Nano antibody for resisting porcine epidemic diarrhea virus S protein, fusion protein and application of nano antibody and fusion protein

Abstract

The invention relates to the technical field of biology, in particular to a nano antibody capable of being used for resisting porcine epidemic diarrhea virus S protein, fusion protein and application of the nano antibody and the fusion protein. The heavy chain variable region comprises three complementary determining regions CDR1, CDR2 and CDR3; the amino acid sequence of the CDR1, CDR2 and CDR3 of the nano antibody is selected from any one of the following items: (a) the amino acid sequence of the CDR1 is a sequence as shown in SEQ ID NO: 2, the amino acid sequence of the CDR2 is a sequence as shown in SEQ ID NO: 3, and the amino acid sequence of the CDR3 is a sequence as shown in SEQ ID NO: 4; (b) a variant comprising at most 5 amino acid substitutions in any one or more of the CDR1 region, CDR2 region and CDR3 region as compared to the amino acid sequence in (a). The nano antibody provided by the invention can be combined with the porcine epidemic diarrhea virus S protein with high affinity, and can be used for preparing medicines for preventing or treating porcine epidemic diarrhea virus infectious diseases.

Description

A nanobody, fusion protein, and its application against porcine epidemic diarrhea virus S protein. Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a nanobody, fusion protein, and their applications that can be used to combat the S protein of porcine epidemic diarrhea virus. Background Technology

[0002] Porcine Epidemic Diarrhea Virus (PEDV) belongs to the genus Alphacoronavirus in the family Coronaviridae. It is an enveloped, single-stranded, positive-sense RNA virus. This virus causes an intestinal infectious disease in pigs, leading to severe diarrhea in infected pigs. Pigs of all ages are susceptible, but it is particularly deadly to suckling piglets, with a mortality rate as high as 80%–100%, causing enormous economic losses. PEDV is mainly transmitted through the fecal-oral route. After infection, the main symptoms are watery diarrhea, vomiting, anorexia, dehydration, and depression. Piglets often die rapidly due to severe dehydration and electrolyte imbalance. A large-scale outbreak of PEDV occurred in South China at the end of October 2010, which then rapidly spread throughout the country, resulting in the death of over 1 million piglets. Since then, the incidence rate has remained high. Currently, the main circulating strains in my country are GIIa, GIIb, and GIIc strains.

[0003] The surface of PEDV viral particles contains a spike protein, or S protein. It is the most important structural and functional protein of coronaviruses, playing a central role in viral infection and immunity. First, the S protein is responsible for recognizing and binding to specific receptors on the surface of host intestinal epithelial cells, mediating the fusion of the viral envelope with the cell membrane, thereby initiating the viral infection process. This is the first and most crucial step in the viral life cycle. Second, the S protein is the most important antigenic protein on the viral surface, capable of stimulating the host to produce neutralizing antibodies. Furthermore, variations in the receptor-binding domain (RBD) of the S protein directly affect viral virulence, cell tropism (primarily targeting intestinal cells), and the ability to evade the immune protection of existing vaccines. Due to the central role of the S protein in viral infection and immunity, it has become a target for clinical diagnostic testing and a core target for vaccine development.

[0004] Currently, the main treatment for porcine epidemic diarrhea (PEDV) caused by PEDV relies on vaccines. However, the PEDV RNA genome is prone to mutation and recombination, leading to continuous viral evolution and the emergence of strains with varying virulence and antigenicity. This can render vaccines targeting specific antigens ineffective, posing a significant challenge to vaccine prevention and control. Furthermore, it takes time for animals to develop antibodies after vaccination to resist pathogen invasion. PEDV infection can still occur before the immunization program is completed or even without immunization. Therefore, there is an urgent need to develop alternative strategies to prevent and control PEDV infection. Neutralizing antibodies produced by viral infection are highly specific, fast-acting, and provide immediate passive immune protection, possessing high preventative and therapeutic value. Additionally, with the development of genetic engineering technology, alpaca-derived nanobodies have stronger antigen targeting and binding affinity compared to traditional monoclonal antibodies, while also avoiding the drawbacks of traditional monoclonal antibodies such as large size and poor stability. In conclusion, since there are currently no antibody-related biological products for the prevention and treatment of PEDV infection, developing anti-PEDV antibody drugs more suitable for the porcine immune system has significant scientific and practical value. Summary of the Invention

