Shark source VNAR nano antibody aiming at porcine deltacoronavirus S protein and application of shark source VNAR nano antibody

Abstract

The invention relates to the technical field of veterinary biological products, and discloses a shark-derived VNAR nano antibody aiming at porcine deltacoronavirus S protein and application thereof. The VNAR nano antibody specifically recognizes a conformation dependent epitope of a PDCoV spike protein receptor binding domain, the molecular weight is 12-15kDa, the value is M, the neutralization value is 2.3 nM, the pH is 2.0-8.0, the VNAR nano antibody is stable, the activity retention is greater than 80% after the VNAR nano antibody is treated at 80 DEG C for 30 minutes, and the VNAR nano antibody has the advantages of high specificity, high sensitivity, high sensitivity and the like. The kit can be used for PDCoV diagnosis detection and treatment prevention.

Description

Technical Field

[0001] This invention relates to the field of veterinary biological products technology, specifically to a shark-derived variable neoantigen receptor nanobody that specifically recognizes the receptor-binding domain of the spike protein of porcine deltacoronavirus and its application in the diagnosis, detection, treatment, and prevention of porcine deltacoronavirus. Background Technology

[0002] Porcine deltacoronavirus (PDCoV) belongs to the genus Deltacoronavirus in the family Coronaviridae. It is an enveloped, single-stranded, positive-sense RNA virus. PDCoV primarily infects piglets, causing intestinal symptoms such as acute diarrhea, vomiting, and dehydration. The mortality rate of infected piglets can be as high as 40% or more, resulting in severe economic losses to the pig farming industry.

[0003] The PDCoV genome is approximately 25.4 kb in length and encodes four major structural proteins: the spike protein (S protein), membrane protein (M protein), envelope protein (E protein), and nucleocapsid protein (N protein). Among these, the S protein is the most abundant glycoprotein on the viral surface and plays a crucial role in viral invasion of host cells. The S protein can be divided into two subunits, S1 and S2. The S1 subunit is responsible for binding to host cell receptors. Its C-terminal domain (S1-CTD) contains the receptor-binding domain (RBD), a key functional region for viral infection and an important target for neutralizing antibodies.

[0004] CN113336844A discloses a shark single-domain antibody targeting the N protein of SARS-CoV-2. A phage display library was constructed by immunizing striped bamboo sharks, and VNAR antibodies targeting the N protein were obtained through screening. However, this patent targets the N protein of SARS-CoV-2, which differs significantly from the S protein of PDCoV in sequence and structure. Furthermore, the N protein is primarily used for diagnostic detection and is unlikely to achieve virus neutralization. Currently, shark-derived VNAR nanobodies targeting the RBD region of the PDCoV S protein have not been reported.

[0005] Existing antibodies against PDCoV are mainly traditional monoclonal antibodies, which have the following technical problems: large molecular weight, about 150kDa, poor tissue penetration, and difficulty in reaching the intestinal infection site; high production cost, requiring glycosylation modification by mammalian cell expression system; limited stability, sensitive to environmental conditions such as high temperature and extreme pH, which is not conducive to the storage and transportation of veterinary preparations; and limited epitope recognition type, mainly recognizing linear epitopes, making it difficult to bind to hidden conformation-dependent epitopes.

[0006] Therefore, there is an urgent need to develop a novel antibody with small molecular weight, high stability, low production cost, and the ability to recognize conformation-dependent epitopes for efficient diagnosis, detection, and treatment of PDCoV. Summary of the Invention

[0007] The purpose of this invention is to address the problems of large molecular weight, poor stability, high production cost, and difficulty in recognizing conformation-dependent epitopes in existing PDCoV antibodies, and to provide a shark-derived VNAR nanobody targeting the porcine deltacoronavirus S protein, as well as the application of this antibody in the diagnosis, detection, treatment, and prevention of PDCoV.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] A first aspect of the present invention provides a shark-derived VNAR nanobody targeting the porcine deltacoronavirus S protein, wherein the VNAR nanobody specifically recognizes a conformation-dependent epitope of the receptor-binding domain of the PDCoV spike protein, and the VNAR nanobody comprises complementarity-determining regions CDR1 and CDR3, wherein the sequence of the CDR3 region is shown in SEQ ID NO:1 and the sequence of the CDR1 region is shown in SEQ ID NO:2.

[0010] Furthermore, the VNAR nanobody also includes framework regions FR1, FR2 and FR3, and the complete amino acid sequence of the VNAR nanobody is shown in SEQ ID NO:3.

[0011] Furthermore, the VNAR nanobody has a molecular weight of 12-15 kDa, preferably 13.2 kDa.

[0012] Furthermore, the dissociation constant of the VNAR nanobody with the PDCoV S protein RBD... Value is M to M, preferably M.

[0013] Furthermore, the VNAR nanobody exhibits neutralizing activity against PDCoV. The value is 0.5-10 nM, preferably 2.3 nM, with a neutralization efficiency of over 95%.

[0014] Furthermore, the VNAR nanobody remains stable in the pH range of 2.0-8.0, with an activity retention rate of greater than 85%.

[0015] Furthermore, the VNAR nanobody retains more than 80% of its activity after being treated at 80°C for 30 minutes.

[0016] Furthermore, the VNAR nanobody maintains cross-binding activity against PDCoV mutants, with a binding activity retention rate of more than 70% compared to the wild type.

[0017] A second aspect of the present invention provides a nucleic acid molecule encoding the VNAR nanobody, the nucleotide sequence of which is shown in SEQ ID NO:4.

[0018] A third aspect of the invention provides a recombinant expression vector comprising the nucleic acid molecule.

