A nanobody specifically recognizing recombinant monkeypox virus a35r protein

Abstract

This invention belongs to the field of molecular biology technology, specifically disclosing a nanobody that specifically recognizes the recombinant monkeypox virus A35R protein. The nanobody provided by this invention comprises at least three sets of complementarity-determining regions (CDRs); these CDRs include CDR1 shown in SEQ ID NO:1, CDR2 shown in SEQ ID NO:2, and CDR3 shown in SEQ ID NO:3. The nanobody of this invention possesses unique CDR1, CDR2, and CDR3 sequences, enabling it to specifically recognize and bind to the recombinant monkeypox virus A35R protein, while exhibiting no reaction with other non-specific cross-reactive proteins. The nanobody provided by this invention also possesses high tolerance, high stability, and high affinity, exhibiting highly specific recognition and binding ability to monkeypox virus, and has promising applications in the preparation of reagents for the prevention, treatment, or detection of monkeypox virus.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology technology, and specifically relates to a nanobody that specifically recognizes the A35R protein of recombinant monkeypox virus. Background Technology

[0002] Recently, monkeypox has become a global concern. Monkeypox is an acute exanthematous zoonotic disease caused by the monkeypox virus, which previously mainly occurred in Africa. This outbreak of monkeypox is unusual, occurring in multiple non-endemic countries and regions, and showing signs of human-to-human transmission, raising serious concerns. Currently, there is no specific treatment for monkeypox virus infection; treatment mainly focuses on symptomatic support and management of complications. Early diagnosis is crucial for the timely detection of infected patients and the control of monkeypox outbreaks.

[0003] Monkeypox virus (MPXV) is an enveloped double-stranded DNA virus with two infectious forms: extracellular enveloped virus (EV) and intracellular mature virus (MV). MV is the most abundant and stable form in the environment, and is the main form of viral transmission between hosts. More than 20 structural proteins have been identified, among which A29L is a membrane protein of the monkeypox virus MV that binds to glycosaminoglycans on the cell surface and mediates virus-cell fusion; M1R is also a membrane protein of MV and is highly conserved among known poxviruses; the envelope glycoprotein A35R on the surface of EV can induce the production of neutralizing antibodies, blocking viral transmission between cells. These proteins are considered important targets for monkeypox virus research.

[0004] Currently, the main detection technologies for recombinant monkeypox virus protein A35R include enzyme-linked immunosorbent assay (ELISA), serum neutralization assay, and SPA co-agglutination assay. The antibodies used in these tests are mostly monoclonal and polyclonal antibodies. However, the research and production process of monoclonal antibodies is extremely cumbersome and complex; the source of polyclonal antibodies is limited, resulting in high testing costs and making them unsuitable for large-scale primary screening and point-of-care rapid testing in primary hospitals. Therefore, providing an antibody with high specificity, good resistance, and strong stability is of great significance for the early detection and diagnosis of monkeypox virus. Summary of the Invention

[0005] In view of this, the present invention provides a nanobody that specifically recognizes the recombinant monkeypox virus A35R protein. This nanobody has good specificity, good resistance and strong stability, providing a basis for the detection and development of drugs for monkeypox virus.

[0006] To solve the above-mentioned technical problems, the technical solution provided by the present invention is as follows:

[0007] In a first aspect, the present invention provides a nanobody that specifically recognizes the recombinant monkeypox virus A35R protein, characterized in that the nanobody comprises at least three sets of complementary determinant regions;

[0008] The complementary decision regions include CDR1 shown in SEQ ID NO:1, CDR2 shown in SEQ ID NO:2, and CDR3 shown in SEQ ID NO:3.

[0009] Compared to existing technologies, the nanobody provided by this invention has unique CDR1, CDR2 and CDR3 region sequences, which enables the nanobody to have specific recognition and binding ability to recombinant monkeypox virus A35R protein, while it does not react with other non-specific cross-reactive proteins; the nanobody provided by this invention also has the characteristics of high tolerance, high stability and high affinity.

[0010] The amino acid sequence of CDR1 shown in SEQ ID NO:1 is as follows:

[0011] GRAFRDYA;

[0012] The amino acid sequence of CDR2 shown in SEQ ID NO:2 is as follows:

[0013] VSKTGYAI;

[0014] The amino acid sequence of CDR3 shown in SEQ ID NO:3 is as follows: AADRTHRLGYSLRVDPLGYDD.