[0005] In response to the clinical situation in the current pig farming industry where vaccines cannot completely prevent or treat porcine epidemic diarrhea (PED) infection in pig herds, this invention provides a nanobody, fusion protein, and its application that can be used to prevent or treat the S protein of porcine epidemic diarrhea virus.

[0006] Specifically, the technical solution to the technical problem solved by this application relates to the following aspects: In a first aspect of the present invention, a nanobody against porcine epidemic diarrhea virus S protein is provided, the nanobody comprising a heavy chain variable region, the heavy chain variable region comprising three complementarity-determining regions CDR1, CDR2 and CDR3: the amino acid sequences of the CDR1, CDR2 and CDR3 of the nanobody are selected from any one of the following: (a) the amino acid sequence of CDR1 is as shown in SEQ ID NO: 2, the amino acid sequence of CDR2 is as shown in SEQ ID NO: 3, and the amino acid sequence of CDR3 is as shown in SEQ ID NO: 4; (b) compared with the amino acid sequence in (a), any one or more of the CDR1 region, CDR2 region and CDR3 region contains a variant with up to 5 amino acid substitutions, and the variant still retains the activity of binding porcine epidemic diarrhea virus S protein.

[0007] Furthermore, the amino acid sequence of the variable region of the heavy chain of the nanobody is as shown in SEQ ID NO: 1, or has at least 85%, 88%, 90%, 92% or 95% sequence identity with SEQ ID NO: 1.

[0008] In a second aspect of the invention, a nanobody fusion protein is provided, comprising the nanobody as described in the first aspect, and an immunoglobulin Fc segment fused to the C-terminus of the nanobody.

[0009] Furthermore, the immunoglobulin Fc segment is selected from any one of human IgG Fc segment, porcine IgG Fc segment, and porcine IgA Fc segment.

[0010] In one embodiment of the present invention, the Fc segment is a human IgG Fc segment, the amino acid sequence of which is shown in SEQ ID NO: 5.

[0011] In one embodiment of the present invention, the Fc segment is a porcine IgG Fc segment, the amino acid sequence of which is shown in SEQ ID NO: 6.

[0012] In one embodiment of the present invention, the Fc segment is a porcine IgA Fc segment, the amino acid sequence of which is shown in SEQ ID NO: 7.

[0013] The fusion protein of nanobody and porcine immunoglobulin Fc fragment provided by this invention is a conventional modification of the nanobody in the field of application. The purpose includes, but is not limited to, prolonging the half-life, increasing stability, enhancing the effector functions of CDC, ADCC, etc., and simplifying preparation. Those skilled in the art can also choose other modification strategies based on the application purpose, such as fusing with human immunoglobulin Fc fragment. These modified fusion proteins still maintain the specific binding ability of the anti-porcine epidemic diarrhea virus S protein nanobody provided by this invention. Therefore, the above-mentioned fusion protein modification strategies should not be regarded as a limitation on the anti-porcine epidemic diarrhea virus S protein nanobody provided by this invention.

[0014] In a third aspect of the invention, a polynucleotide is provided that encodes a nanobody against the S protein of porcine epidemic diarrhea virus as described in the first aspect, or a nanobody fusion protein as described in the second aspect.

[0015] In a fourth aspect of the invention, a recombinant expression vector is provided, the expression vector comprising the polynucleotides as described in the third aspect. The vector is used to clone and / or express the encoding gene of the nanobody or nanobody fusion protein against the porcine epidemic diarrhea virus S protein. In one specific embodiment of the invention, the vector is pcDNA3.4.