[0019] Furthermore, the recombinant expression vector is pET-30a, pET-28a, or pComb3XSS vector.

[0020] A fourth aspect of the invention provides a recombinant host cell comprising the recombinant expression vector.

[0021] Furthermore, the recombinant host cell is either Escherichia coli BL21(DE3) strain or Escherichia coli Rosetta(DE3) strain.

[0022] A fifth aspect of the present invention provides a method for preparing the VNAR nanobody, comprising the following steps:

[0023] Step 1: Immunize striped bamboo sharks with recombinant PDCoV S protein RBD at a dose of 50-200 μg / shark, with an immunization interval of 10-21 days, and 4-8 immunizations, obtaining immune serum with a titer greater than [missing value]. Immune sharks.

[0024] Step 2: Isolate immune shark peripheral blood lymphocytes, extract total RNA, reverse transcribe to obtain cDNA, and amplify the VNAR gene fragment by nested PCR using cDNA as a template.

[0025] Step 3: Ligate the VNAR gene fragment to the phage display vector, transform competent cells, and construct a library with a capacity greater than [missing information]. CFU VNAR phage display library.

[0026] Step 4: Using the recombinant PDCoV S protein RBD as the target, perform 3-5 rounds of affinity panning on the phage library to screen for positive phage clones that specifically bind to the S protein RBD.

[0027] Step 5: Perform Phage-ELISA verification and sequencing analysis on positive clones to obtain the VNAR gene sequence.

[0028] Step 6: The VNAR gene is cloned into a prokaryotic expression vector, transformed into the expression strain, and then purified by IPTG-induced expression and affinity chromatography to obtain VNAR nanobodies with a purity greater than 95%.

[0029] A sixth aspect of the present invention provides the application of the VNAR nanobody in the preparation of PDCoV diagnostic reagents.

[0030] Furthermore, the diagnostic reagent is an ELISA test kit, an immunochromatographic test strip, or an immunofluorescence test reagent.

[0031] Furthermore, the ELISA detection kit uses the VNAR nanobody as the detection antibody or a competitive antibody, with a detection limit of 0.01-0.1 ng / mL and a linear range of 0.1-100 ng / mL.

[0032] A seventh aspect of the invention provides the use of the VNAR nanobody in the preparation of pharmaceutical compositions for the prevention or treatment of PDCoV infection.

[0033] Furthermore, the dosage form of the pharmaceutical composition is an injection, an oral preparation, or a spray.

[0034] Furthermore, the pharmaceutical composition also comprises a pharmaceutically acceptable carrier and adjuvant.

[0035] The shark-derived VNAR nanobody targeting the porcine deltacoronavirus S protein provided by this invention has the following beneficial effects:

[0036] The VNAR nanobody of this invention has a molecular weight of only 12-15 kDa, which is about 1 / 10 of that of traditional IgG antibodies. It has excellent tissue penetration and can effectively reach the intestinal infection site to exert a neutralizing effect.

[0037] The VNAR nanobody of this invention has a unique convex ring structure in its CDR3 region, which can penetrate deep into the recessed area on the surface of the S protein RBD, recognize conformation-dependent epitopes that are difficult for traditional antibodies to access, and improve the neutralization efficiency and coverage of the antibody.

[0038] The affinity of the VNAR nanobody of this invention for S protein RBD Value reached M, neutralizing PDCoV The value is 2.3 nM, and the neutralization efficiency is over 95%, which is significantly better than existing traditional monoclonal antibodies.

[0039] The VNAR nanobody of this invention remains stable in a wide pH range of 2.0-8.0, and its activity retention rate is still greater than 80% after high temperature treatment at 80°C for 30 min, making it suitable for the storage and transportation of veterinary preparations under various environmental conditions.

[0040] The VNAR nanobody of this invention can exert its activity without glycosylation modification, and can be efficiently prepared by the Escherichia coli prokaryotic expression system, with an expression level of 50-100 mg / L. The production cost is only 1 / 10-1 / 5 of that of the mammalian cell expression system.

[0041] The VNAR nanobody of this invention maintains good cross-binding activity against PDCoV mutants, with a binding activity retention rate of more than 70%, and has broad-spectrum protective potential. Attached Figure Description

[0042] Figure 1 This is a graph showing the enrichment rate changes of the VNAR phage display library after three rounds of panning in Example 1 of the present invention.

[0043] Figure 2 This is a diagram showing the SDS-PAGE gel electrophoresis analysis results of the VNAR nanobody in Example 2 of this invention.

[0044] Figure 3 This is an ELISA detection curve of the binding of VNAR nanobody to PDCoV S protein RBD in Example 3 of the present invention.

[0045] Figure 4 This is a curve showing the neutralizing activity of the VNAR nanobody against the PDCoV pseudovirus in Example 4 of this invention.

[0046] Figure 5 This is a graph showing the thermal stability analysis results of the VNAR nanobody in Example 5 of the present invention.

[0047] Figure 6 This is a graph showing the pH stability analysis results of the VNAR nanobody in Example 6 of the present invention. Detailed Implementation

[0048] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto. Unless otherwise specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments used, unless otherwise specified by the manufacturer, are all commercially available conventional products.

[0049] Example 1: Construction of VNAR phage display library

[0050] This embodiment constructs a shark-derived VNAR phage display library targeting the PDCoV S protein RBD.

