Recombinant nanobodies against Japanese encephalitis virus EDIII protein and their applications

Abstract

This invention discloses a recombinant nanobody targeting the EDIII protein of Japanese encephalitis virus (JEV) and its applications, belonging to the fields of biomedicine and immunology. The recombinant nanobody is either Nb12-Fc or Nb18-Fc, both formed by fusing an alpaca-derived VHH fragment with a human IgG1 antibody Fc fragment. It specifically binds to the JEV EDIII protein and exhibits highly efficient neutralizing activity against JEV and other related flaviviruses. This invention is the first to disclose a recombinant nanobody targeting the JEV EDIII protein, filling a technological gap in this field both domestically and internationally. It also provides a complete and reproducible preparation process, offering a novel biological agent with strategic potential for the prevention and treatment of JEV. This has significant theoretical and practical value for promoting the development of anti-JEV drugs and improving the JEV prevention and control system.

Description

Technical Field

[0001] This invention belongs to the fields of biomedicine and immunology, specifically relating to recombinant nanobodies against the EDIII protein of Japanese encephalitis virus and their applications. Background Technology

[0002] Japanese encephalitis virus (JEV) is a mosquito-borne zoonotic pathogen that primarily affects the human central nervous system and the reproductive system of pigs, causing inflammation of the human central nervous system and reproductive disorders in pigs. It poses a significant threat to public health and the healthy development of the livestock industry. Currently, there are no specific antiviral drugs against JEV in clinical practice, making it impossible to effectively treat infected cases. Although vaccination is an effective means of preventing JEV infection, the prevalence of JEV has continued to expand in recent years, and its dominant genotype has gradually shifted from the traditional type III to type I. Existing vaccines have limited effectiveness against the new dominant genotype, presenting new and severe challenges to the prevention and control of Japanese encephalitis. Therefore, developing novel and efficient JEV prevention and control technologies and related products has become an urgent technical challenge to be solved in this field.

[0003] JEV belongs to the genus Flaviviridae in the family Flaviviridae. It is a single-stranded positive-sense RNA virus whose genome encodes three structural proteins (C protein, prM protein, and E protein) and seven non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5). Among these, the E protein is a key functional molecule for JEV invasion of host cells, primarily mediating core processes such as viral adsorption and membrane fusion with host cells. It is also a major target for the production of neutralizing antibodies and a core target for anti-JEV drug development. The E protein can be further divided into three independent domains: EDI, EDII, and EDIII. EDIII, as the viral receptor-binding domain, is rich in multiple conserved neutralizing epitopes, making it an ideal target for inducing the production of neutralizing antibodies. Notably, the amino acid sequences of the E protein among different members of the Flaviviridae genus exhibit a certain degree of conservation. This not only endows antibodies targeting EDIII with highly efficient JEV neutralizing activity but also gives them the potential for broad-spectrum cross-neutralization with other Flaviviridae, providing an important theoretical basis and technical support for the development of broad-spectrum anti-Flavorida drugs.

[0004] Nanobodies (Nb) are heavy chain antibodies naturally occurring in camel species such as alpacas and dromedaries, lacking the light chain. Their core functional fragment is the variable domain of the heavy chain antibody (VHH), making them the smallest known antibody fragments with complete antigen-binding activity. Compared to traditional monoclonal antibodies, nanobodies offer significant advantages: small molecular weight (approximately 15 kDa), easily penetrating tissue barriers; high stability, maintaining activity under extreme temperature and pH conditions; low immunogenicity, less likely to trigger immune rejection; and ease of modification, expression, and purification through genetic engineering, showing broad application prospects in molecular diagnostics, tumor immunotherapy, drug delivery, and antiviral therapy. Currently, there are no reports of recombinant nanobodies targeting the EDIII protein of Japanese encephalitis virus (JEV), and there is a lack of nanobodily agents suitable for JEV treatment. Summary of the Invention

[0005] Technical problem solved: In view of the lack of effective anti-JEV drugs and the limitations of existing prevention and control methods, this invention provides recombinant nanobodies against the EDIII protein of Japanese encephalitis virus and their applications. By screening, recombinant nanobodies that specifically recognize the JEV EDIII protein and have highly efficient neutralizing activity are obtained, providing innovative biological agents and technical solutions for the prevention and treatment of JEV, which have important clinical application value and industrial prospects.