[0016] In a fifth aspect of the invention, a host cell is provided, the host cell comprising the polynucleotide as described in the third aspect or the recombinant expression vector as described in the fourth aspect. The host cell is used to express nanobodies for obtaining the above-described anti-porcine epidemic diarrhea virus S protein or the above-described anti-porcine epidemic diarrhea virus S protein nanobodies fusion protein. In a specific embodiment of the invention, the host cell is a CHO cell.

[0017] In a sixth aspect of the invention, the use of nanobodies against the porcine epidemic diarrhea virus (PEDV) S protein as described in the first aspect or any fusion protein as described in the second aspect in the preparation of medicaments for the prevention and / or treatment of PEDV infectious diseases is provided. The nanobodies against the PEDV S protein provided by this invention exhibit excellent neutralizing ability against PEDV and can be used for the prevention / treatment of PEDV infection in piglets.

[0018] In a seventh aspect of the invention, a pharmaceutical composition is provided comprising a nanobody as described in the first aspect, or a nanobody fusion protein as described in the second aspect, and pharmaceutically acceptable excipients.

[0019] In an eighth aspect of the invention, a reagent or kit for diagnosing porcine epidemic diarrhea virus infection is provided, comprising nanobodies as described in the first aspect, or nanobodies fusion proteins as described in the second aspect.

[0020] This invention offers the following technical advantages: By immunizing alpacas with the porcine epidemic diarrhea virus (PEDV) S protein and constructing an antibody library, and then using phage display technology, nanobodies specifically binding to the PEDV S protein were successfully screened. These nanobodies possess advantages such as low immunogenicity, good solubility, and excellent stability. Surface plasmon resonance (SPR) technology confirmed that the nanobodies provided by this invention can bind to the PEDV S protein with high affinity. Furthermore, in virus neutralization experiments, they can neutralize different subtypes of PEDV with high neutralizing activity, improving the therapeutic effect against PEDV infection in piglets. The nanobodies and nanobodies fusion proteins provided by this invention offer new biological products for the clinical application of preventing and treating PEDV infection in piglets. Attached Figure Description

[0021] Figure 1 shows the purified humanized anti-porcine epidemic diarrhea virus S protein P6-04-hFc fusion protein as detected by SDS-PAGE electrophoresis. Lane R: P6-04-hFc (reducing conditions), Lane NR: P6-04-hFc (non-reducing conditions).

[0022] Figure 2 is a schematic diagram of the ELISA assay for the binding activity of the nanobody fusion protein P6-04-hFc to the antigen.

[0023] Figure 3 shows the SPR (Symptom Reactivity Test) results of the affinity between the nanobody fusion protein P6-04-hFc and the antigen.

[0024] Figure 4 shows the results of the in vitro neutralizing activity assay of the nanobody fusion protein P6-04-hFc.

[0025] Figure 5 shows the purified porcine-derived anti-porcine epidemic diarrhea virus S protein P6-04-pIgGFc and P6-04-pIgAFc fusion protein detected by SDS-PAGE electrophoresis. Figure 6 shows the survival curve of piglets after the second challenge.

[0026] Figure 7 shows the viral load in the duodenum, jejunum, ileum, and cecum of three groups of piglets on the fourth day after the first challenge (three samples were taken from each organ for statistical analysis).

[0027] Figure 8 shows the viral load in the duodenum, jejunum, ileum, and cecum of three groups of piglets on the fourth day after the second challenge (three samples were taken from each organ for statistical analysis). Detailed Implementation

[0028] The present invention will be further explained in conjunction with the following embodiments. The embodiments given are only for illustrating the present invention, but do not limit the present invention in any way.

[0029] Unless otherwise specified, the raw materials and chemical reagents used in the examples are all commercially available products, and the technical means used are conventional means known to those skilled in the art.

[0030] Unless otherwise specified, the materials, reagents, instruments and methods used in the following examples are all conventional materials, reagents, instruments and methods in the art and can be obtained through commercial channels.