[0051] Preparation of recombinant PDCoV S protein RBD: Based on the S protein sequence of PDCoV strain CHN-AH-2004 in GenBank accession number KX022756, a gene fragment encoding the S1-CTD region (amino acids 294-455) was designed and synthesized, cloned into the pET-30a vector, and transformed into Escherichia coli strain BL21(DE3). Positive clones were picked and inoculated into LB medium (containing 50 μg / mL kanamycin) and cultured at 37℃ and 200 rpm until... When the concentration reached 0.6-0.8, IPTG was added to a final concentration of 0.5 mM, and expression was induced at 25°C for 16 h. The bacterial cells were collected, sonicated, and then... The target protein was purified using an NTA affinity chromatography column, and the purity was greater than 90% as determined by SDS-PAGE. The protein concentration was determined by the BCA method, and the protein was aliquoted and stored at -80℃ for later use.

[0052] Immunization of striped bamboo sharks: Six healthy striped bamboo sharks, weighing approximately 2-3 kg, were selected and immunized after two weeks of acclimatization. The initial immunization consisted of 100 μg of recombinant PDCoV S protein RBD emulsified with an equal volume of complete Freund's adjuvant and injected subcutaneously at multiple sites. A booster immunization was administered 14 days later, with a dose of 50 μg of protein emulsified using incomplete Freund's adjuvant. A total of six immunizations were given. Fourteen days after the last immunization, 1 mL of tail vein blood was collected to determine serum antibody titers. Sharks with titers greater than [missing value] were selected. Two sharks were taken, and 10 mL of blood was collected from their tail veins for subsequent experiments.

[0053] Isolation of peripheral blood lymphocytes: The collected shark peripheral blood was diluted with an equal volume of PBS and carefully stacked on the surface of the lymphocyte separation solution. It was then centrifuged at 300×g for 30 min. The grayish-white lymphocyte layer was collected, washed three times with PBS, and centrifuged at 350×g for 10 min each time to obtain purified lymphocytes.

[0054] Total RNA extraction and cDNA synthesis: Total RNA was extracted from lymphocytes using TRIzol reagent. The procedure is as follows: [The text abruptly ends here, likely due to an incomplete sentence or missing information.] Add 1 mL of TRIzol reagent to each lymphocyte, lyse thoroughly, then add 200 μL of chloroform, vortex vigorously for 15 s, incubate at room temperature for 5 min, and centrifuge at 12000 × g, 4 °C for 15 min. Transfer the supernatant to a new centrifuge tube, add an equal volume of isopropanol, incubate at room temperature for 10 min, and centrifuge at 12000 × g, 4 °C for 10 min. Discard the supernatant, wash the RNA precipitate with 1 mL of 75% ethanol, and centrifuge at 7500 × g, 4 °C for 5 min. Discard the supernatant, air dry at room temperature for 5 min, and dissolve in 30 μL of DEPC water. Determine the RNA concentration using a NanoDrop spectrophotometer. The ratio was between 1.8 and 2.0, and RNA integrity was detected by 1% agarose gel electrophoresis.

[0055] Reverse transcription was performed using the PrimeScript RT reagent Kit. The reaction mixture consisted of: 4 μL of 5×PrimeScript Buffer, 1 μL of PrimeScript RT Enzyme Mix, 1 μL of Oligo dT Primer, 1 μL of Random 6 mers, 5 μg of Total RNA, and DEPC water to a final volume of 20 μL. The reaction program was: 37℃ for 15 min, 85℃ for 5 s, and storage at 4℃ to obtain the first strand of cDNA.

[0056] Amplification of the VNAR gene: Nested PCR was used to amplify the VNAR gene. The primers for the first round of PCR were: F1-VNAR: 5'-ATGAATATTTTCTTGTTTTCGGGCC-3', R1-VNAR: 5'-ATAGTATCCGCTAATTAGACAAA-3'. The reaction mixture consisted of: 25 μL of 2×Phusion High-Fidelity PCR Master Mix, 1 μL of F1-VNAR (10 μM), 1 μL of R1-VNAR (10 μM), 2 μL of cDNA, and water to a final volume of 50 μL. The reaction program was: 98℃ for 3 min; 98℃ for 10 s, 55℃ for 30 s, 72℃ for 30 s, for a total of 35 cycles; 72℃ for 5 min.

[0057] The primers for the second round of PCR were: F2-VNAR: 5'-CGTGGCCCAGGCGGCCGGGCCCCCTGGTTACCAAATGT-3', R2-VNAR: 5'-CGTGGCCCAGGCGGCCGGGCCCTTTGCCAGGTTTCACAGTCAG-3'. Both primers contained Sfi I restriction sites at both ends. Using the first round of PCR products as templates, the reaction system was the same as above. The reaction program was: 98℃ for 3 min; 98℃ for 10 s, 62℃ for 30 s, 72℃ for 30 s, for a total of 30 cycles; 72℃ for 5 min. The products were detected by 2% agarose gel electrophoresis, and the target band of approximately 360-400 bp was recovered by gel excision.

[0058] Construction of the phage display library: The VNAR gene fragment and pComb3XSS vector were double-digested with Sfi I restriction enzyme. The digestion system was: 5 μg DNA, 20 U Sfi I, 5 μL CutSmart Buffer, and water to a final volume of 50 μL. The digestion was incubated overnight at 50°C. After purification by agarose gel electrophoresis, the digested products were ligated using T4 DNA ligase. The ligation system was: 100 ng vector, 300 ng VNAR fragment, 2 μL T4 DNA Ligase, 2 μL 10×T4 Ligase Buffer, and water to a final volume of 20 μL. Ligation was incubated overnight at 16°C.

[0059] After purification of the ligation product, XL1-Blue competent cells were electroporated at 1800V for 5ms. Immediately after electroporation, 1mL of SOCG medium was added, and the cells were incubated at 37℃ and 180rpm for 60min. 100μL of the bacterial culture was then serially diluted (…). to The sample was plated on LB agar plates containing 100 μg / mL ampicillin and incubated overnight at 37°C. The library size was then calculated. The VNAR phage display library obtained in this example had a library size of [missing information]. CFU meets the requirements for subsequent screening.

