantages of the present application, the present application will be further described below in combination with specific examples.

[0031] Expression and identification of nanobody Nb1

[0032] 1. Construction of nanobody Nb1 and expression vector PPICZαA-Nb1

[0033] The coding gene of Nb1 (the amino acid sequence of which is shown as SEQ ID NO. 1) shown in SEQ ID NO. 2 is fused with a C-terminal histidine tag (6*His) and inserted into the sequence region of 1191-1270 of the commercial vector ppiczαA, i.e. between the two enzyme cutting sites of XhoI and XbaI. The PPICZαA-Nb1 recombinant plasmid with the Nb1 gene is transformed into E. coli DH5α competent cells, and a bleomycin-resistant clone is selected. The plasmid is extracted and subjected to PCR identification. The plasmid correctly identified by PCR is sent to Beijing Norsen Genomics Research Center Co., Ltd. for sequencing identification. The correctly identified recombinant plasmid is named as PPICZαA-Nb1.

[0034] The PCR identification map of PPICZαA-Nb1 is shown in Figure 1 . A Nb1 band (the length of the target gene is 342 bp, and the distance between the two ends of the sequencing primer and the target gene is about 100 bp, so the total length of the PCR product is 591 bp) with a length of about 591 bp is obtained. Figure 1 In the figure, lanes 1, 2 and 3 are three monoclonal strains of Nb1 amplified by PCR, and lane M is Trans 2K Plus DNA Marker (BioMed company BM111). It can be seen that a band with the size of the target gene is successfully amplified. The recombinant plasmid is further sequenced, indicating that the vector construction is successful.

[0035] 2. Expression and purification of nanobody Nb1 in Pichia pastoris expression system

[0036] (1) Small-scale induced expression of Nb1

[0037] The PPICZαA-Nb1 plasmid is linearized by single-point enzyme cutting of the recombinant plasmid using the restriction enzyme SacI, and the linearized plasmid is subjected to nucleic acid electrophoresis to verify whether it is linearized. Figure 2Lane M is Trans 2K Plus DNA Marker (Bio-Rad, BM111), Lane 1 is the original plasmid, and Lane 2 is the plasmid after single-point enzyme digestion, which can be seen that the plasmid is completely cut open and linearized successfully. 190 μL of linearized plasmid was mixed with 20 μL of 3M NaAc and 440 μL of ice ethanol in an ice bath for 2 h. Centrifugation was performed at 4°C and 14000 rpm for 5 mins, the supernatant was aspirated, and the precipitate was reserved. 1 ml of 70% ice ethanol was added for washing, and centrifugation was performed at 14000 rpm for 5 mins. After the supernatant was discarded, it was placed in a clean bench for 10 mins, 20 μL of deionized water was added for resuspension, and it was stored at 4°C for standby. The purified linearized plasmid was electroporated into GS115 Pichia pastoris competent cells to obtain the recombinant yeast strain PPICZαA-Nb1.

[0038] The single clone colonies were inoculated into 3 mL of YPD medium, and cultured at 30°C for 24 hours. The bacterial liquid was transferred into fresh 5 mL of BMGY medium at a volume ratio of 1:1000, and the remaining bacterial liquid was stored in 15% glycerol bacteria and frozen at -80°C. At the same time, a negative control group (NV) without inducer was set, and the culture was shaken at 30°C for 24 h. The culture was induced with 1% V / V of methanol for the first time when the OD600 of the bacterial liquid was between 1.5 and 2.0. The culture was induced with 1% V / V of methanol for the second time after 24 h of continuous culture. The culture was induced with 1% V / V of methanol for the third time after 24 h of continuous culture. After the third induction, the yeast liquid was centrifuged at 4°C and 10000 rpm for 10 min, and the supernatant was collected. Each sample was prepared in two parts for SDS-PAGE electrophoresis. One part was used for Coomassie Brilliant Blue R-250 staining, and the other part was used for Western blot identification. The 6*His tag fused to the C-terminal of the antibody molecule was used for antibody identification using His-Tag Monoclonal Antibody, HRP Conjugated (Bio-Rad, BD-PM2096). Figure 3 、 Figure 4 In the figure, lane M is a protein Marker (Gene Star, M221), and lanes 1-5 are the selected single clone strains. The results show that the target protein is expressed in the yeast culture supernatant. Figure 3 The target protein is marked by a red box in the figure.

[0039] (2) Purification of Nb1

[0040] According to the expression results in (1), the glycerol bacteria of the high-expression clone were selected for recovery, and 1L of triangular flask was used for large-scale culture according to the same method above. 200 mL of culture medium was added to each flask, and a total of 800 mL of yeast culture supernatant was obtained. The sample was filtered using a 0.45 μm filter membrane for preparation for purification.

[0041] The 6*His tag fused at the C-terminal of the antibody molecule was used to purify the yeast culture supernatant by Ni affinity chromatography. The column used for purification was His Trap HP (cytiva: 17524802), which was a pre-packed column with a column volume of 5 mL. Three purification columns were used in series. The purification was performed according to the following method: the purification column was equilibrated with 3-5 column volumes of binding buffer Buffer A at a flow rate of 5 mL / min; after the column was equilibrated, the yeast culture supernatant was loaded at a flow rate of 5 mL / min; after loading, the column was washed with 5 column volumes of binding buffer Buffer A at a flow rate of 5 mL / min; after the column was equilibrated, the protein was eluted using elution buffer Buffer B (50 mM HEPES, 500 mM NaCl, 500 mM imidazole, pH 7.5) at concentrations of 20%, 40%, 80%, and 100% (corresponding to 100 mM, 200 mM, 400 mM, and 500 mM imidazole, pH 7.5) at a flow rate of 5 mL / min, respectively, and the elution peaks of each gradient were collected. The purified samples collected were subjected to SDS-PAGE electrophoresis for identification, and the results are shown in Figure 5 Figure 5 It can be seen that the antibody molecule has the highest purity in the 80% Buffer B eluate. The collected 80% Buffer B eluate was placed in a dialysis bag and dialyzed in PBS buffer at 4°C to replace the buffer of the antibody with PBS. After replacement, PEG8000 was used to concentrate the antibody, and the concentrated antibody was subjected to SDS-PAGE electrophoresis, Figure 6 showing that the antibody band after purification and concentration is uniform and has high purity. The antibody was filtered to remove bacteria using a 0.22 μm filter, and after concentration determination, it was labeled, divided, and stored at -20°C.