[0031] The term "nanobody" in this article refers to a single-domain antibody, a unique type of naturally occurring antibody found in camels and sharks that lacks a light chain. This type of antibody contains only one variable heavy chain domain (VHH), unlike other antibodies which naturally lack a light chain.

[0032] As is known to those skilled in the art, each heavy chain variable region can consist of three complementarity-determining regions (CDR1 / 2 / 3) and four conserved framework regions (FR1 / 2 / 3 / 4). The complementarity-determining regions (CDRs, typically CDR1, CDR2, and CDR3) are the regions in the variable region that have the greatest impact on the antibody's affinity and specificity. In some embodiments, the variable region of the heavy chain, from the N-terminus to the C-terminus, includes FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. Because nanobodies do not have an Fc fragment and cannot produce cytotoxic effects such as ADCC / CDC like conventional antibodies, VHH antibodies are often fused with an Fc fragment to construct Fc-VHH fusion proteins to increase ADCC and CDC activity. Compared to ordinary antibodies, these forms of nanobodies can be widely used in the treatment of various diseases.

[0033] Example 1: Alpaca Immunization and Nanobody Library Construction. 400 μg of porcine epidemic diarrhea virus (PEDV) S protein (Genbank: ALS35469.1) was mixed with adjuvant at a 1:1 ratio and administered subcutaneously at multiple sites near the lymph nodes on both sides of the alpaca's neck. Immunization was performed every two weeks for a total of two immunizations. One week after each immunization, 5 mL of alpaca blood was collected, and the supernatant serum was used for ELISA antibody titer determination. One week after the second immunization, approximately 50-60 mL of alpaca blood was collected, and PBMCs (peripheral blood mononuclear cells) were isolated, and total RNA was extracted. The total RNA was reverse-engineered into cDNA. Using the cDNA as a template, a first nested PCR amplification was performed using specific primers. The approximately 700 bp band was excised and purified using a gel, and then used as a template for a second round of nested PCR amplification to obtain a VHHs gene fragment of approximately 400 bp. The recovered and purified VHHs nanobody fragments were ligated into the pComb3XSS vector, electroporated into E. coli TG1, and after incubation at 37°C for 60 min, plated onto LB agar plates containing ampicillin for overnight growth, thus completing the construction of the antibody library. The library size was determined to be 3.12 × 10⁻⁶. 9 PFU / mL.

[0034] Example 2: Screening for Specific Nanobodies Using Phage Display Technology. The bacterial culture of the anti-porcine epidemic diarrhea virus S protein nanobody library prepared in Example 1 was cultured at 37°C until the OD600nm reached 0.5. Then, helper phage M13K07 was added at a ratio of approximately 20 for 30 min of further culture. After overnight culture, the bacterial culture was centrifuged at 4°C. The supernatant was transferred to a new centrifuge tube, and 5×PEG8000 / NaCl was added. After incubation on ice for 30 min, the tube was centrifuged at 4°C for 10 min, the supernatant was discarded, and the bacterial cell pellet was obtained. The pellet was resuspended in PBS to obtain the phage nanobody display library.

[0035] Using a solid-phase panning method, the above-mentioned phage nanobody display library was panned three times using porcine epidemic diarrhea (PED) S protein as the antigen. 30 μg, 20 μg, and 10 μg of PED S protein (different antigen concentrations in three rounds of panning) were added to 2 mL of PBS and incubated overnight at 4°C to coat the immunotubes. The coating solution was discarded the next day, and then 5% BSA, 1% casein, or 2% protein-free blocking solution was added as blocking solution (different blocking solutions in three rounds of panning), and the tubes were blocked at 37°C for 2 h. The blocking solution was discarded, and the tubes were washed three times with 0.1% PBST, followed by three washes with PBS. The phage nanobody display library was diluted to an appropriate concentration with the blocking solution, added to the immunotubes, and incubated at room temperature for 2 h, followed by rotation incubation for 20 min. The tubes were washed 5-11 times with 0.1% PBST, followed by two washes with PBS. The phages were then eluted with 0.2 M Glycine-HCl (pH 2.1) and neutralized with Tris-HCl (pH 9.1). The phage library obtained after three rounds of elution and panning was identified by ELISA using porcine epidemic diarrhea S protein as the antigen, confirming the enrichment of phages expressing specific nanobodies.