[0006] Technical solution: In a first aspect, the present invention provides a recombinant nanobody against Japanese encephalitis virus EDIII protein. The recombinant nanobody is Nb12-Fc or Nb18-Fc, both of which are formed by fusing alpaca-derived VHH fragments with human IgG1 antibody Fc fragments. It can specifically bind to Japanese encephalitis virus EDIII protein and has highly efficient neutralizing activity against JEV and other related normal flaviviruses.

[0007] Preferably, the amino acid sequence of the recombinant nanobody Nb12-Fc is shown in SEQ ID NO.1, and the amino acid sequence of the recombinant nanobody Nb18-Fc is shown in SEQ ID NO.2.

[0008] SEQ ID NO.1: DVQLQESGGGLVQAGGSLRLSCAASGRTDDIHAMGWFRQAPGKEREFVAVIRWSSDFTYYSSSVTGRFTISRDNAKNTGYLQMNRLSPEDTAVYYCATQDPTTNSYSYNWPDTYDHWGQGTQVTVSSGRGGGGSGGGGSTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREE MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0009] SEQ ID NO.2: DVQLQESGGGLVQPGGSLRLSCAASGITFYGYIMGWYRQAPGKPRELVATISTGGNTNYADSLKGRFTISRDNAKNTVYLQMNKLEPEDTAVYYCYTDGAYYDGRYHSQDYWGQGTQVTVSSGRGGGGSGGGGSTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEM TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0010] Furthermore, the nucleotide sequence encoding the recombinant nanobody Nb12-Fc is shown in SEQ ID NO.3; the nucleotide sequence encoding the recombinant nanobody Nb18-Fc is shown in SEQ ID NO.4.

[0011] SEQ ID NO.3:

[0012] SEQ ID NO.4:

[0013] In a second aspect, the present invention provides a method for preparing the recombinant nanobody described in the first aspect, comprising the following steps: (1) Using the constructed alpaca natural VHH phage display library, antibody library solid-phase screening technology was used to specifically screen for JEVEDIII protein as antigen to obtain phage clones that specifically bind to JEVEDIII protein; positive phage clones were sequenced to obtain the nucleotide sequences of the variable region (VHH) fragments of two specific heavy chain antibodies, which were named Nb-12 and Nb-18, respectively. (2) The nucleotide sequence encoding Nb-12 or Nb-18 was recombined with the Fc fragment encoding sequence of human antibody IgG1 to construct a fusion gene; the fusion gene was cloned into the pCAGGS-sec-HA-Strep-Tag II eukaryotic expression vector to construct the recombinant expression vectors pCAGGS-sec-Nb12-hIgG1-Fc-HA-Strep-Tag II and pCAGGS-sec-Nb18-hIgG1-Fc-HA-Strep-Tag II, and the correct vector construction was verified by sequencing. (3) The two verified recombinant expression vectors were transfected into HEK-293T eukaryotic cells to perform transient expression of recombinant nanobodies. On the 5th day after transfection, the cell supernatant was collected and the recombinant nanobodies Nb12-Fc and Nb18-Fc in the supernatant were purified by Strep-Tag II magnetic bead affinity purification method to obtain high-purity recombinant nanobodies. The purification effect was verified by SDS-PAGE electrophoresis.

[0014] Thirdly, the present invention provides recombinant expression vectors containing recombinant nanobody encoding genes. Specifically, the recombinant expression vector pCAGGS-sec-Nb12-hIgG1-Fc-HA-Strep-Tag II contains the encoding gene of the recombinant nanobody Nb12-Fc, and the recombinant expression vector pCAGGS-sec-Nb18-hIgG1-Fc-HA-Strep-Tag II contains the encoding gene of the recombinant nanobody Nb18-Fc. Both recombinant expression vectors can be used for the efficient expression of recombinant nanobodies.

[0015] Fourthly, the present invention provides the application of the recombinant nanobody described in the first aspect in the preparation of anti-Japanese encephalitis virus related products.