[0042] Example 2 Detection of the reactivity of nanobody Nb1 to SARS-CoV-2 RBD

[0043] ​The indirect ELISA method was used to detect the reactivity of the nanobody Nb1 of Example 1 with SARS-CoV-2 RBD, and the specific steps were as follows: SARS-CoV-2 RBD-Fc protein (SARS-CoV-2 S protein RBD functional region fused with human IgG Fc fragment, the amino acid sequence is shown as SEQ ID NO. 5) and MERS-CoV-2 RBD-Fc protein (MERS coronavirus S protein RBD functional region fused with human IgG Fc fragment, the amino acid sequence is shown as SEQ ID NO. 6) and bovine serum albumin (Thermo, 23209) were used to coat 96-well enzyme-coated plates, the coating concentration was 2 μg / mL, 50 μL per well; different concentrations of the protein Nb1 obtained in Example 1 were used as the primary antibody; His-Tag Monoclonal Antibody, HRP Conjugated (Biolegend, BD-PM2096) was used as the secondary antibody.

[0044] The ELISA detection results are shown in Figure 7 As shown in

[0045] Example 3: Detection of neutralizing activity of nanobody Nb1 against SARS-CoV-2

[0046] In this example, the neutralization test of pseudovirus was used to detect the neutralizing activity of the nanobody Nb1 of Example 1 against SARS-CoV-2, and the specific steps were as follows: the diluted pseudovirus (500000 RLU) was mixed with gradient-diluted nanobody Nb1, incubated at 37°C for 1 h, the hACE2-293T cell supernatant was discarded, the mixed solution was added, incubated at 37°C for 48 h, the cell lysis solution was added, and the lysis was carried out at room temperature for 30 min, the lysis product was added to the white plate, the luciferase substrate was added, and the relative luciferase activity was determined using Infinite F500 (Tecan).

[0047] The results are shown in Figure 8 As shown in

[0048] Example 4 Detection of the neutralization activity of Nanobody Nb1-Fc against live SARS-CoV-2 virus

[0049] The live virus neutralization test was performed using Nanobody Nb1-Fc (amino acid sequence as shown in SEQ ID NO. 7) fused with C-terminal human IgG Fc fragment: 50 μL of gradient diluted Nanobody Nb1-Fc was mixed with 50 μL of SARS-CoV-2 live virus (200 plaque forming units) and incubated at 37 °C for 1 h, then transferred to a 96-well plate seeded with Vero E6 cells and incubated at 37 °C for 1 h for virus infection. After infection, the virus solution was discarded and DMEM virus maintenance solution containing 1.2% (v / v) sodium carboxymethyl cellulose was added. After 24 h, the cells were fixed with 4% (v / v) paraformaldehyde and cell permeability was increased with 0.2% (v / v) Triton X-100. Immunostaining was performed with SARS-CoV-2 S protein rabbit monoclonal antibody (Sinobiological, 40143-T62) and peroxidase-labeled goat anti-rabbit antibody (H+L) (Jackson, 111-035-144), respectively. Finally, KPL TrueBle Peroxidase Substrate (SeraCare Life Science, 5510-0030) was used to show the plaques of virus infection, and CTL Immuno Spot S6 (Ccell Technology Limited) was used to count the virus plaque units. The results are shown in Figure 9 Figure 6, which shows that Nanobody Nb1 can still effectively neutralize more than 50% of four different SARS-CoV-2 mutant live viruses at a concentration of 1.1 μg / mL.

[0050] Finally, it should be noted that the above examples are only used to illustrate the technical solutions of the present application and not to limit the protection scope of the present application. Although the present application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present application can be modified or replaced by equivalents without departing from the essence and scope of the technical solutions of the present application.

Claims

1. An anti-SARS-CoV-2 neutralizing activity nanobody, characterized in that, The nanobody has an amino acid sequence as shown in SEQ ID NO. 1; the nanobody can recognize the receptor binding domain of SARS-CoV-2.

2. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the nanobody of claim 1.

3. The nucleic acid molecule of claim 2, wherein, The nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO.

2.

4. A biomaterial comprising the nucleic acid molecule of any one of claims 2-3, wherein, The biological material is a recombinant vector, a transgenic cell line, an expression cassette or an engineered bacterium.

5. A recombinant antibody, characterized in that, The recombinant antibody is composed of the nanobody of claim 1 and IgG Fc.

6. Use of the nanobody of claim 1, the nucleic acid molecule of claim 2 or 3, the biological material of claim 4 or the recombinant antibody of claim 5 in the preparation of a drug against SARS-CoV-2.

7. Use of the nanobody of claim 1, the nucleic acid molecule of claim 2 or 3, the biological material of claim 4 or the recombinant antibody of claim 5 in the preparation of a reagent or kit for detecting SARS-CoV-2.

8. A reagent or kit for detecting SARS-CoV-2, characterized in that, The nanobody of claim 1, the nucleic acid molecule of claim 2 or 3, the biological material of claim 4 or the recombinant antibody of claim 5.

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