[0060] Affinity selection: Dilute the recombinant PDCoV S protein RBD to 50 μg / mL, coat 100 μL per well of a 96-well microplate, and incubate overnight at 4°C. The next day, discard the coating solution, wash three times with PBS, add 2% BSA-PBS for blocking, and incubate at 37°C for 2 hours. Discard the blocking solution and add... 100 μL of phage library supernatant (pfu / mL) was incubated at 37°C for 2 h. Unbound phages were discarded, and the sample was washed 10 times with PBST (PBS containing 0.05% Tween-20) and 3 times with PBS. 100 μL of glycine-hydrochloric acid elution buffer (0.1 M, pH 2.2) was added, and the sample was eluted at room temperature for 10 min. The eluted phages were immediately neutralized with an equal volume of 1 M Tris-HCl (pH 8.0). The eluted phages were added to XL1-Blue bacterial culture in logarithmic growth phase, and the culture was incubated at 37°C for 30 min. After plating and culture, the bacterial cells were collected and amplified with helper phage VCSM13 for the next round of panning.

[0061] Three rounds of affinity panning were performed, and the phage output titer was calculated after each round. The results showed that the phage enrichment folds in the first to third rounds were 12-fold, 85-fold, and 320-fold, respectively. Figure 1 This indicates that phages that specifically recognize the PDCoV S protein RBD were effectively enriched.

[0062] Example 2: Screening, Expression, and Purification of VNAR Nanobodies

[0063] In this embodiment, high-affinity VNAR nanobodies were screened from a phage display library and then expressed and purified in prokaryotes.

[0064] Screening of positive clones: 96 single clones were randomly selected from the third round of panning plates and inoculated into 96-well plates with 400 μL of 2×YT medium (containing 100 μg / mL ampicillin and 50 μg / mL kanamycin) per well, and incubated overnight at 37°C and 200 rpm. The next day, 400 μL of 2×YT medium containing VCSM13 helper phage was added to each well of a new 96-well plate, and 40 μL of bacterial culture was transferred from each well of the overnight incubation plate. After incubation at 37°C and 150 rpm for 2 h, the plates were centrifuged at 4000 rpm for 20 min, the supernatant was discarded, and the cells were resuspended in 400 μL of 2×YT medium. The plates were then incubated overnight at 37°C and 250 rpm with shaking. The next day, the plates were centrifuged at 4000 rpm for 20 min, and the supernatant was used for Phage-ELISA detection.

[0065] Phage-ELISA assay: Recombinant PDCoV S protein RBD was diluted to 2 μg / mL, and 100 μL was coated per well of a 96-well ELISA plate. The plate was incubated overnight at 4°C. After washing three times with PBS, the plate was blocked with 2% BSA-PBS for 1 h. 100 μL of phage supernatant was added, and the plate was incubated at 37°C for 1.5 h. After washing five times with PBST, HRP-labeled anti-M13 antibody (1:5000 dilution) was added, and the plate was incubated at 37°C for 1 h. After washing five times with PBST, 100 μL of TMB chromogenic buffer was added, and the plate was incubated at room temperature in the dark for 15 min. Finally, 50 μL of 2M... Terminate the reaction and read the results using the ELISA reader. value.

[0066] by A positive criterion was a value greater than 1.0 and more than three times that of the negative control; a total of 23 positive clones were obtained through screening. Sequencing analysis of the positive clones revealed 8 unique VNAR sequences after removing repetitive sequences. Further validation using competitive ELISA and Western blot determined that one VNAR clone with the highest affinity and neutralizing activity was finally identified and named VNAR-PS1.

[0067] Sequence analysis of VNAR-PS1: Sequencing results showed that the CDR3 region sequence of VNAR-PS1 is CRGSGHDYYWGCGVGPLSSV (SEQ ID NO:1), the CDR1 region sequence is CTYPGVS (SEQ ID NO:2), and the complete amino acid sequence is ARVDQTPRTVTKETGESLTINCVLRDGSFYLCNTYPGVSNWYRFKSGATNKESINGGRYVETLNSKASSFSLLISDLRVADSAVYYCRGSGHDYYWGCGVGPLSSVRGQGTTVTVNA (SEQ ID NO:3, a total of 117 amino acid residues). The nucleotide sequence is shown in SEQ ID NO:4. The theoretical molecular weight of VNAR-PS1 is 13.2 kDa, and the isoelectric point pI is 6.85.

[0068] Construction of prokaryotic expression vector: Based on the amino acid sequence of VNAR-PS1, the coding gene was optimized using E. coli codons. After the whole gene was synthesized, it was cloned into the pET-30a vector between the Nde I and Xho I sites, transformed into BL21(DE3) competent cells, and positive clones were verified by colony PCR and sequencing.

[0069] Induction of VNAR nanobody expression: Positive clones were selected and inoculated into 5 mL LB medium (containing 50 μg / mL kanamycin) and cultured overnight at 37°C and 200 rpm. The next day, they were transferred to 500 mL LB medium at a 1:100 ratio and cultured at 37°C and 200 rpm until... When the concentration reached 0.6-0.8, IPTG was added to a final concentration of 0.5 mM, and expression was induced at 25°C and 180 rpm for 16 h. The bacterial cells were collected by centrifugation at 4000 rpm and 4°C for 20 min, washed once with PBS, and stored at -80°C.