[0036] Specific nanobody positive clones were identified using a phage-linked immunosorbent assay (Phage-ELISA). The phages selected from the three rounds of panning were cultured again and plated. 176 single clones were randomly selected each time for ELISA verification. BSA-coated clones served as a control group. OD450 > 0.15 was considered positive, and OD450 < 0.1 was considered negative. Positive clones were sequenced to obtain the P6-04 nanobody sequence.

[0037] The amino acid sequence of the variable region of the heavy chain of nanobody P6-04 is shown in SEQ ID NO: 1, which contains three complementarity-determining regions CDR1, CDR2 and CDR3: the amino acid sequence of CDR1 is shown in SEQ ID NO: 2, the amino acid sequence of CDR2 is shown in SEQ ID NO: 3 and the amino acid sequence of CDR3 is shown in SEQ ID NO: 4.

[0038] Example 3: Expression and Purification of Nanobodies The nanobodies P6-04 obtained in Example 2 were cloned into the expression vector pcDNA3.4 with hFc (hFc amino acid sequence number as shown in SEQ ID NO: 5) to obtain two recombinant plasmids. These two recombinant plasmids were electroporated into CHO cells. Approximately 5 mL of electroporation buffer was added to the cells, mixed well, and then an appropriate amount of plasmid (concentration above 500 ng / μL) was added. After thorough mixing of the cell-plasmid suspension, 10 mL was added to a 10 mL electroporation tube and electroporated. The electroporated cells were then aliquoted into shake flasks containing 100 mL of culture medium and incubated statically for 40 min. After incubation, the shake flasks were placed in an incubator at 37°C, 110 rpm, and 8% CO2 for 24 h. Sodium butyrate was added as feed, and the cells were cultured for another 3-7 days. The supernatant was collected, centrifuged at 5000 rpm for 30 min, and then filtered through a 0.22 μm filter to remove cell debris.

[0039] The target protein in the collected cell supernatant was purified using a Protein A affinity chromatography column. The column was equilibrated with 20 mL of 1×PBS at a flow rate of 1 mL / min. The cell supernatant was loaded at the same flow rate of 1 mL / min and washed with 20 mL of 1×PBS to remove impurities. Elution was then performed with 50 mM sodium acetate at pH 3.8. Approximately 1000 μL of the eluted target protein was collected in aliquots. The high-concentration protein was then transferred to a dialysis bag and dialyzed in a beaker containing 1×PBS.

[0040] The target protein was identified by SDS-PAGE, as shown in Figure 1, and a relatively pure nanobody fusion protein P6-04-hFc was obtained.

[0041] Example 4: Determination of the binding activity characteristics of the nanobody fusion protein P6-04-hFc prepared in Example 3. The binding activity of the nanobody to the antigen was detected using ELISA. Porcine epidemic diarrhea virus S protein was diluted to 1 μg / mL and coated onto a 96-well ELISA plate (100 μL per well) overnight at 4°C. The coating solution was discarded, and the plate was washed 6 times with PBST (0.1% Tween-20). 100 µL of blocking buffer was added to each well, and the plate was incubated at 37°C for 1 h. The blocking buffer was discarded, and the plate was washed 6 times with PBST. 100 μL of nanobody fusion protein P6-04-hFc, serially diluted 4-fold starting from 100 nM, was added to each well, and the plate was incubated at 37°C for 1 h. The primary antibody was discarded, and the plate was washed 5 times with PBST. 100 μL of Goat Anti-Human IgG-Fc, HRP diluted 1:10000 was used as the secondary antibody, and the plate was incubated at 37°C for 1 h. Discard the secondary antibody, wash the plate 5 times with PBST, add 100 µl of TMB single-component chromogenic solution to each well, and incubate at room temperature in the dark for 15 min. Finally, add 50 µl of stop solution to each well to terminate the reaction, and measure the OD450 value using a microplate reader. P4B-1 antibody was used as a positive control, and Anti-HEL human IgG1 was used as a negative control. The binding ability was evaluated by calculating the concentration (EC50) required for the antibody to bind 50% of the antigen protein. The results are shown in Figure 2; the half-maximal effective concentration (EC50) for binding the nanobody fusion protein P6-04-hFc was 0.1455 nM.