[0015] Preferably, the nanobody comprises an amino acid sequence having at least 90% sequence identity with any of the sequences in SEQ ID NO:4.

[0016] Preferably, the nanobody contains the amino acid sequence shown in SEQ ID NO:4.

[0017] The amino acid sequence of SEQ ID NO:4 is as follows: AVQLVESGGGLAQTGGSLRLSCAASGRAFRDYAMAWFRQAPGKEREFVAAV SKTGYAIRYEDSVKGRFTISRDNAKNTVSLQMASLKVEDTAVYYCAADRTHRLGYSLRVDPLGYDDWGQGTQVTVSS.

[0018] In a second aspect, the present invention provides a nucleic acid molecule encoding the aforementioned nanobody.

[0019] Preferably, the nucleic acid molecule comprises the nucleotide sequence shown in SEQ ID NO:5.

[0020] The nucleotide sequence of SEQ ID NO.5 is shown below:

[0021]

[0022] A third aspect of the present invention provides an expression vector comprising the above-mentioned nucleic acid molecules and their expression regulatory elements.

[0023] A fourth aspect of the present invention provides a host cell comprising and expressing the aforementioned nucleic acid molecules.

[0024] A fifth aspect of the present invention provides a pharmaceutical composition comprising the above-described nanobody or its antigen-binding fragment and a pharmaceutically acceptable carrier and / or excipient.

[0025] The sixth aspect of the present invention provides the use of the above-described nanobody in the preparation of kits or medicaments for the prevention, treatment or diagnosis of monkeypox virus infection.

[0026] A seventh aspect of the present invention provides a kit for detecting monkeypox virus, comprising the above-mentioned nanobody.

[0027] The nanobody provided by this invention has a highly specific recognition and binding ability for monkeypox virus, and has the potential to be used in the preparation of reagents for the prevention, treatment or detection of monkeypox virus. Attached Figure Description

[0028] Figure 1 This is a schematic diagram of the identification results of VHH-A35R nanobody provided in an embodiment of the present invention;

[0029] Figure 2 The image shows the ELISA results of the VHH-A35R nanobody against monkeypox virus A35 protein provided in this embodiment of the invention.

[0030] Figure 3 This is a diagram showing the specificity test results of the VHH-A35R nanobody provided in an embodiment of the present invention;

[0031] Figure 4 Affinity assay of VHH-A35R nanobody provided in this embodiment of the invention at a KD value of 54±6 nM;

[0032] Figure 5 The activity assay of the VHH-A35R nanobody provided in this embodiment of the invention after being treated at different temperatures for 2 hours and placed at 4°C for 2 days is shown in the figure.

[0033] Figure 6 The activity assay of the VHH-A35R nanobody provided in this embodiment of the invention was obtained after treatment at different pH values ​​for 2 hours and incubation at 4°C for 2 days. Detailed Implementation

[0034] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments and accompanying drawings. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0035] Example 1

[0036] Selection and Identification of VHH-A35R Nanobodies

[0037] 1. Nanobodies against MPXV A35R were obtained from a natural phage library using a solid-phase affinity panning method. 100 μL of MPXV A35R protein diluted in PBS was added to each well. Antigen-coated immunotubes (50 μg / tube, CBS, pH 9.6, 2 mL / tube) were incubated overnight at 4°C with slow rotation. The liquid in the overnight-coated immunotubes was discarded, and the tubes were washed three times with 2 mL of PBS buffer at room temperature, rotating for 5 min each time. 2 mL of blocking buffer (3% milk) was added, and the tubes were blocked by rotation at room temperature for 2 h. The liquid in the immunotubes was discarded, and the tubes were washed three times with 2 mL of PBS buffer at room temperature, rotating for 5 min each time. The washing buffer was discarded, and 2 mL of PBS buffer was added. 100 μL of the prepared phage library was added as the screening input phage library, and the tubes were incubated by rotation at room temperature for 1 h. The liquid in the immunotubes was discarded, and 2 mL of PBS buffer was added. Wash the immunoassay tubes 20 times for 5 min each time with PBST (1×PBS plus 0.1% Tween-20) buffer at room temperature; discard the liquid in the immunoassay tubes, add 1 mL of 0.25 mg / mL Trypsin solution, and elute by rotation at room temperature for 30 min; add 10 mL of 10% AEBSF to stop elution, transfer the solution in the immunoassay tubes to a new 1.5 mL centrifuge tube, take 10 μL for titer determination, amplify 500 μL of the eluent for the next round of panning, and store the remainder at 4℃;

