Nanobody h9 targeting the e protein of japanese encephalitis virus and uses thereof

Abstract

The application discloses a nanobody H9 targeting Japanese encephalitis virus E protein and an application thereof, and belongs to the technical field of biotechnology. Amino acid sequences of complementarity determining regions CDR1, CDR2 and CDR3 of a heavy chain variable region of the nanobody H9 are respectively shown as SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3. The application further provides an H9-Fc fusion protein formed by fusion of the nanobody and a human Fc protein. The nanobody and the fusion protein thereof can specifically bind to Japanese encephalitis virus E protein, have significant neutralization activity on Japanese encephalitis virus, and can effectively relieve weight loss caused by Japanese encephalitis virus infection and significantly reduce virus load in blood and tissues in an animal model, and show excellent prevention and treatment effects. The application further provides polynucleotides encoding the nanobody, vectors and host cells containing the polynucleotides, and an application of the nanobody or the fusion protein in preparation of a medicine for treating and / or preventing Japanese encephalitis virus disease.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically to a nanobody H9 targeting the E protein of Japanese encephalitis virus and its application. Background Technology

[0002] Japanese encephalitis virus (JEV) is a major cause of viral encephalitis in Asia, belonging to the Flaviviridae family and Flavivirus genus. Currently, although preventative vaccines exist, immunization coverage remains low in developing countries, and there are still no specific antiviral drugs for JEV infection. Critically ill patients mainly rely on symptomatic and supportive treatment, resulting in a persistently high mortality rate.

[0003] The envelope protein (E protein) of JEV is the most important structural protein on the surface of the viral particle, containing approximately 500 amino acid residues. The E protein consists of three domains (Domain I, II, and III), mediating viral adsorption and membrane fusion with host cells, and is also a major target of host neutralizing antibodies. Domain III (EDIII) folds independently into an immunoglobulin-like conformation, exposed on the viral particle surface, and contains key type-specific neutralizing epitopes, making it an important region for neutralizing antibody recognition. Therefore, neutralizing antibodies targeting the E protein (especially EDIII) are considered the most promising direction for anti-JEV biologics.

[0004] Traditional monoclonal antibodies (IgG, with a molecular weight of approximately 150 kDa) face multiple bottlenecks in antiviral therapy. First, their large molecular weight makes it difficult to effectively cross the blood-brain barrier and reach lesions in the central nervous system. Since the core pathological damage of JEV occurs precisely in the central nervous system, the inability of antibodies to effectively enter brain tissue severely restricts their therapeutic efficacy. Second, traditional antibody preparation is time-consuming and costly, and animal-derived antibodies are prone to triggering immune rejection in humans. Furthermore, some flavivirus antibodies also carry the risk of antibody-dependent enhancement of infection (ADE).

[0005] Nanobodies are naturally occurring "heavy chain antibodies" lacking light chains, derived from the serum of camel-dwelling animals. The variable domain of heavy chain (VHH) of this type of antibody serves as the smallest functional unit for antigen recognition, with a molecular weight of approximately 12-14 kDa. It is the smallest complete antigen-binding fragment and is named a single-domain antibody or nanobody.

[0006] Compared to traditional antibodies, nanobodies possess unique structural characteristics and functional advantages: They have simple structures, making them easy to prepare and express; they exhibit high stability, as the conserved disulfide bonds within their structure allow them to maintain conformational stability even under harsh conditions such as high temperatures, extreme pH levels, and the presence of proteases; they possess strong affinity, with the relatively long CDR3 of VHH facilitating the formation of a more stable and compact antigen-binding interface, making their affinity comparable to that of ordinary antibodies; they are small in size, enabling them to effectively penetrate dense tissue barriers and even the blood-brain barrier; and they have low immunogenicity, making them easy to humanize, thus showing broad application prospects in disease diagnosis and treatment. These characteristics give VHH and its recombinant fragments molecular advantages that are difficult for traditional antibodies to match, and they have become important research tools in the fields of immunotherapy and immunodiagnostics.