[0042] Example 5: Determination of the affinity of the nanobody fusion protein P6-04-hFc prepared in Example 3. The affinity constant of P6-04-hFc was determined using a Protein A chip via SPR analysis on a Biacore 8K protein interaction analyzer. HBS-EP+ buffer was used as the running buffer. The nanobody fusion protein P6-04-hFc was diluted to 1 μg / mL and loaded onto the Protein A chip at a flow rate of 10 μL / min for 120 s. Porcine epidemic diarrhea virus S protein was serially diluted 2-fold from 200 nM to 3.125 nM and loaded onto the Protein A chip containing the captured antibody at a flow rate of 30 μL / min, binding for 120 s, dissociating for 1000 s, in 1:1 binding mode. Finally, the affinity value (KD) of the antibody was calculated using the instrument's analysis software. The KD value of the nanobody fusion protein P6-04-hFc was 1.97 × 10⁻⁶. -10 M, the results are shown in Figure 3, indicating that there is good affinity between the antigen and the antibody.

[0043] Example 6: Determination of the neutralizing activity of the nanobody fusion protein P6-04-hFc prepared in Example 3. The neutralizing level of the nanobody fusion protein P6-04-hFc was determined by plaque reduction neutralization assay (PRNT). One to two days before the experiment, Vero cells in logarithmic growth phase were seeded at an appropriate density into 12-well cell culture plates. The cell culture plates were incubated at 37°C in a 5% CO2 incubator until the cells grew into a dense, uniform monolayer with approximately 90%-100% confluence. Porcine epidemic diarrhea virus was diluted to a viral load of 200 PFU / mL. Simultaneously, the nanobody fusion protein P6-04-hFc was serially diluted 1:4 to 1:128. Equal volumes of the diluted virus solution and antibody were added to a sterile test tube, mixed thoroughly, and incubated at 37°C in a 5% CO2 incubator for 60-90 min. The seeded cell culture plates were washed three times with PBS to remove residual serum and cell metabolites. After the virus-antibody mixture has incubated, add 500 μL to each well of a cell culture plate. Set up a virus control (virus only) and a blank control (no virus or antibody). Place the cell culture plate in a 37°C, 5% CO2 incubator for 60-90 min. During this time, gently shake the plate every 15-20 min to ensure even distribution of the liquid and that the virus-antibody mixture fully contacts all cells. After adsorption, mix 2% low-melting-point agarose with an equal volume of 2×DMEM and apply 1 mL to each well, covering the cells. Allow the mixture to solidify completely before returning the plate to the incubator for further culture. Three to five days later, when clearly visible plaques appeared in the virus control wells, fixation and staining were performed. The number of plaques in each well was observed and recorded, and the plaque reduction rate was statistically analyzed. The results are shown in Figure 4. The IC50 (half-maximum inhibitory concentration) was calculated and plotted. The IC50 value of the nanobody fusion protein P6-04-hFc was 0.02941 mg / ml, indicating that it has a good neutralizing effect on porcine epidemic diarrhea virus.