[0038] 2. After three rounds of selection, randomly selected monoclonal antibodies were tested using monoclonal ELISA to obtain positive bacterial supernatants expressing antibody-displaying variable region phage particles. The binding activity and specificity of these supernatants were then determined using indirect phage-ELISA. A control group was included in the experiment. Specific sample addition procedures are shown in Table 1.

[0039] Indirect Phage-ELISA Sample Loading Table

[0040]

[0041] ELISA-positive clones were sent to a biotechnology service company for sequencing to obtain the DNA sequence of the inserted fragment, which encodes a nanobody targeting MPXV A35R.

[0042] Example 2

[0043] Large-scale preparation of VHH-A35R nanobodies

[0044] 1. Obtaining the DNA fragment encoding the VHH-A35R nanobody: This was achieved using data from Novizan Biotechnology. MaxSuper-Fidelity DNA Polymeras were used to obtain the variable region encoding gene of the heavy chain antibody by PCR (primers used are shown in Table 2), and the VHH-A35R nanobody gene was recovered by agarose gel electrophoresis.

[0045] Table 2 Primers for amplifying the variable region of heavy chain antibodies

[0046]

[0047] Note: The underlined sequence is the restriction enzyme site, and the italicized part is the 6×His tag.

[0048] 2. The obtained VHH-A35R nanobody gene fragment was cloned into the expression vector pNFCG1-Fc Ig G1-FC, and the VHH-A35R nanobody was constructed by PCR and enzyme digestion identification.

[0049] 3. Transform the expression plasmid into competent E. coli DH5α, and then extract plasmid DNA; transfect HEK293 suspension cells with sterile plasmid DNA; add 1 mL KPM (serum-free cell transfection buffer) and 20 μg sterile plasmid DNA to one centrifuge tube, and gently pipette to mix; add 1 mL KPM and 100 mL TA-293 transfection reagent to another centrifuge tube, and gently pipette to mix; transfer all liquid from the centrifuge tube containing the transfection reagent to the centrifuge tube containing the plasmid, and gently pipette to mix; incubate at room temperature for 10 minutes to prepare the plasmid-vector complex; remove HEK293 cells (2 × 10⁶ cells / year) from the constant temperature shaker. 6 Add the prepared plasmid-vector complex (20 mL / mL) while shaking, and return to the CO2 constant temperature shaker for incubation. After 3 hours, add 20 μL of penicillin-streptomycin mixture. 24 hours after transfection, add 120 μL of 0.6% 293 cell protein expression enhancer (KE-293) (0.6 mL / 100 mL) and 400 μL of transient transfection nutrient additive (KT-Feed 50×). After transfection, incubate at 37℃ and 120 r / min for 5-6 days, collect cell supernatant at 1000 rpm for 10 min to determine product expression level, and perform affinity chromatography purification and SDS-PAGE electrophoresis analysis.

[0050] Example 3

[0051] Reactivity assay of VHH-A35R nanobody

[0052] 1. The reactivity of VHH-A35R nanobody was determined by ELISA. The experimental method is as follows:

[0053] Add 100 μL of MPXV A35R protein diluted with CBS to each well, incubate overnight at 4°C, remove the coating solution, wash the plate 5 times with PBST (containing 0.1% Tween-20), add 100 μL of PBS M (PBS containing 2% skim milk powder) to each well, and block at 37°C for 1 h; wash the plate 5 times with PBST (containing 0.1% Tween-20), add 100 μL of VHH-A35R nanobody (10 μg / mL, 1 μg / mL, 10 μg / mL) -1 μg / mL, 10 -2 μg / mL, 10 -3 μg / mL, 10 -4 μg / mL, 10 -5 μg / mL, 10 -6 Incubate overnight at 4°C with 100 μg / mL anti-6×His-tagged mouse monoclonal antibody; aspirate unbound VHH-A35R nanobody, wash 3-5 times with PBST (containing 0.1% Tween-20), add 100 μL anti-6×His-tagged mouse monoclonal antibody, incubate at 37°C for 1 h; wash 3-5 times with PBST (containing 0.1% Tween-20), add 100 μL Goat anti-mouse IgG (HRP), incubate at 37°C for 1 h; wash 3-5 times with PBST (containing 0.1% Tween-20), add 100 μL TMB chromogenic solution to each well, develop color at 37°C for 30 min, stop the reaction with 100 μL 1M H2SO4 to each well, and measure OD450.

[0054] The results are as follows Figure 2 As shown, the maximum dilution concentration of the VHH-A35R nanobody can reach 10. -2 μg / mL, exhibiting good reactivity.

[0055] Example 4

[0056] Specificity test of VHH-A35R nanobody

[0057] The specificity of the VHH-A35R nanobody was determined by ELISA, and the experimental method is as follows:

[0058] In this experiment, in addition to coating MPXV A35R protein, 3% BSA was selected as a control, and TIM3 was used as an unrelated protein for specificity experiments.

[0059] Add 100 μL of MPXV A35R protein diluted with CBS and 3% BSA protein to each well, incubate overnight at 4°C, remove the coating solution, wash the plate 5 times with PBST (containing 0.1% Tween-20), add 100 μL of PBSM (PBS containing 2% skim milk powder) to each well, and block at 37°C for 1 h; wash the plate 5 times with PBST (containing 0.1% Tween-20), add 100 μL of VHH-A35R nanobody, and incubate overnight at 4°C; remove unbound VHH-A35R nanobody, wash the plate 3-5 times with PBST (containing 0.1% Tween-20), add 100 μL of anti-6×His-tagged mouse monoclonal antibody, and incubate at 37°C for 1 h; wash the plate 3-5 times with PBST (containing 0.1% Tween-20), and add 100 μL of Goat anti-mouse... IgG (HRP) was incubated at 37°C for 1 hour. The plate was washed 3-5 times with PBST (containing 0.1% Tween-20), and 100 μL of TMB chromogenic solution was added to each well. The plate was incubated at 37°C for 30 minutes. The reaction was terminated by adding 100 μL of 1M H2SO4 to each well, and the OD450 was measured.

[0060] The results are as follows Figure 3 As shown, the VHH-A35R nanobody specifically and effectively recognizes the MPXV A35R protein, while not reacting with other proteins.

[0061] Example 5

[0062] Affinity assay of VHH-A35R nanobody

[0063] The affinity between VHH-A35R nanobody and MPXV A35R protein was determined by an affinity assay. The experimental method is as follows:

[0064] 1. Prepare VHH-A35R nanobody (1 ng / μL), MPXV A35R protein (10 ng / μL, 5 ng / μL, 2.5 ng / μL, 1.25 ng / μL, 0.625 ng / μL), all diluted with PBST (containing 0.02% Tween-20, 0.01% BSA). The ProA probe required for the experiment needs to be pre-wetted for at least 10 minutes.

[0065] 2. Experimental setup: Baseline, 60s; Loading, 300s; Baseline 2, 120s; Association, 300s; Dissciation, 300s; Regeneration, 30s;

[0066] 3. The experimental results were obtained through analysis using Octet Analysis Studio 12.2.