[0070] Purification of VNAR nanobodies: Resuspend the frozen bacterial bodies in lysis buffer (50 mM). Add 1 mg / mL lysozyme to a solution of 300 mM NaCl, 10 mM imidazole, pH 8.0, and incubate on ice for 30 min. Sonicate the mixture (400 W, 3 s sonication, 5 s intermittent sonication, total 30 min), centrifuge at 12000 × g, 4 °C for 30 min, and collect the supernatant. Filter the supernatant through a 0.45 μm filter membrane and load it onto a pre-equilibrated solution. -NTA affinity chromatography column. Wash with wash buffer containing 20 mM imidazole until... If the concentration is less than 0.02, elute the target protein with elution buffer containing 250 mM imidazole. Collect the elution peak, dialyze to PBS buffer, and concentrate to 1-5 mg / mL using Amicon ultrafiltration. Determine protein concentration using BCA method and analyze purity using SDS-PAGE.

[0071] The results show that ( Figure 2 The purified VNAR-PS1 nanobody showed a single band on an SDS-PAGE gel, with a molecular weight of approximately 13 kDa, consistent with the theoretical value, and a purity greater than 95%. Approximately 32 mg of purified protein was obtained from 500 mL of culture medium, with an expression level of approximately 64 mg / L.

[0072] Example 3 Affinity determination of VNAR nanobodies

[0073] In this embodiment, the binding affinity between VNAR-PS1 nanobody and PDCoV S protein RBD was determined using ELISA and biomembrane interferometry.

[0074] Indirect ELISA assay: Recombinant PDCoV S protein RBD was diluted to 4 μg / mL, and 100 μL was coated per well of a 96-well ELISA plate and incubated overnight at 4°C. The plate was washed three times with PBS and blocked with 2% BSA-PBS for 1 h. VNAR-PS1 nanobody was serially diluted 2-fold starting at 1000 nM, for a total of 12 concentration gradients, with 3 replicates per concentration. 100 μL of the diluted antibody solution was added, and the plate was incubated at 37°C for 1.5 h. The plate was washed five times with PBST, and HRP-labeled anti-His tag antibody (1:5000 dilution) was added, and the plate was incubated at 37°C for 1 h. The plate was washed five times with PBST, and TMB was used for color development for 15 min. The plate was then incubated with 2M... Terminate the reaction and read value.

[0075] Nonlinear fitting analysis was performed using GraphPad Prism software, and the combined curve was plotted. Figure 3 The results showed that VNAR-PS1 binds to the PDCoV S protein RBD in a dose-dependent manner, and the calculated... The value is 3.8 ± 0.4 nM.

[0076] Biofilm layer interferometry (BLI) assay: Kinetic analysis was performed using the Octet RED96e system. Biotin-labeled PDCoV S protein RBD (20 μg / mL) was immobilized on the surface of a streptavidin sensor for 600 s. VNAR-PS1 nanobody was diluted to different concentrations (200 nM, 100 nM, 50 nM, 25 nM, 12.5 nM), and binding (association) kinetics were measured for 300 s and dissociation (dissociation) for 600 s. The data were fitted using a 1:1 binding model using Octet Data Analysis software to calculate the binding rate constant. Dissociation rate constant and equilibrium dissociation constant value.

[0077] The results showed that the VNAR-PS1 nanobody Value is , Value is Calculations yielded Value is M indicates that VNAR-PS1 binds to the PDCoV S protein RBD with high affinity.

[0078] Example 4: Evaluation of the virus neutralizing activity of VNAR nanobodies

[0079] This embodiment uses sham virus neutralization experiments and live virus neutralization experiments to evaluate the neutralizing activity of VNAR-PS1 nanobody against PDCoV.

[0080] Preparation of pseudoviruses: The VSV pseudovirus system was used. HEK293T cells were transfected with a plasmid expressing the PDCoV S protein, and infected with a replication-deficient VSV virus carrying a luciferase reporter gene 24 h later. Cell supernatant was collected after 48 h, filtered through a 0.45 μm filter, aliquoted, and stored at -80℃. The pseudovirus titer was determined using LLC-PK1 cells, using a titer of [missing value]. Neutralization experiments were conducted using pseudoviruses with a TCID50 / mL concentration.

[0081] Pseudovirus neutralization assay: VNAR-PS1 nanobody was serially diluted 3-fold starting at 1000 nM, with 4 replicates per concentration. 50 μL of diluted antibody was mixed with 50 μL of pseudovirus (approximately 100 TCID50) and incubated at 37°C for 1 h. The antibody-virus mixture was then added to LLC-PK1 cells pre-seeded in 96-well plates (approximately...). (cells / well), 37℃, 5% Incubate for 48 hours. Aspirate the supernatant, add 100 μL of luciferase substrate to each well, and incubate at room temperature in the dark for 10 minutes. Detect the luminescence value (RLU) using a multi-mode microplate reader. Set up a positive control (sham virus only) and a negative control (cells only).

[0082] Neutralization rate calculation formula: Neutralization rate (%) = ;

[0083] The neutralization curve was plotted using GraphPad Prism software. Figure 4 ),calculate Value. The results showed that VNAR-PS1 was effective against PDCoV pseudoviruses. The value was 2.3 ± 0.3 nM, and the neutralization rate reached over 95% when the antibody concentration was greater than 50 nM.

[0084] Live virus neutralization assay: Performed in a BSL-2 laboratory. The VNAR-PS1 nanobody was serially diluted 2-fold starting at 500 nM. 50 μL of the diluted antibody was mixed with 50 μL of PDCoV CHN-AH-2004 strain virus solution (100 TCID50) and incubated at 37°C for 1 h. The mixture was then seeded into monolayer LLC-PK1 cells and incubated at 37°C with 5% [unspecified concentration]. Cultured for 72 hours. Cytopathic effect (CPE) was observed using crystal violet staining, and viral neutralization titer was calculated.