[0007] Currently, although Japanese encephalitis (JEV) vaccines are available, vaccine coverage varies across different regions, and there is still a lack of specific antiviral drugs against JEV infection. Therefore, developing novel and highly effective anti-JEV biologics has significant clinical importance and application value. Summary of the Invention

[0008] To address the aforementioned problems, this invention aims to provide a nanobody H9 targeting the E protein of Japanese encephalitis virus (JEV) and its application. This invention involves immunizing alpacas with JEV and its E protein, and obtaining the nanobody H9 targeting the JEV E protein using phage library technology. The nanobody H9 exhibits excellent performance in terms of affinity, binding activity, and neutralizing activity against JEV. Furthermore, this invention provides its application in anti-JEV prevention and treatment drugs.

[0009] To achieve the above objectives, the present invention adopts the following technical solution: This invention provides a nanobody H9 that targets the E protein of Japanese encephalitis virus.

[0010] The amino acid sequences of the complementarity-determining regions CDR1, CDR2, and CDR3 of the heavy chain variable region of the nanobody H9 targeting the E protein of Japanese encephalitis virus are shown in SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3, respectively.

[0011] The described anti-Japanese encephalitis virus nanobody H9 is an antibody consisting only of a heavy chain, which includes a variable region and a constant region. The variable region has three complementarity determining regions (CDRs): CDR1, CDR2, and CDR3, exhibiting high variability and diversity. The sequence diversity of the CDR regions determines the antibody's specificity and affinity, as they recognize and bind to specific antigenic determinants through interaction with the antigen.

[0012] In the preferred embodiment, the amino acid sequence of the heavy chain variable region of the nanobody H9 targeting the E protein of Japanese encephalitis virus is shown in SEQ ID NO.4.

[0013] The amino acid sequences of the complementarity-determining regions CDR1, CDR2 and CDR3 of the nanobody H9 targeting the E protein of Japanese encephalitis virus are shown at positions 26-35, 49-59 and 97-116 of SEQ ID NO.4, respectively.

[0014] In a preferred embodiment, the nucleotide sequence of the polynucleotide encoding the nanobody H9 targeting the Japanese encephalitis virus E protein is shown in SEQ ID NO. 5.

[0015] In this invention, the polynucleotide with the sequence shown in SEQ ID NO.5 is a polynucleotide encoding the variable region of the H9 heavy chain of the nanobody with the amino acid sequence shown in SEQ ID NO.4 according to a specific embodiment. Based on the triplet codon rule, any polynucleotide encoding the variable region of the H9 heavy chain of the nanobody with the amino acid sequence shown in SEQ ID NO.4 belongs to the polynucleotide encoding the above-mentioned nanobody targeting the E protein of Japanese encephalitis virus as defined in this invention.

[0016] In a preferred embodiment, a vector containing the polynucleotide encoding the nanobody H9 targeting the Japanese encephalitis virus E protein is used to clone and / or express the encoding gene of the nanobody targeting the Japanese encephalitis virus E protein.

[0017] In a further preferred embodiment, the vector is pTT5. Other vectors known to those skilled in the art, especially eukaryotic cell expression vectors, can also be used for the cloning and expression of the coding genes described in this invention.

[0018] In a further preferred embodiment, the vector is encoded using Expi293F cells. Other host cells, especially eukaryotic host cells, known to those skilled in the art, can also be used for the expression of the nanobodies described in this invention.

[0019] This invention also provides a fusion protein containing the nanobody H9 targeting the E protein of Japanese encephalitis virus. Based on different application requirements such as extending the half-life of antibodies in vivo, traversing cell or tissue structures, targeting specific regions, exercising cellular effects, dimer or multimer formation, and purification processes, the nanobody provided by this invention can be fused with other proteins to form a fusion protein. In the fusion protein, the nanobody provided by this invention can exert its unique characteristic of specifically targeting and binding to the Japanese encephalitis virus.