[0067] Example 6

[0068] Effect of temperature on the stability of VHH-A35R nanobody

[0069] VHH-A35R nanobody (0.01 μg / mL) was prepared and treated at -80℃, -20℃, 4℃, 37℃, 50℃, and 80℃ for two hours. The stability of the VHH-A35R nanobody was then determined by ELISA: 100 μL of MPXV A35R protein diluted with CBS and 3% BSA protein were added to each well. The mixture was incubated overnight at 4℃. The coating solution was removed, and the plate was washed 5 times with PBST (containing 0.1% Tween-20). 100 μL of PBSM (PBS containing 2% skim milk powder) was added to each well, and the plate was blocked at 37℃ for 1 hour. The plate was then washed 5 times with PBST (containing 0.1% Tween-20), and 100 μL of the treated solution was added to each well. VHH-A35R nanobody, incubated overnight at 4℃; aspirate unbound VHH-A35R nanobody, wash the plate 3-5 times with PBST (containing 0.1% Tween-20), add 100μL anti-6×His-tagged mouse monoclonal antibody, incubate at 37℃ for 1h; wash the plate 3-5 times with PBST (containing 0.1% Tween-20), add 100μL Goat anti-mouse IgG (HRP), incubate at 37℃ for 1h; wash the plate 3-5 times with PBST (containing 0.1% Tween-20), add 100μL TMB chromogenic solution to each well, incubate at 37℃ for 30min, stop the reaction by adding 100μL 1M H2SO4 to each well, and measure OD450.

[0070] Experimental results are as follows Figure 5 As shown, the VHH-A35R nanobody provided by this invention exhibits good stability between -80℃ and 50℃.

[0071] Example 7

[0072] Effect of pH on the stability of VHH-A35R nanobody

[0073] The stability of VHH-A35R nanobodies was determined by ELISA after treating them at different pH values ​​for 2 hours.

[0074] Prepare VHH-A35R nanobody (0.01 μg / mL). Adjust the pH of PBS to pH=2, pH=4, pH=6, pH=10, and pH=12 respectively for two hours, then incubate at 4℃ for two days. The stability of the VHH-A35R nanobody was determined by ELISA: Add 100 μL of MPXV A35R protein diluted with CBS and 3% BSA protein to each well, incubate overnight at 4℃, remove the coating solution, wash the plate 5 times with PBST (containing 0.1% Tween-20), add 100 μL of PBSM (PBS containing 2% skim milk powder) to each well, block at 37℃ for 1 hour; wash the plate 5 times with PBST (containing 0.1% Tween-20), add 100 μL of the treated solution... VHH-A35R nanobody, incubated overnight at 4℃; aspirate unbound VHH-A35R nanobody, wash 3-5 times with PBST (containing 0.1% Tween-20), add 100 μL anti-6×His-tagged mouse monoclonal antibody, incubate at 37℃ for 1 h; wash 3-5 times with PBST (containing 0.1% Tween-20), add 100 μL Goat anti-mouse IgG (HRP), incubate at 37℃ for 1 h; wash 3-5 times with PBST (containing 0.1% Tween-20), add 100 μL TMB chromogenic solution to each well, incubate at 37℃ for 30 min, stop the reaction by adding 100 μL 1M H2SO4 to each well, and measure OD450.

[0075] Experimental results are as follows Figure 6 As shown, pH=4, pH=6, pH=10, and pH=12 have little effect on the activity of VHH-A35R nanobody, which also indicates that the VHH-A35R provided by the present invention has excellent acid and alkali resistance.

[0076] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A nanobody that specifically recognizes the A35R protein of recombinant monkeypox virus, characterized in that, The nanobody comprises CDR1 shown in SEQ ID NO:1, CDR2 shown in SEQ ID NO:2, and CDR3 shown in SEQ ID NO:

3.

2. The nanobody that specifically recognizes the recombinant monkeypox virus A35R protein as described in claim 1, characterized in that, The nanobody contains an amino acid sequence that has at least 90% sequence identity with the sequence of SEQ ID NO:

4.

3. The nanobody that specifically recognizes the recombinant monkeypox virus A35R protein as described in claim 1, characterized in that, The nanobody contains the amino acid sequence shown in SEQ ID NO:

4.

4. A nucleic acid molecule encoding the nanobody according to any one of claims 1-3.

5. An expression vector comprising the nucleic acid molecule and its expression regulatory elements as described in claim 4.

6. A host cell comprising and expressing the nucleic acid molecule of claim 4.

7. A pharmaceutical composition, characterized in that, Includes the nanobody as described in claim 1 or 2 and a pharmaceutically acceptable carrier and / or excipient.

8. Use of a nanobody that specifically recognizes the recombinant monkeypox virus A35R protein as described in claim 1 or 2 in the preparation of a kit for diagnosing monkeypox virus infection.

9. A kit for detecting monkeypox virus, characterized in that, It includes the nanobody as described in any one of claims 1-3.

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