[0016] Preferably, the product includes a neutralizing antibody drug against Japanese encephalitis virus (JEV), a diagnostic reagent for JEV, and a vaccine adjuvant for JEV. The neutralizing antibody drug can be used directly for the treatment of JEV infection, the diagnostic reagent can be used for rapid detection and screening of JEV, and the vaccine adjuvant can enhance the immune effect of existing JEV vaccines and expand the scope of prevention and control.

[0017] Beneficial effects: (1) High affinity: The equilibrium dissociation constant K between Nb12-Fc and JEVEDIII protein was verified by surface plasmon resonance (SPR) experiment. D =9.14×10 -9 The equilibrium dissociation constant K of M, Nb18-Fc and JEV EDIII protein D =2.59×10 -9 M, both reached the nanomolar level, indicating that the two antibodies can bind specifically and efficiently to JEV EDIII protein; (2) High inhibitory activity: The half-maximal inhibitory concentration (IC50) of the two recombinant nanobodies against JEV was verified by virus neutralization assay. 50 The effective inhibitors of JEV replication in host cells were 6.61 nM (Nb12-Fc) and 7.12 nM (Nb18-Fc), respectively, which showed good therapeutic potential. (3) Broad-spectrum antiviral activity: Experimental verification showed that the two recombinant nanobodies not only had highly efficient neutralizing activity against JEV, but also had significant neutralizing activity against other pathogenic normal flaviviruses (such as West Nile virus WNV, Murray Valley encephalitis virus MVEV, and Usutu virus USUV), which can achieve cross-control of multiple normal flaviviruses and have a wider range of applications. (4) The recombinant nanobody of the present invention can be efficiently expressed in eukaryotic cells, the purification process is simple, and it has good stability and low immunogenicity, making it easy to industrialize and apply in clinical practice; This invention discloses for the first time a recombinant nanobody targeting the EDIII protein of Japanese encephalitis virus, filling a technological gap in this field both domestically and internationally. It also provides a complete and reproducible preparation process, offering a novel biological agent with strategic potential for the prevention and treatment of Japanese encephalitis virus. This invention has significant theoretical and practical value for promoting the development of anti-JEV drugs and improving the JEV prevention and control system. Attached Figure Description

[0018] Figure 1 The image shows the results of Western blotting analysis of the specific reaction between recombinant nanobodies Nb12-Fc and Nb18-Fc and JAVE protein. Figure 2 The image shows the SPR (Symptom Reactivity Test) results of the affinity between the recombinant nanobody Nb12-Fc and JEV EDIII protein. Figure 3 The graph shows the SPR (Symptom Reactivity Test) results of the affinity between the recombinant nanobody Nb18-Fc and JEV EDIII protein. Figure 4 The graph shows the results of detecting the neutralizing activity of recombinant nanobodies Nb12-Fc and Nb18-Fc against JEV. Figure 5 The graph shows the results of detecting the neutralizing activity of recombinant nanobodies Nb12-Fc and Nb18-Fc against WNV. Figure 6 The graph shows the results of detecting the neutralizing activity of recombinant nanobodies Nb12-Fc and Nb18-Fc against MVEV. Figure 7 The graph shows the results of detecting the neutralizing activity of recombinant nanobodies Nb12-Fc and Nb18-Fc against USUV. Figure 8 Map of the recombinant expression vector pET28a-EDIII-His plasmid; Figure 9 Map of the recombinant expression vector pCAGGS-sec-Nb12-hIgG1-Fc-HA-Strep-Tag II plasmid; Figure 10 The image shows the plasmid map of the recombinant expression vector pCAGGS-sec-Nb18-hIgG1-Fc-HA-Strep-Tag II. Detailed Implementation

[0019] The present invention will be described in detail below with reference to specific embodiments: The reagents, methods and equipment used in the embodiments of the present invention are all conventional reagents, methods and equipment in this technical field. Unless otherwise specified, the reagents and materials used are all commercially available products, and the experimental operations follow conventional experimental specifications.