[0020] In a preferred embodiment, the fusion protein is an H9-Fc fusion protein obtained by fusing the H9 nanobody with a human Fc fragment protein.

[0021] In a further preferred embodiment, the amino acid sequence of the H9-Fc fusion protein is shown in SEQ ID NO.6.

[0022] In a further preferred embodiment, the nucleic acid coding sequence of the H9-Fc fusion protein is shown in SEQ ID NO.7.

[0023] The present invention also provides an application of the aforementioned nanobody H9 and / or fusion protein.

[0024] The use of the nanobody H9, or its fusion protein, in the preparation of drugs for the treatment and / or prevention of Japanese encephalitis virus.

[0025] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. The H9-Fc fusion protein provided by this invention has significant neutralizing activity against JEV, and exhibits a clear dose-response relationship, with a half-maximal inhibitory concentration (IC50) of 1 / 3. 50 The concentration reached 2.38 μg / mL (see Example 6). Figure 4 This indicates that it can achieve efficient virus blocking at a lower dose, solving the problem of insufficient neutralizing activity in existing technologies.

[0026] 2. In the prevention model, administration of only 1 mg / kg of H9-Fc one day before challenge effectively alleviated weight loss in mice and significantly reduced viral load in the blood, liver, spleen, kidneys, and brain (see Example 7). Figure 5 In the treatment model, administration of 1 mg / kg H9-Fc one day after challenge significantly improved the survival status of mice and cleared the virus from tissues (see Example 8). Figure 6 This demonstrates that the nanobodies and their fusion proteins provided by this invention have broad application prospects in the preparation of drugs for the treatment and / or prevention of Japanese encephalitis virus disease. Attached Figure Description

[0027] Figure 1 This is the result of SDS-PAGE quantitative detection of JEV virus.

[0028] Figure 2 This is the result of SDS-PAGE analysis of JEV E protein (separating gel concentration 10%).

[0029] Figure 3 This is the SDS-PAGE result of H9-Fc (separating gel concentration 10%).

[0030] Figure 4This is the neutralization activity curve of H9-Fc against JEV virus.

[0031] Figure 5 This is the result of the evaluation of the protective activity of H9-Fc against JEV in animals using a prophylactic administration regimen.

[0032] Figure 6 This is the result of the evaluation of the protective activity of H9-Fc against JEV in animals using therapeutic administration methods. Detailed Implementation

[0033] The technical solution of the present invention will be further described and illustrated below through examples. All raw materials used in the examples are commercially available or prepared using conventional methods.

[0034] Example 1 The purification and quantification of JEV virus includes the following steps: Aedes albopictus C6 / 36 cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum at 28°C and 5% CO2. When cell confluence reached approximately 90%, JEV SA14 cells were inoculated at a multiplicity of infection (MOI) of 3 and cultured at 28°C for 4 days. The virus-containing culture medium was centrifuged at 6000 rpm for 1 h to remove impurities. 40% (w / v) PEG8000 NTE buffer (10 mM Tris-HCl, pH 8.0; 120 mM NaCl; 1 mM EDTA) was added to a final concentration of 8%. The culture was incubated overnight at 4°C to precipitate the virus, followed by centrifugation at 9000 rpm for 1 h. The virus-containing precipitate was collected, resuspended in NTE buffer, purified first using a 24% (w / v) sucrose pad, and then further separated using a linear 10%–30% (w / v) potassium tartrate gradient. The viral bands were located and extracted using light scattering, then replaced with NTE buffer, and subsequently concentrated using a concentration device with a molecular weight cutoff of 100 kDa.

[0035] The concentration of JEV virus was characterized by the concentration of the E protein, and identified using SDS-PAGE. Figure 1 As shown, using bovine serum albumin solutions of different known concentrations as standards, the target main band was clearly visible in the JEV sample lanes, with only trace amounts of small molecular weight bands (impurities) below, indicating that the target protein had been well enriched and purified.

