Nano antibody aiming at feline infectious peritonitis virus and application thereof

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to a nanometer antibody aiming at feline infectious peritonitis virus and application of the nanometer antibody. According to the invention, specific nano antibodies 2S-29 and 2S-39 of two novel FIPV S proteins are obtained by screening through a phage display technology, and a double-antibody sandwich ELISA method for detecting FIPV S antigens is provided. The two antibodies have excellent specificity and affinity to the feline infectious peritonitis virus FIPV S protein. A double-antibody sandwich ELISA method established based on the nano antibody has good specificity, repeatability and sensitivity, the lowest detection limit reaches 4 ng / mL, the FIPV S antigen can be rapidly and accurately detected, and a reliable means is provided for early diagnosis of feline infectious peritonitis.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to nanobodies against feline infectious peritonitis virus and their applications. Background Technology

[0002] Feline infectious peritonitis virus (FIPV) originates from feline enteric coronavirus (FECV). Both are biotypes of FCoV and are single-stranded positive-sense RNA viruses. Currently, most diagnostic methods struggle to distinguish between them. FIPV virus particles are approximately 90-100 nm in diameter, with a genome size of about 30 kb. The 5' end of the genome (approximately 20 kb) contains overlapping ORF1a / 1b, encoding a polypeptide that, upon enzymatic cleavage, yields 16 non-structural proteins (NSPs), primarily acting as replicases in viral RNA synthesis. The 3' end of the genome contains 9 ORFs, encoding 4 structural proteins (spike protein S, membrane protein M, envelope protein E, nucleocapsid protein N) and 5 accessory proteins with unknown functions (3a, 3b, 3c, 7a, 7b). Among these, the S protein possesses a receptor-binding domain, mediating cell receptor junctions and membrane fusion, and is a key protein for viral invasion.

[0003] Feline infectious peritonitis (FIP) caused by feline infectious peritonitis (FIPV) is a highly fatal feline disease with a mortality rate approaching 100%. With the development of China's pet industry, the diagnosis and treatment of FIP are receiving increasing attention. While there are reports of drugs such as GS441524 and GC376 being effective against FIP, there are currently no standardized, domestically produced FIP-specific drugs in China. Immunohistochemistry is the gold standard for FIP diagnosis; a diagnosis cannot be made based solely on non-specific symptoms. Combining laboratory diagnostic methods to improve accuracy is crucial for treatment.

[0004] Nanobody (VHH) libraries mainly fall into three categories: natural libraries, immune libraries, and synthetic libraries, used for targeted screening of VHHs for specific antigens. Natural libraries obtain cDNA from unimmunized healthy alpaca B cells via RT-PCR, amplify the VHH gene, and clone it into a phage vector for display. Theoretically, a sufficiently large library can screen for antibodies against any antigen. Phage display technology is the mainstream method for preparing VHHs; in addition to solid / liquid phase screening, protein chip, in vivo, tissue, and cell screening methods are increasingly being used.

[0005] Given the limitations of existing diagnostic methods and the advantages of nanobodies, there is an urgent need to develop specific nanobodies and efficient detection methods for FIPV. Summary of the Invention

[0006] Existing diagnostic methods for feline infectious peritonitis virus (FIPV) suffer from cumbersome procedures, insufficient accuracy, and a lack of highly specific and affinity antibodies for detection and prevention. This invention aims to obtain specific nanobodies against the FIPV S protein through phage display technology and establish a rapid and accurate double-antibody sandwich ELISA detection method based on these nanobodies. This addresses the shortcomings of existing diagnostic techniques and provides a powerful tool for the diagnosis of FIPV and the prevention and treatment of related diseases.