[0044] Example 7: Porcine Derivative Modification of Nanobodies. The Fc segment in the nanobody fusion protein P6-04-hFc prepared in Example 3 was replaced with porcine IgG Fc or porcine IgAFc. pIgGFc or pIgAFc was ligated after the P6-04 coding sequence of this application and cloned into the pcDNA3.4 expression vector to obtain two recombinant plasmids. The remaining specific steps are the same as in Example 3. The obtained proteins were identified by SDS-PAGE, as shown in Figure 5, yielding relatively pure nanobody fusion proteins P6-04-pIgGFc and P6-04-pIgAFc. Figure 5A shows the SDS-PAGE electrophoresis detection of P6-04-pIgGFc. Lane R: P6-04 pIgGFc (reducing conditions), Lane NR: P6-04-pIgGFc (non-reducing conditions). Figure 5B shows the SDS-PAGE electrophoresis detection of P6-04-pIgAFc. Lane R: P6-04-pIgAFc (reduction condition), Lane NR: P6-04-pIgAFc (non-reduction condition).

[0045] In the fusion protein P6-04-pIgGFc, the C-terminus of the nanobody P6-04 is fused with the amino acid sequence of the porcine immunoglobulin IgG Fc segment as shown in SEQ ID NO: 6.

[0046] In the fusion protein P6-04-pIgAFc, the C-terminus of the nanobody P6-04 is fused with the amino acid sequence of the porcine immunoglobulin IgA Fc segment as shown in SEQ ID NO: 7.

[0047] Example 8 Evaluation of the protective effect of nanoantibodies against PEDV infection in piglets Nine 3-day-old piglets were selected and divided into 3 groups.

[0048] Group 1, 3 piglets, were given the nano-antibody fusion protein P6-04-pIgAFc prepared in Example 7; Group 2, 3 piglets, were given the nano-antibody fusion protein P6-04-pIgGFc prepared in Example 7; Group 3, 3 piglets, were given PBS; these served as the control group.

[0049] Administration: After 2 days of acclimatization, three piglets in group 1 were orally administered 50 mg of the nanobody fusion protein P6-04-pIgAFc, three piglets in group 2 were orally administered 50 mg of the nanobody fusion protein P6-04-pIgGFc, and three piglets in group 3 were orally administered the same volume of PBS. Two hours later, nine piglets were orally administered 2 × 10⁻⁶ PEDV GⅡb strain. 4 PFU / mL.

[0050] After challenge, piglets' body weight and temperature were monitored daily. Group 1 (3 piglets) received 25 mg of the nanobody fusion protein P6-04-pIgAFc orally daily, Group 2 (3 piglets) received 25 mg of the nanobody fusion protein P6-04-pIgGFc orally daily, and the control group received the same volume of PBS orally daily. On day 4, one piglet from each of the three groups was randomly selected for dissection, and intestinal samples were collected for viral load analysis.

[0051] Meanwhile, on day 4, the remaining two piglets in group 1 were orally administered 50 mg of the nanobody fusion protein P6-04-pIgAFc, and the remaining two piglets in group 2 were orally administered 50 mg of the nanobody fusion protein P6-04-pIgGFc. The remaining two piglets in the control group were given the same volume of PBS. Two hours later, the remaining two piglets in each of the three groups were again orally challenged with 2 × 10⁻⁶ PEDV GⅡb strain. 4 PFU / mL.

[0052] After the second challenge, the piglets' body weight and temperature were monitored daily. The remaining two piglets in group 1 were given 25 mg of the nanobody fusion protein P6-04-pIgAFc orally daily, and the remaining two piglets in group 2 were given 25 mg of the nanobody fusion protein P6-04-pIgGFc orally daily. The two piglets in the control group were given the same volume of PBS orally daily. After 4 days, all the piglets in the control group died. All piglets in the control group and the treated group were necropsyed, and their intestines were collected for viral load analysis.