[0036] Example 2 JEV E protein was expressed and purified through the following steps: JEV-E protein was expressed using *E. coli* BL21(DE3). First, the JEV-E protein gene sequence (GenBank: U14163.1, nucleotides 978 to 2477) was optimized according to *E. coli* codon preference, and a 6-histidine tag was added to the N-terminus. Then, the gene fragment was synthesized artificially and cloned into the pET21a vector (*E. coli* expression vector) using a seamless cloning method. The recombinant plasmid was transformed into BL21(DE3) competent cells. Single colonies were picked and cultured in 300 mL of LB agar containing ampicillin at 37°C and 220 rpm until the OD600 reached 0.6. Then, 1 mM IPTG was added, and the cells were shaken for another 6 hours before centrifugation to collect the cells. The cells were sonicated, the precipitate was collected, resuspended, and sonicated again. This process was repeated three times. The final sonication was performed with 6 M guanidine hydrochloride to dissolve the protein, resulting in a protein concentration of 30 mg / mL. Add 2 mL of protein denaturation buffer to 500 mL of protein renaturation buffer (400 mM MArginine, 100 mM Tris-pH 8.0, 2 mM ETDA, 10% glycerol, 5 mM GSH, 0.5 mM GSSG), and renaturate overnight at 4°C. After concentration and buffer replacement, concentrate the protein using a Superdex 200 increase solution to obtain the final protein.

[0037] Its expression and purity were identified by SDS-PAGE, such as Figure 2 As shown, the purified JEV-E protein has a molecular weight of about 55 kDa, which is in line with expectations and the purity meets the requirements.

[0038] Example 3 The establishment of an alpaca immune and VHH nanobody library includes the following steps: S1. Take 0.1 mg of JEV virus prepared in Example 1 and 0.25 mg of JEV E protein prepared in Example 2, dilute with PBS to a final volume of 1 mL, mix with an equal volume of 1 mL Freund's complete adjuvant and emulsify thoroughly, and then administer the first immunization to alpacas via subcutaneous multi-point injection. Thereafter, administer booster immunizations every three weeks. S2. After the fifth immunization, serum antibody titers were detected by ELISA, and the results showed that the serum titer was greater than 10. 6 Then, 100 mL of peripheral blood was collected, and peripheral blood mononuclear cells (PBMCs) were isolated. S3. Total RNA was extracted from the extracted PBMCs according to the instructions of Omega Total RNA Kit I. Using the obtained total RNA as a template, cDNA was synthesized by reverse transcription according to the instructions of TAKARA PrimeScript™ II 1st Strand cDNA Synthesis Kit. S4. Using the cDNA obtained by reverse transcription as a template, the IgG2VHH fragment was amplified with AlpVh-L and AlpVHH-R1 as upstream and downstream primers; the IgG3VHH fragment was amplified with AlpVh-L and AlpVHH-R2 as upstream and downstream primers (primer sequences are shown in Table 1). The target fragment was recovered after agarose gel electrophoresis. S5. Using the target fragment recovered in the first round as a template, the IgG2 VHH fragment was amplified in the second round using AlpVh-F1 and AlpVHH-R1 as upstream and downstream primers; the IgG3 VHH fragment was amplified in the second round using AlpVh-F1 and AlpVHH-R2 as upstream and downstream primers (primer sequences are shown in Table 1). The VHH gene fragment was recovered, and the recovered IgG2 and IgG3 VHH fragments were mixed for subsequent library construction steps. S6. The recovered IgG2 and IgG3 VHH fragment mixture and pComb3XSS vector were digested with Sfi I restriction endonuclease. The digested fragments were directly purified using a gel purification column. The purified VHH gene fragments were mixed with the linearized vector and ligated overnight at 4°C using T4 ligase. The ligation product was purified and mixed with TG1 competent cells. Electroporation was performed at 1800V. The electroporated mixture was transferred to 2XYT medium containing ampicillin and kanamycin and brought to a final volume of 200 mL. The medium was then incubated at 37°C and 250 rpm for 1 h with shaking to obtain the VHH nanobody library.