[0085] The results showed that VNAR-PS1 achieved a neutralizing titer of 100% against live PDCoV. The results show that a 100 nM antibody concentration can completely inhibit virus-induced CPE, further confirming that VNAR-PS1 has excellent PDCoV neutralizing activity.

[0086] Example 5: Thermal stability analysis of VNAR nanobodies

[0087] This embodiment evaluates the stability of VNAR-PS1 nanobodies under different temperature conditions.

[0088] The VNAR-PS1 nanobody solution (1 mg / mL, PBS buffer) was aliquoted into multiple EP tubes and treated for 30 min at 4 °C, 25 °C, 37 °C, 50 °C, 60 °C, 70 °C, 80 °C, and 90 °C, respectively. The treated samples were then cooled on ice, and residual binding activity was detected using ELISA.

[0089] ELISA detection method: The recombinant PDCoV S protein RBD was diluted to 4 μg / mL and coated onto an ELISA plate. After blocking, VNAR-PS1 antibody solutions before and after heat treatment (final concentration 100 nM) were added. The remaining steps were the same as in Example 3. The untreated sample stored at 4℃ was used as a control (activity set to 100%), and the relative activity retention rate after each temperature treatment was calculated.

[0090] The results show that ( Figure 5 The activity retention rate of VNAR-PS1 was 95.2% after treatment at 60℃ for 30 min, 90.8% after treatment at 70℃, 82.3% after treatment at 80℃, and 65.4% after treatment at 90℃. This indicates that VNAR-PS1 has excellent thermal stability, with an activity retention rate greater than 80% after treatment at 80℃ for 30 min.

[0091] Differential scanning fluorescence (DSF) assay: DSF analysis was performed using a Real-Time PCR instrument to determine the thermal denaturation temperature (Tm value) of VNAR-PS1. 5 μg / mL VNAR-PS1 was mixed with 5×SYPRO Orange dye, and the temperature was gradually increased from 25℃ to 95℃ (heating rate 0.5℃ / min), and changes in fluorescence signal were detected. The Tm value was determined using a first derivative curve of fluorescence intensity versus temperature.

[0092] The results showed that the Tm value of VNAR-PS1 was 78.5±0.8℃, which was significantly higher than the Tm value of traditional antibodies (usually 50-70℃), further confirming its excellent thermal stability.

[0093] Example 6 pH stability analysis of VNAR nanobodies

[0094] This embodiment evaluates the stability of VNAR-PS1 nanobodies under different pH conditions.

[0095] Prepare buffer systems with different pH values: pH 2.0 (50mM glycine-HCl), pH 3.0 (50mM citric acid-sodium citrate), pH 4.0 (50mM citric acid-sodium citrate), pH 5.0 (50mM acetic acid-sodium acetate), pH 6.0 (50mM MES), pH 7.0 (50mM phosphate buffer), pH 8.0 (50mM Tris-HCl), pH 9.0 (50mM sodium carbonate-sodium bicarbonate), and pH 10.0 (50mM sodium carbonate-sodium bicarbonate).

[0096] The VNAR-PS1 nanobody solution was mixed with the above-mentioned different pH buffers at a volume ratio of 1:9 to achieve a final protein concentration of 0.1 mg / mL, and incubated at 37°C for 2 h. After incubation, the sample was neutralized to pH 7.0 with PBS, and residual binding activity was detected by ELISA.

[0097] The results show that ( Figure 6 VNAR-PS1 maintained high binding activity across a wide pH range of 2.0–8.0, with the following activity retention rates: 86.5% at pH 2.0, 92.3% at pH 3.0, 95.8% at pH 4.0, 97.2% at pH 5.0, 98.5% at pH 6.0, 100% (control) at pH 7.0, and 96.8% at pH 8.0. At pH 9.0 and pH 10.0, the activity retention rates decreased to 72.3% and 58.6%, respectively. This indicates that VNAR-PS1 exhibits good stability over a broad pH range of 2.0–8.0, with activity retention rates consistently exceeding 85%.

[0098] Example 7 Epitope Specificity Analysis of VNAR Nanobodies

[0099] This embodiment analyzes the epitope types identified by VNAR-PS1 using competitive ELISA and Western blot analysis.

[0100] Competitive ELISA assay: A competitive ELISA was performed using pre-denatured and non-denatured PDCoV S protein RBD. Denaturation treatment: The S protein RBD was boiled at 100℃ for 10 min and then rapidly cooled in an ice bath; Non-denaturation treatment: stored at room temperature. The non-denatured and denatured S protein RBDs were coated onto ELISA plates, and VNAR-PS1 antibody was added to detect binding activity.

[0101] The results showed that VNAR-PS1 binds to the non-denatured S protein RBD. The value is 2.35, while the binding to the denatured S protein RBD... The value is only 0.18, indicating that VNAR-PS1 mainly recognizes the conformation-dependent epitopes of the S protein RBD.

[0102] Western blot analysis: PDCoV S protein RBD under both reducing and non-reducing conditions was subjected to SDS-PAGE electrophoresis and transferred to a PVDF membrane. VNAR-PS1 (5 μg / mL) was used as the primary antibody, and HRP-labeled anti-His antibody (1:5000) was used as the secondary antibody. ECL staining was performed.

[0103] The results showed that VNAR-PS1 specifically binds to the S protein RBD only under non-reducing conditions, while no obvious binding signal is observed under reducing conditions, further confirming that VNAR-PS1 recognizes a conformation-dependent epitope.

[0104] Example 8: Evaluation of the cross-reactivity of VNAR nanobodies against PDCoV variants

[0105] This embodiment evaluates the cross-binding activity of VNAR-PS1 against different PDCoV isolates and variants.