[0007] To achieve the above objectives, the technical solution of the present invention is as follows: The first aspect of the present invention provides nanobodies against feline infectious peritonitis virus, said nanobodies including 2S-29 antibody and 2S-39 antibody; The nanobody has a heavy chain variable region comprising three complementarity-determining regions HCDR1, HCDR2, and HCDR3 and four framework regions HFR1, HFR2, HFR3, and HFR4, arranged in the order HFR1, HCDR1, HFR2, HCDR2, HFR3, HCDR3, and HFR4. For the 2S-29 antibody: the amino acid sequence of HCDR1 is shown in SEQ ID NO:1.1; the amino acid sequence of HCDR2 is shown in SEQ ID NO:2.1; the amino acid sequence of HCDR3 is shown in SEQ ID NO:3.1; the amino acid sequence of HFR1 has at least 80% identity with the sequence shown in SEQ ID NO:4.1; the amino acid sequence of HFR2 has at least 80% identity with the sequence shown in SEQ ID NO:5.1; the amino acid sequence of HFR3 has at least 80% identity with the sequence shown in SEQ ID NO:6.1; the amino acid sequence of HFR4 has at least 80% identity with the sequence shown in SEQ ID NO:7.1. For the 2S-39 antibody: the amino acid sequence of HCDR1 is shown in SEQ ID NO:1.2; the amino acid sequence of HCDR2 is shown in SEQ ID NO:2.2; the amino acid sequence of HCDR3 is shown in SEQ ID NO:3.2; the amino acid sequence of HFR1 has at least 80% identity with the sequence shown in SEQ ID NO:4.2; the amino acid sequence of HFR2 has at least 80% identity with the sequence shown in SEQ ID NO:5.2; the amino acid sequence of HFR3 has at least 80% identity with the sequence shown in SEQ ID NO:6.2; and the amino acid sequence of HFR4 has at least 80% identity with the sequence shown in SEQ ID NO:7.2. It should be noted that at least 80% identity means identity ≥80%, including 85%, 90%, 95%, or 100%; of course, it should be understood that sequences with at least 80% identity have similar functions to the sequences described above.

[0008] In one optional embodiment, the amino acid sequence of the 2S-29 antibody is shown in SEQ ID NO:8.1; the amino acid sequence of the 2S-39 antibody is shown in SEQ ID NO:8.2.

[0009] In one optional embodiment, the nanobody has a heavy chain constant region containing a sequence having at least 70% identity with the sequence shown in SEQ ID NO:9. It should be noted that at least 70% identity means identity ≥70%, such as 75%, 80%, 85%, 90%, 95%, or 100%; of course, it should be understood that sequences with at least 70% identity have similar functions to the aforementioned sequences.

[0010] The second aspect of the present invention provides any one of the following substances: (i) A polynucleotide molecule comprising a nucleotide sequence encoding the nanobody described in the first aspect; specifically, the polynucleotide molecule of the present invention is obtained by translating the nanobody of the present invention according to conventional methods; alternatively, it may be a nucleotide sequence obtained by further modifying the sequence obtained by translating the above-described amino acid sequence; the modification method is a method known in the art to increase expression efficiency or other nucleotide modification methods for specific purposes. (ii) An expression vector, including the polynucleotide molecule in (i); specifically, the expression vector in this invention may be selected from any one of lentiviral expression vectors, retroviral expression vectors, adenoviral expression vectors, adeno-associated virus expression vectors, DNA vectors, RNA vectors, and plasmids. Lentiviral vectors may be selected from the following group: human immunodeficiency virus 1 (HIV-1), human immunodeficiency virus 2 (HIV-2), visna-maedivirus (VMV), caprine arthritis-encephalitis virus (CAEV), equine infectious anemia virus (EIAV), feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), and simian immunodeficiency virus (SIV); (iii) Engineered bacteria, including the expression vector in (ii); specifically, the engineered bacteria in this invention refers to bacteria that can assist the above-mentioned expression vector in expression, such as Escherichia coli, and the Escherichia coli including the expression vector here is the Escherichia coli after the expression vector is inserted. (iv) Host cells, including the expression vector in (ii); specifically, the host cells in this invention refer to cells that can assist the above-mentioned expression vectors in expression, such as yeast cells; (v) Products for detecting feline infectious peritonitis virus, including the nanobodies described in the first aspect and / or the polynucleotide molecules in (i) and / or the expression vector in (ii) and / or the engineered bacteria in (iii) or the host cells in (iv); specifically, the nanobodies of the present invention have a strong binding effect with the surface proteins of feline infectious peritonitis virus, and products for detecting feline infectious peritonitis virus can be prepared based on this; the products can be detection reagents or detection kits, such as reagents or kits based on immunoblotting, or reagents or kits based on immunoenzyme technology, etc. (vi) A pharmaceutical composition comprising the nanobody described in the first aspect and / or the polynucleotide molecule in (i) and / or the expression vector in (ii) and / or the engineered bacteria in (iii) and / or the host cell in (iv); specifically, as described above, based on the strong binding interaction between the nanobody of the present invention and the surface protein of feline infectious peritonitis virus, a pharmaceutical composition for the prevention or treatment of feline infectious peritonitis virus infection can be prepared; the pharmaceutical composition here refers to a pharmaceutical composition consisting of the nanobody of the present invention combined with other small molecule compounds, peptides, antibodies or proteins that can be effectively used for the prevention or treatment of feline infectious peritonitis virus infection; for example, bispecific or polyclonal antibodies combined with other antibodies, ADCs combined with small molecule compounds, or fusion proteins combined with other proteins; the choice of which to use may be made according to specific circumstances; (vii) A pharmaceutical formulation comprising the nanobody described in the first aspect and / or the polynucleotide molecule in (i) and / or the expression vector in (ii) and / or the engineered bacteria in (iii) and / or the host cell in (iv) and / or the pharmaceutical composition in (vi). Specifically, the above-mentioned nanobody and the pharmaceutical composition can be prepared into different dosage forms to meet different clinical needs by adding a pharmaceutically acceptable carrier; the dosage form may include a spray, oral liquid, tablet, nebulizer, granule, capsule or ointment; of course, the choice of pharmaceutically acceptable carrier varies depending on the dosage form, and can be prepared according to methods known in the art.