[0020] Example 1: Construction and expression of recombinant nanobodies Nb12-Fc and Nb18-Fc 1. Expression of JEV EDIII protein The coding sequence of JEV E protein domain III (EDIII) was artificially synthesized (as shown in SEQ ID NO.5), and cloned into the pET28a prokaryotic expression vector with a His tag at the C-terminus to construct the recombinant expression vector pET28a-EDIII-His (plasmid map shown). Figure 8 (As shown); the recombinant vector was transformed into E. coli BL21(DE3) competent cells, and positive single clones were picked and inoculated into LB medium containing kanamycin and cultured at 37°C and 200 rpm until OD. 600=0.6-0.8; Add IPTG to a final concentration of 0.5 mM, and induce expression at 16℃ for 16-18 h; Collect bacterial cells, resuspend in PBS buffer, sonicate, centrifuge at 12000 rpm for 30 min at 4℃, and collect the supernatant; Purify the His-tagged EDIII protein in the supernatant using nickel column affinity chromatography, and then replace the purified protein with PBS buffer (pH 7.4) by ultrafiltration and store at -80℃ for later use. The purity of the protein was verified by SDS-PAGE electrophoresis.

[0021] SEQ ID NO.5: ACCTATGGCATGTGCACAGAAAAATTCTCGTTCGCGAAAAAATCCGGCGGACACTGGTCACGGAACAGTTGTCATTGAACTCTCCTACTCTGGGAGTGATGGCCCCTGCAAAATTCCGATTGTCTCCGTTGCGAGCCTCAATGACATGACCCC CGTTGGGCGGCTGGTGACAGTGAACCCCTTCGTCGCGACTTCCAGTGCCAATTCAAAGGTGCTGGTCGAGATGGAACCCCCTCGGAGACTCCTACATCGTAGTTGGAAGGGGAGACAAGCAGATCAACCACCATTGGCACAAAAGCTGGA.

[0022] 2. Screening of specific nanobodies (1) Phage panning: The purified JEV EDIII protein was diluted to 50 µg / tube with CBS buffer (pH 9.6) and added to the immunoassay tube for overnight coating; the coating solution was removed the next day, and the tube was washed and blocked with 3% BSA; the blocking solution was discarded, and 1×10⁻⁶ phages were added to the immunoassay tube. 12 The pfu alpaca natural nanobody phage library was incubated at room temperature by rotation for 1 h. Subsequently, the immunotubes were washed 20 times with 0.1% PBST solution, 1 mL of 0.25 mg / mL Trypsin solution was added, and the tubes were eluted by rotation at room temperature for 30 min. The eluent was collected, and 10 μL of 10% AEBSF was added immediately to stop the elution. The titer of the eluent was then determined.

[0023] (2) Phage elution amplification: The elution buffer was re-infected with TG1 Escherichia coli in the logarithmic growth phase. The bacterial culture was collected the next day, and M13KO7 helper phage was added for amplification for 6 h. Subsequently, the amplified phage was purified using PEG8000 and its titer was determined to obtain the enriched nanobody library. This library can be directly used for the next round of screening.

[0024] (3) After three rounds of repeated panning, single clones from the third round of elution were selected and cultured in 96-well plates, and IPTG was added to induce the expression of VHH-pIII fusion protein (i.e., nanobody-pIII protein). After 24 h of induction, the supernatant of the bacterial culture containing the nanobody was collected, and the binding of the nanobody to the antigen was detected by enzyme-linked immunosorbent assay (ELISA). Based on the ELISA detection data, positive single clones were selected and sequenced to obtain the nucleotide sequences of two specific VHH fragments, which were named Nb-12 and Nb-18, respectively.

[0025] 3. Construction, expression, and purification of recombinant nanobodies The coding sequence of the artificially synthesized human IgG1 antibody Fc fragment (as shown in SEQ ID NO. 6) was recombined with the Nb-12 and Nb-18 nucleotide sequences obtained by sequencing to construct Nb12-Fc and Nb18-Fc fusion genes, respectively. The two fusion genes were then cloned into the pCAGGS-sec-HA-Strep-Tag II eukaryotic expression vector to obtain the recombinant expression vector pCAGGS-sec-Nb12-hIgG1-Fc-HA-Strep-Tag II (plasmid map shown in...). Figure 9 (as shown) and pCAGGS-sec-Nb18-hIgG1-Fc-HA-Strep-Tag II (plasmid map as shown) Figure 10 As shown in the figure, sequencing confirmed that the vector was constructed correctly, with no base mutations or reading frame shifts.

[0026] SEQ ID NO.6: .