[0106] Recombinant S protein RBDs were obtained from PDCoV strains isolated from different geographical origins and time periods, including: CHN-AH-2004 strain (wild type, GenBank: KX022756), USA-OH-2014 strain (GenBank: KJ462462), Korea-2016 strain (GenBank: KY019623), and Thailand-2015 strain (GenBank: KU051641). Recombinant expression vectors were constructed based on the sequences of each strain, and the S protein RBDs were expressed and purified.

[0107] The binding activity of VNAR-PS1 to the S protein RBD of different strains was detected by ELISA, with wild-type CHN-AH-2004 strain as the control (binding activity was set to 100%).

[0108] The results showed that the relative binding activities of VNAR-PS1 to the S protein RBD of each strain were as follows: CHN-AH-2004 strain 100%, USA-OH-2014 strain 92.5%, Korea-2016 strain 88.3%, and Thailand-2015 strain 85.7%. This indicates that VNAR-PS1 maintains good cross-binding activity against different PDCoV variants, with a binding activity retention rate of over 70%, demonstrating broad-spectrum recognition potential.

[0109] Example 9: Development of an ELISA detection kit for VNAR nanobodies

[0110] This embodiment describes the development of a double-antibody sandwich ELISA kit for detecting PDCoV S protein based on VNAR-PS1.

[0111] Preparation of coating antibody: VNAR-PS1 was labeled with NHS-Biotin as the coating antibody. VNAR-PS1 (2 mg / mL) was reacted with 10 times molar excess of NHS-Biotin in PBS at room temperature for 2 h, and the free label was removed by dialysis.

[0112] Preparation of detection antibody: HRP-labeled VNAR-PS1 was used as the detection antibody. The procedure was followed according to the HRP labeling kit instructions, and the antibody was purified by dialysis after labeling.

[0113] Establishment of a double-antibody sandwich ELISA method: 96-well plates were coated with streptavidin, and biotin-labeled VNAR-PS1 (5 μg / mL) was added and incubated at 37°C for 1 h. After washing, different concentrations of PDCoV S protein RBD standards (0.01-100 ng / mL) were added, and the plates were incubated at 37°C for 1 h. After washing, HRP-labeled VNAR-PS1 (1:2000 dilution) was added, and the plates were incubated at 37°C for 1 h. After washing, TMB was developed, and the readings were recorded. value.

[0114] Standard curve plotting: The vertical axis represents the sum of the values ​​and the horizontal axis represents the logarithm of the S protein RBD concentration. A four-parameter logistic model was used to fit the standard curve. The results showed that the limit of detection (LOD) was 0.05 ng / mL, the linear range was 0.1-50 ng / mL, and the correlation coefficient was [missing value]. =0.998.

[0115] Sample Validation: Ten PDCoV-positive and ten PDCoV-negative pig fecal samples were collected for validation. Results showed a 100% concordance rate for positive samples and 100% specificity for negative samples, indicating that the kit has good sensitivity and specificity.

[0116] Comparative Example 1: Preparation and Performance Comparison of Traditional Monoclonal Antibodies

[0117] This comparative study prepared a conventional mouse monoclonal antibody against the PDCoV S protein RBD and compared its performance with that of the VNAR nanobody.

[0118] BALB / c mice were immunized with recombinant PDCoV S protein RBD, and hybridoma cell fusion technology was used to screen for hybridoma cell lines that secreted monoclonal antibodies against S protein RBD. The monoclonal antibodies were purified by Protein A affinity chromatography, and their purity was identified by SDS-PAGE.

[0119] The performance comparison results are as follows:

[0120] In terms of molecular weight, traditional monoclonal antibodies are about 150 kDa, while VNAR-PS1 is only 13.2 kDa, which is about 1 / 11 of that of traditional monoclonal antibodies.

[0121] Regarding affinity, the BLI assay was used to determine the affinity of traditional monoclonal antibodies. Value is M, VNAR-PS1 is M,VNAR-PS1 has approximately four times the affinity of traditional monoclonal antibodies.

[0122] Regarding neutralizing activity, traditional monoclonal antibodies The value was 18.6 nM, while VNAR-PS1 was 2.3 nM. The neutralizing activity of VNAR-PS1 was approximately 8 times that of conventional monoclonal antibodies.

[0123] In terms of thermal stability, the activity retention rate of traditional monoclonal antibodies after treatment at 80℃ for 30 min was 28.5%, while that of VNAR-PS1 was 82.3%, indicating that VNAR-PS1 has significantly better thermal stability than traditional monoclonal antibodies.

[0124] Regarding pH stability, the activity retention rate of traditional monoclonal antibodies was 15.2% at pH 2.0, while that of VNAR-PS1 was 86.5%, indicating that VNAR-PS1 has significantly better acid stability than traditional monoclonal antibodies.

[0125] In terms of epitope type, traditional monoclonal antibodies mainly recognize linear epitopes, while VNAR-PS1 recognizes conformation-dependent epitopes.

[0126] In terms of production costs, traditional monoclonal antibodies require expression in mammalian cells, and the cost is about 5-10 times that of E. coli expression systems.

[0127] The above comparison results show that the VNAR nanobody of the present invention is significantly superior to traditional monoclonal antibodies in terms of affinity, neutralizing activity, stability and production cost.

[0128] Comparative Example 2: The impact of different immunization strategies on the quality of VNAR libraries

[0129] This comparative study compares the effects of different immunization doses and immunization times on the quality of VNAR phage display libraries.

[0130] Three different immunization strategies were set up: Group A used 25 μg / tail, immunized 4 times; Group B used 100 μg / tail, immunized 6 times (preferred scheme of the present invention); Group C used 200 μg / tail, immunized 8 times. The remaining operation steps are the same as in Example 1.