[0053] The evaluation results of the therapeutic protection of nanobodies against PEDV infection in piglets are shown in Figures 6, 7, and 8. P6-04-pIgGFc and P6-04-pIgAFc represent the two groups treated with nanobodies, while the Challenged control group is the positive control group treated with PBS. Figure 6 shows the survival curves of piglets after the second challenge; Figure 7 shows the viral load in the duodenum, jejunum, ileum, and cecum of the three groups of piglets on day 4 after the first challenge; Figure 8 shows the viral load in the duodenum, jejunum, ileum, and cecum of the three groups of piglets on day 4 after the second challenge. The results show that the viral load in the duodenum, jejunum, ileum, and colon of the two groups of piglets treated with the nanobodies was lower than that of the group not treated with the nanobodies, indicating that the anti-porcine epidemic diarrhea virus S protein nanobodies prepared in Example 7 of this invention has good preventive and therapeutic effects against diseases caused by porcine epidemic diarrhea virus infection.

[0054] The above are merely embodiments of the present invention and do not limit the scope of the patent. Any equivalent modifications made based on the content of this specification, or direct or indirect applications in other related technical fields, are similarly included within the scope of patent protection of the present invention.

Claims

1. A nanobody against the S protein of porcine epidemic diarrhea virus, characterized in that, The nanobody comprises a heavy chain variable region, which includes three complementarity-determining regions CDR1, CDR2, and CDR3: the amino acid sequences of the CDR1, CDR2, and CDR3 of the nanobody are selected from any one of the following: (a) the amino acid sequence of CDR1 is as shown in SEQ ID NO: 2, the amino acid sequence of CDR2 is as shown in SEQ ID NO: 3, and the amino acid sequence of CDR3 is as shown in SEQ ID NO: 4; (b) compared with the amino acid sequence in (a), any one or more of the CDR1, CDR2, and CDR3 regions contain a variant with up to 5 amino acid substitutions, and the variant still retains the activity of binding porcine epidemic diarrhea virus S protein.

2. The nanobody according to claim 1, characterized in that, The amino acid sequence of the variable region of the heavy chain of the nanobody is as shown in SEQ ID NO: 1, or has at least 85%, 88%, 90%, 92% or 95% sequence identity with SEQ ID NO:

1.

3. A nanobody fusion protein, characterized in that, It comprises the nanobody as described in claim 1 or 2, and an immunoglobulin Fc segment fused to the C-terminus of the nanobody.

4. The nanobody fusion protein according to claim 3, characterized in that, The immunoglobulin Fc segment is selected from any one of human IgG Fc segment, porcine IgG Fc segment, and porcine IgA Fc segment.

5. The nanobody fusion protein according to claim 4, characterized in that, The immunoglobulin Fc segment is selected from any one of the following: 1) human IgG Fc segment, the amino acid sequence of which is shown in SEQ ID NO: 5; 2) porcine IgG Fc segment, the amino acid sequence of which is shown in SEQ ID NO: 6; 3) porcine IgA Fc segment, the amino acid sequence of which is shown in SEQ ID NO:

7.

6. A polynucleotide, characterized in that, The polynucleotide encoding the nanobody against the S protein of porcine epidemic diarrhea virus as described in claim 1 or 2, or the nanobody fusion protein as described in any one of claims 3-5.

7. A recombinant expression vector, characterized in that, The expression vector comprises the polynucleotide as described in claim 6.

8. A host cell, characterized in that, The host cell contains the polynucleotide as described in claim 6 or the recombinant expression vector as described in claim 7.

9. The use of the nanobody against the porcine epidemic diarrhea virus S protein as described in claim 1 or 2, or the fusion protein as described in any one of claims 3-5, in the preparation of drugs for the prevention and / or treatment of porcine epidemic diarrhea virus infectious diseases.

10. A pharmaceutical composition, characterized in that, It includes the nanobody as described in claim 1 or 2, or the nanobody fusion protein as described in any one of claims 3-5, and pharmaceutically acceptable excipients.

11. A reagent or kit for diagnosing porcine epidemic diarrhea virus infection, characterized in that, It comprises the nanobody as described in claim 1 or 2, or the nanobody fusion protein as described in any one of claims 3-5.

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