[0039] Table 1. List of reaction primer sequences

[0040] Example 4 The phage display technology is used to screen for specific H9 nanobodies, including the following steps: S1. After culturing the nanobody library obtained in Example 3 at 37°C and 220 rpm for 1 h, KM13 helper phage was added, and the mixture was allowed to infect for 30 min. After culturing at 37°C and 200 rpm for 1 h, the bacterial pellet was collected by centrifugation and resuspended in 100 mL of 2XYT medium containing ampicillin and kanamycin. The mixture was then incubated overnight at 30°C and 220 rpm to amplify and express the phage. S2. The next day, the culture supernatant was collected, 4% PEG8000 and 3% NaCl were added, the mixture was centrifuged after being placed on ice, the phage pellet was collected, resuspended in PBS (pH 7.4), filtered through a 0.2 μm filter to remove bacteria, and the phage library was obtained. Phage library concentration determination: Take 10 μL of the filtered library and perform 100-fold serial dilutions to 10⁻⁶ using 2YT medium. 8 10 μL of the diluted solution was used to infect 100 μL of TG1 strain. After 30 minutes, the solution was spread onto 2XYT semi-solid medium plates. The number of single colonies was counted the next day, and the phage concentration of the amplified library was calculated. 7% DMSO was added to the filtered library, mixed well, and stored at -80 °C. S3. Inactivated Japanese encephalitis virus particles were coated with a high-affinity enzyme-linked immunosorbent assay (ELISA) plate. After coating at 4°C overnight, the plate was washed with 0.1% PBST, and 5% skim milk powder was added to block the plate at 37°C for 1 hour. The blocking solution was discarded and the plate was washed again. S4. Add approximately 1 × 10⁻⁶ to the coated ELISA plate. 11 Phage libraries of pfu and CFU were incubated at 37°C for 2 hours. After washing with 0.1% PBST, 100 µL of glycine-HCl elution buffer was added to each well. The plates were incubated at room temperature for 10 minutes, and the elution buffer was aspirated and then quickly neutralized with an equal volume of 1 M Tris-HCl (pH 7.4). S5. Mix the neutralized eluent with E. coli TG1, incubate at 37°C for 30 min, then shake and culture. After phage rescue, culture at 30°C overnight with shaking to obtain the first-round amplified antibody library. Repeat the above process to obtain the second-round library. S6. Ninety-six monoclonal antibodies were randomly selected from the bacterial plates after the second round of screening. These antibodies were induced to express VHH antibodies via IPTG to prepare a soluble crude extract of recombinant VHH antibodies. The reactivity of this crude extract with inactivated Japanese encephalitis virus particles was detected by ELISA, using OD200 as the metric. 450 A value more than 3 times higher than the PBS control was considered a positive clone. S7. Sequencing of a portion of the bacterial culture from the positive clone yields the nanobody H9 targeting the E protein of Japanese encephalitis virus.

[0041] The nanobody H9, which was screened to fight the JEV virus E protein in this embodiment, has amino acid lengths of FR1, FR2, FR3 and FR4 of 25, 13, 37 and 10, respectively, and amino acid lengths of CDR1, CDR2 and CDR3 of 10, 11 and 20, respectively. Its amino acid sequence is shown in SEQ ID NO.4.

[0042] The nucleotide sequence encoding the polynucleotide of the nanobody H9 against JEV virus E protein is shown in SEQ ID NO.5.

[0043] Example 5 The expression and purification of the H9-Fc fusion protein includes the following steps: Based on the core coding sequence of the H9 nanobody obtained in Example 3, a human IgG1 Fc fragment was fused to its C-terminus. The fusion gene was cloned into the pTT5 eukaryotic expression vector via homologous recombination. The amino acid sequence of the constructed H9-Fc fusion protein is shown in SEQ ID NO. 6, and the corresponding nucleotide coding sequence is shown in SEQ ID NO. 7. The recombinant plasmid was transfected into Expi293F suspension cells and cultured at 37°C and 8% CO2 for 4 days. The cell culture supernatant was then collected. The supernatant was centrifuged at 10000 g for 5 min at 4°C to remove cell debris, and then filtered through a 0.22 μm filter for clarification. The target protein in the supernatant was purified using a Protein A affinity chromatography column. After binding, washing, and elution steps, the eluted fraction was collected to obtain the purified H9-Fc fusion protein.