[0131] The results showed that the library size of group A was [missing information]. CFU, after three rounds of screening, the positive clone rate was 8.3%; the group B library capacity was... CFU, positive clone rate was 23.9%; group C library capacity was [missing information]. CFU, with a positive clone rate of 21.5%.

[0132] Group A had insufficient immunization intensity, resulting in low library size and positive rate; while Group C had a slightly higher library size, its positive rate was comparable to Group B, and the increased number of immunizations led to a longer cycle and increased costs. Therefore, this invention preferably uses an immunization strategy of 100 μg / tail, with 6 immunizations.

[0133] Comparative Example 3: Effect of different expression temperatures on soluble VNAR expression

[0134] This comparative study compares the effects of different induction temperatures on the soluble expression of VNAR nanobodies.

[0135] Three different expression temperatures were set: Group A was induced at 37℃, Group B at 25℃ (preferred solution of this invention), and Group C at 16℃. The remaining operations were the same as in Example 2. After 16 hours of induction, bacterial cells were collected, and the contents of soluble protein and inclusion body protein were measured.

[0136] The results showed that the soluble protein expression level in group A was 8 mg / L, with inclusion bodies accounting for more than 85%; the soluble protein expression level in group B was 64 mg / L, with inclusion bodies accounting for less than 20%; and the soluble protein expression level in group C was 35 mg / L, with inclusion bodies accounting for about 30%.

[0137] High-temperature induction at 37°C leads to the formation of numerous inclusion bodies in the protein, resulting in high total expression levels but low soluble protein content. While low-temperature induction at 16°C increases the soluble protein content, it significantly reduces the expression level. Therefore, this invention preferably induces expression at 25°C, achieving high protein yield while ensuring soluble expression.

[0138] Sequence information:

[0139] SEQ ID NO:1 (Amino acid sequence of CDR3 region of VNAR-PS1, 20 amino acids): CRGSGHDYYWGCGVGPLSSV

[0140] SEQ ID NO:2 (Amino acid sequence of CDR1 region of VNAR-PS1, 7 amino acids): CTYPGVS

[0141] SEQ ID NO:3 (Complete amino acid sequence of VNAR-PS1, 117 amino acids): ARVDQTPRTVTKETGESLTINCVLRDGSFYLCNTYPGVSNWYRFKSGATNKESINGGRYVETLNSKASSFSLLISDLRVADSAVYYCRGSGHDYYWGCGVGPLSSVRGQGTTVTVNA

[0142] SEQ ID NO:4 (nucleotide sequence encoding VNAR-PS1, 351bp): GCGCGTGTTGATCAGACCCCGCGTACCGTTACCAAAGAAACCGGTGAATCTCTGACCATCAACTGCGTTCTGCGTGATGGTTCTTTCTATCTGTGCAACACCTATCCGGGTGTTTCTAACTGGTATCGTTTCAAATCTGGTGCGACCAACAAAGAATCTATCAACGGTGGTCGT TATGTTGAAACCCTGAACTCTAAAGCGTCTTCTTTCTCTCTGCTGATTTCTGATCTGCGTGTTGCTGATTCTGCGGTTTTACTGCCGTGGTTCTGGTCATGATTATTACTGGGGTGTGGGTTCTGCGCTGTCTTCTGTTCGTGGTCAGGGTACCACCGTTACCGTTAACGCG

[0143] The embodiments described above are merely illustrative of specific implementations of the present invention, and while the descriptions are detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. A shark-derived VNAR nanobody targeting the porcine deltacoronavirus S protein, characterized in that: The VNAR nanobody specifically recognizes the conformation-dependent epitope of the receptor-binding domain of the PDCoV spike protein. The VNAR nanobody contains complementarity-determining regions CDR1 and CDR3, the sequence of which is shown in SEQ ID NO:1 and the sequence of which is shown in SEQ ID NO:

2.

2. The VNAR nanobody as described in claim 1, characterized in that: The VNAR nanobody also includes framework regions FR1, FR2 and FR3, and the complete amino acid sequence of the VNAR nanobody is shown in SEQ ID NO:

3.

3. The VNAR nanobody as described in claim 1, characterized in that: The VNAR nanobody has a molecular weight of 12-15 kDa.

4. The VNAR nanobody as described in claim 1, characterized in that: The dissociation constant of the VNAR nanobody and the PDCoV S protein RBD Value is M to M.

5. The VNAR nanobody as described in claim 1, characterized in that: The neutralizing activity of the VNAR nanobody against PDCoV The value is 0.5-10 nM, and the neutralization efficiency is over 95%.

6. The VNAR nanobody as described in claim 1, characterized in that: The VNAR nanobody remains stable in the pH range of 2.0-8.0, with an activity retention rate of greater than 85%; after being treated at 80℃ for 30 min, the activity retention rate of the VNAR nanobody is greater than 80%.

7. The use of the VNAR nanobody according to any one of claims 1 to 6 in the preparation of PDCoV diagnostic reagents or pharmaceutical compositions for the prevention and treatment of PDCoV infection.

8. The application as described in claim 7, characterized in that: The diagnostic reagent is an ELISA kit, an immunochromatographic test strip, or an immunofluorescence assay kit; the ELISA kit uses the VNAR nanobody as the detection antibody or a competitive antibody, and the detection limit is 0.01-0.1 ng / mL.

9. The application as described in claim 7, characterized in that: The dosage form of the pharmaceutical composition is an injection, an oral preparation, or a spray.

10. The application as described in claim 7, characterized in that: The pharmaceutical composition also includes a pharmaceutically acceptable carrier and adjuvant.

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