[0027] The recombinant expression vectors were transfected into HEK-293T cells for expression. On the fifth day after transfection, the cell supernatant was collected, and the antibodies were purified using the Strep-Tag II magnetic bead affinity purification method to obtain the recombinant nanobodies Nb12-Fc and Nb18-Fc.

[0028] Example 2: Binding activity analysis of recombinant nanobodies Nb12-Fc and Nb18-Fc (1) The binding specificity of recombinant nanobodies Nb12-Fc and Nb18-Fc to JEV E protein was verified by Western Blot: JEV Beijing / 2020-1 strain (preserved in our laboratory, GenBank No. OP588746) was used to infect BHK-21 cells at 0.1 MOI. After 24 h, protein samples were collected by RIPA lysis buffer. The purified recombinant nanobodies Nb12-Fc and Nb18-Fc were used as primary antibodies, and HRP-human-IgG1-FC antibody was used as secondary antibody.

[0029] The results are as follows Figure 1 As shown, the target band of E protein appears at 55 kDa, indicating that the two recombinant nanobodies can specifically react with JEV E protein.

[0030] (2) The affinity of Nb12-Fc and Nb18-Fc for JEV EDIII protein was determined by surface plasmon resonance (SPR) assay. A CM5 chip was placed in a Biacore T200 microarray, and the chip was activated for 7 min with an equal volume mixture of EDC [1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride] and NHS (N-hydroxysuccinimide). JEV-EDIII protein was then coupled to the CM5 chip as a ligand. PBS was used as the running buffer at a flow rate of 30 μL / min. Antibodies Nb12-Fc and Nb18-Fc were serially diluted 2-fold with PBS, with an initial concentration of 200 nM. The solutions were flowed sequentially through the chip, binding for 300 s and dissociation for 420 s. After each cycle, the cells were regenerated with 10 mM glycine-hydrochloric acid (pH 1.5) for 30 s. All data were processed using Biacore software.

[0031] The results are as follows Figure 2 and Figure 3 As shown, the fitted line represents the time variation of binding and dissociation between recombinant nanobodies Nb12-Fc or Nb18-Fc and JEV-EDIII protein. Analysis results indicate that the Kc of Nb12-Fc... D =9.14×10 -9 M, Nb12-Fc K D =2.59×10 -9 M. This indicates that antibodies Nb12-Fc or Nb18-Fc have high affinity for JEV-EDIII protein, and the dissociation constant (K) is [missing value]. D It has reached the nanomolar level.

[0032] Example 3: Determination of the neutralizing activity of recombinant nanobodies (1) Neutralization activity assay of recombinant nanobodies against JEV: Two recombinant nanobodies of serial dilution were incubated with an equal volume of recombinant JEV reporter virus rGI-mCherry (preserved in our laboratory) containing 100 TCID50 at 37°C for 1 hour to allow the nanobodies to bind to the virus. Subsequently, the serum-virus mixture was seeded into a 96-well plate covered with a monolayer of BHK-21 cells, and after incubation for another 1 hour, the supernatant was discarded, and DMEM medium containing 2% FBS was added. The plate was then cultured for another 24 hours. Finally, the expression of mCherry red fluorescent protein at each dilution was statistically analyzed using fluorescence microscopy, and the antibody dilution that could inhibit 50% of mCherry red fluorescent protein expression was calculated.

[0033] The results are as follows Figure 4 As shown, both nanobodies exhibit neutralizing activity, IC50. 50 The values ​​were 6.61 nM (Nb12-Fc) and 7.12 nM (Nb18-Fc), respectively.

[0034] (2) Validation of the broad-spectrum neutralizing activity of recombinant nanobodies: Two recombinant nanobodies were incubated with an equal volume of WNV, MVEV, and USUV chimeric viruses (recombinant viruses carrying WNV, MVEV, and USUV structural proteins respectively with JEV as the backbone) at 37°C for 1 hour to allow the nanobodies to bind to the viruses. Subsequently, the serum-virus mixture was seeded into a 96-well plate covered with a monolayer of BHK-21 cells and cultured for another 24 hours. After fixation with 4% paraformaldehyde and blocking with 5% BSA solution, the cells were stained with Anti-JEV-NS3 antibody as the primary antibody and Alexa Fluor 488 Anti-Mouse IgG antibody as the secondary antibody and observed under a fluorescence microscope. The viral infection status at each dilution was counted, and the antibody dilution that could inhibit 50% of viral infection was calculated.