[0044] Protein expression and purity were detected by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The electrophoresis results are as follows: Figure 3 As shown, under reducing conditions, due to the disruption of disulfide bonds, the fusion protein exhibits a band of approximately 40 kDa, consistent with the expected theoretical molecular weight, indicating that the H9-Fc fusion protein was successfully expressed and has good purity. The amino acid sequence of the H9-Fc fusion protein is shown in SEQ ID NO.6, and the nucleic acid coding sequence of the H9-Fc fusion protein is shown in SEQ ID NO.7.

[0045] Example 6 H9-Fc fusion protein viral neutralizing activity analysis includes the following steps: S1. One day before the experiment, Vero E6 cells were diluted to 2×10⁻⁶ cells with culture medium (DMEM + 10% FBS). 5 The concentration of cells / mL was seeded into 24-well cell culture plates at a seeding volume of 500 μL / well and incubated at 37°C in a 5% CO2 cell culture incubator. S2. On the day of the experiment, JEV virus was diluted to 1000pfu / mL with DMEM, and nanobody H9-Fc was diluted to the initial concentration (100 μg / mL) with the diluted virus. The diluted virus was then diluted 4 times, mixed thoroughly, and incubated in a cell culture incubator for 1 h. S3. Discard the cell culture supernatant in the 24-well plate, add 100 μL of the co-incubated virus-antibody mixed suspension to each well; at the same time, set up a control group, add only 100 μL of diluted virus, and co-incubate in a cell culture incubator for 1 h, then discard the supernatant, add 1 ml of semi-solid culture medium (1.8% methylcellulose + 2% FBS + 1% penicillin antibody), and continue to culture in a 37℃, 5% CO2 cell culture incubator for 60 h; After S4 and 72 h, discard the cell culture supernatant, add 500 μL / well of crystal violet staining solution (50 mg crystal violet, 20 mL anhydrous ethanol, and water to 100 mL), let stand at room temperature for 30 min, discard the staining solution, wash 5 times with water, pat dry the 24-well plate, and take a photo for record. S5. Count the number of plaques in each well. Calculate the antibody inhibition efficiency by dividing the number of plaques in control wells by the number of plaques in experimental wells. Fit the antibody inhibition efficiency and antibody concentration to a curve using GraphPad Prism 8 to calculate the antibody IC50. 50 value.

[0046] Test results as follows Figure 4 The results showed that the H9-Fc fusion protein exhibited strong neutralizing activity against JEV, with a dose-dependent relationship and an IC50 value of [missing information]. 50 It was 2.38 μg / mL.

[0047] Example 7 The protective activity of the H9-Fc fusion protein in animals was analyzed using the following steps: S1. Six IFNARI- / - mice (which are susceptible to JEV and are often used to evaluate the in vivo efficacy of antiviral drugs) were divided into two groups (control group and experimental group), with three mice in each group. S2. In a clean bench, administer an equal volume of PBS buffer the day before infection (day -1). The experimental group was given H9-Fc antibody (1 mg / kg) via intraperitoneal injection, while the control group was given the same volume of PBS via intraperitoneal injection. S3, Day 0: Each mouse was injected intraperitoneally with 1×10 3 PFU JEV#797 was used to attack the virus; S4. From the day before the challenge to the 5th day after the challenge, the weight changes of the mice were monitored daily. Peripheral blood samples were collected on the 3rd and 5th days after infection. The mice were sacrificed on the 5th day after infection, and various tissues and organs were collected for viral load analysis.

[0048] The viral RNA levels in mouse peripheral blood and tissues (liver, spleen, kidney, and brain) were detected using quantitative reverse transcription PCR. Results are expressed as viral RNA copies per gram of tissue or per milliliter of blood. Peripheral blood was collected from male mice on day 5 post-infection, and cytokine expression levels were detected using quantitative reverse transcription PCR.

[0049] Test results as follows Figure 5 The results showed that, compared to the control group, the challenge dose of 1×10 3 PFU and H9-Fc fusion protein can effectively alleviate weight loss, and the viral load in blood and tissues is also significantly lower than that in the control group.

[0050] Example 8 The therapeutic and protective activity of the H9-Fc fusion protein in animals was analyzed using the following steps: S1. Six IFNARI- / - mice were divided into two groups (control group and experimental group) according to sex, with three mice in each group; S2. Administer 1×10⁻⁶ via intraperitoneal injection on day 0 within a clean bench. 3 PFU JEV#797 was used to attack the virus; S3. On the first day after infection, H9-Fc antibody (1 mg / kg) or an equal volume of PBS buffer was administered via intraperitoneal injection. S4. Starting from day 0 of the self-infection challenge, the weight changes of mice were monitored daily. Peripheral blood was collected on day 7 post-infection. The mice were then sacrificed, and various tissues and organs were collected for viral load analysis. S5. Quantitative reverse transcription PCR was used to detect the viral RNA levels in peripheral blood and tissues (liver, spleen, kidney, and brain). The viral RNA content in blood samples was expressed as copies per microliter of blood, and in tissue samples as copies per gram of tissue.

[0051] Test results as follows Figure 6 The results showed that, compared with the control group, the H9-Fc fusion protein could effectively alleviate the weight loss caused by JEV infection and significantly reduce the load of JEV in the blood and tissues.

[0052] It should be understood that the above embodiments are for illustrative purposes only and are not intended to limit the scope of protection of the present invention. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A nanobody H9 targeting the E protein of Japanese encephalitis virus, characterized in that, The amino acid sequences of the complementarity-determining regions CDR1, CDR2, and CDR3 of the heavy chain variable region of the nanobody H9 targeting the Japanese encephalitis virus E protein are shown in SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3, respectively.

2. The nanobody H9 targeting the E protein of Japanese encephalitis virus according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region of the nanobody H9 targeting the E protein of Japanese encephalitis virus is shown in SEQ ID NO.

4.

3. The nanobody H9 targeting the E protein of Japanese encephalitis virus according to claim 1, characterized in that, The nucleotide sequence of the polynucleotide encoding the nanobody H9 targeting the Japanese encephalitis virus E protein is shown in SEQ ID NO.

5.

4. The nanobody H9 targeting the E protein of Japanese encephalitis virus according to claim 3, characterized in that, A vector containing the polynucleotide, the vector being used to clone and / or express the encoding gene of the nanobody targeting the E protein of Japanese encephalitis virus.

5. The nanobody H9 targeting the E protein of Japanese encephalitis virus according to claim 4, characterized in that, The carrier is pTT5.

6. The nanobody H9 targeting the E protein of Japanese encephalitis virus according to claim 4, characterized in that, The host cell used for encoding the vector is the Expi293F cell.

7. A fusion protein, characterized in that, The fusion protein contains the nanobody H9 targeting the Japanese encephalitis virus E protein as described in any one of claims 1 to 6.

8. The fusion protein according to claim 7, characterized in that, The fusion protein is the H9-Fc fusion protein obtained by fusing the nanobody H9 with the human Fc segment protein.

9. The fusion protein according to claim 8, characterized in that, The amino acid sequence of the H9-Fc fusion protein is shown in SEQ ID NO.6; the nucleic acid coding sequence of the H9-Fc fusion protein is shown in SEQ ID NO.

7.

10. The use of a nanobody H9 according to any one of claims 1 to 6, or a fusion protein according to any one of claims 7 to 9, in the preparation of a medicament for treating and / or preventing Japanese encephalitis virus.

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