[0035] The results are as follows Figures 5-7 As shown, both nanobodies exhibit neutralizing activity against other flaviviruses such as WNV, MVEV, and USUV. Nb12-Fc, in particular, shows an IC50 value of [missing information - likely related to neutralization activity against WNV, MVEV, and USUV]. 50 The values ​​are 5.40 nM, 9.68 nM, and 5.51 nM respectively; Nb18-Fc IC for WNV, MVEV, and USUV 50 The values ​​are 10.08 nM, 12.00 nM, and 16.23 nM, respectively.

[0036] This invention verifies the reaction specificity, binding activity, JEV replication inhibition ability, and broad-spectrum antiviral ability of recombinant nanobodies Nb12-Fc and Nb18-Fc using methods such as ELISA, Western Blot, SPR, and neutralization assay, providing a new strategy for the prevention and treatment of JEV and other orthoflaviruses.

[0037] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and 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 recombinant nanobody against the EDIII protein of Japanese encephalitis virus, characterized in that: The recombinant nanobody is either Nb12-Fc or Nb18-Fc, both of which are formed by fusing an alpaca-derived VHH fragment with a human IgG1 antibody Fc fragment.

2. The recombinant nanobody against Japanese encephalitis virus EDIII protein according to claim 1, characterized in that: The amino acid sequence of the recombinant nanobody Nb12-Fc is shown in SEQ ID NO.1, and the amino acid sequence of the recombinant nanobody Nb18-Fc is shown in SEQ ID NO.

2.

3. The recombinant nanobody against Japanese encephalitis virus EDIII protein according to claim 1, characterized in that: The nucleotide sequence encoding the recombinant nanobody Nb12-Fc is shown in SEQ ID NO.3; the nucleotide sequence encoding the recombinant nanobody Nb18-Fc is shown in SEQ ID NO.

4.

4. The method for preparing the recombinant nanobody against Japanese encephalitis virus EDIII protein as described in claim 1, characterized in that, Includes the following steps: (1) Using the constructed alpaca natural VHH phage display library, antibody library solid-phase screening technology was used to specifically screen for JEVEDIII protein as antigen to obtain phage clones that specifically bind to JEVEDIII protein; positive phage clones were sequenced to obtain the nucleotide sequences of the variable region (VHH) fragments of two specific heavy chain antibodies, which were named Nb-12 and Nb-18, respectively. (2) The nucleotide sequence encoding Nb-12 or Nb-18 was recombined with the Fc fragment encoding sequence of human antibody IgG1 to construct a fusion gene; the fusion gene was cloned into the pCAGGS-sec-HA-Strep-Tag II eukaryotic expression vector to construct the recombinant expression vectors pCAGGS-sec-Nb12-hIgG1-Fc-HA-Strep-Tag II and pCAGGS-sec-Nb18-hIgG1-Fc-HA-Strep-Tag II, and the correct vector construction was verified by sequencing. (3) The two verified recombinant expression vectors were transfected into HEK-293T eukaryotic cells to perform transient expression of recombinant nanobodies. On the 5th day after transfection, the cell supernatant was collected and the recombinant nanobodies Nb12-Fc and Nb18-Fc in the supernatant were purified by Strep-Tag II magnetic bead affinity purification method to obtain high-purity recombinant nanobodies. The purification effect was verified by SDS-PAGE electrophoresis.

5. The preparation method according to claim 4, characterized in that: The recombinant expression vector pCAGGS-sec-Nb12-hIgG1-Fc-HA-Strep-Tag II contains the encoding gene of the recombinant nanobody Nb12-Fc, and the recombinant expression vector pCAGGS-sec-Nb18-hIgG1-Fc-HA-Strep-Tag II contains the encoding gene of the recombinant nanobody Nb12-Fc.

6. The use of the recombinant nanobody according to claim 1 in the preparation of anti-Japanese encephalitis virus related products.

7. The application according to claim 6, characterized in that: The products include neutralizing antibody drugs against Japanese encephalitis virus, diagnostic reagents for Japanese encephalitis virus, and adjuvants for Japanese encephalitis vaccines.

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