[0011] The third aspect of this invention provides the application of the nanobodies described in the first aspect, or the polynucleotide molecules, expression vectors, engineered bacteria, host cells, pharmaceutical compositions, or pharmaceutical preparations described in the second aspect, in the preparation of feline infectious peritonitis virus (FIP) detection products. It should be noted that the detection of FIP can be used for the diagnosis of FIP infection diseases; that is, the FIP detection products of this invention are also applicable to the diagnosis of FIP infection diseases, such as avian leukosis. Furthermore, FIP detection products include, but are not limited to, detection reagents, detection kits, reagent cards, or microfluidic chips.

[0012] A fourth aspect of the present invention provides a double-antibody sandwich ELISA method for detecting feline infectious peritonitis virus using the nanobodies described in the first aspect, comprising the following steps: An enzyme-labeled 2S-29 antibody is prepared as a capture antibody, and an HRP-labeled 2S-39 antibody is prepared as a detection antibody. The sample to be tested is added to the capture antibody to allow it to bind, and then the detection antibody is added. After color development, the sample is ready. The double-antibody sandwich ELISA method is used for non-diagnostic detection.

[0013] The beneficial effects of this invention are as follows: The nanobody targeting feline infectious peritonitis virus (FIPV) provided in this invention exhibits excellent specificity and affinity for the FIPVS protein. Compared to traditional antibodies, it offers advantages such as small size, low cost, high solubility, and stability under extreme conditions, making it more suitable for disease diagnosis and prevention. The double-antibody sandwich ELISA method established based on this nanobody demonstrates good specificity, repeatability, and sensitivity, with a detection limit of 4 ng / mL. It can rapidly and accurately detect the FIPVS antigen, providing a reliable means for the early diagnosis of feline infectious peritonitis. Attached Figure Description

[0014] Figure 1 The results of double enzyme digestion identification of pCAGGS-FIPV-S plasmid; Figure 2 The purification and identification of FIPV S protein; Figure 3 This is for identification of overlapping PCR amplification. Figure 4 This describes the enzyme digestion reaction. Figure 5 The positive rate of the phage random peptide library was identified; Figure 6 The results of PCR identification of some single-clone colonies after the third round of phage library screening; Figure 7 PCR amplification of the target fragment for antibody; Figure 8 The results of Western Blot identification of the eukaryotic antibody expression were shown. Figure 9 The results of indirect ELISA identification of eukaryotic antibody expression. Figure 10 The eukaryotic expression and purification status of the recombinant human antibody; Figure 11 IFA identification results for FIPV recombinant antibodies; Figure 12 The results of HRP-labeled antibody titer determination; Figure 13 This represents the optimal antibody pairing. Figure 14 To determine the optimal working concentration of antibodies for capturing antibody 2S-29 and detecting antibody 2S-39; Figure 15 The effect of different types of sealing liquid on absorbance; Figure 16 The effect of antigen incubation time on absorbance; Figure 17 To investigate the effect of nanobody incubation time on absorbance; Figure 18 The results of sensitivity analysis of the ELISA method; Figure 19 This describes the specificity of the ELISA method. Figure 20 This refers to the intra-assay repeatability of the ELISA method. Figure 21 This describes the inter-batch repeatability of the ELISA method. Detailed Implementation

[0015] To facilitate understanding of the present invention, a more comprehensive description will be given below. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0016] Example 1: Construction of a phage random peptide library (I) Expression and purification of FIPV S protein (1) Construction and identification of pCAGGS-FIPV-S plasmid Specific primers were designed using the S gene sequence of the FIPV UU4 strain from the NCBI database. When performing PCR amplification using a synthetically produced FIPV S trimer as a template, the Kozak sequence, a mouse signal peptide, and [other components] were introduced at the 5' end of the extracellular region of the S gene. EcoR I restriction site, and simultaneously insert His tag, double Strep tag, Foldon trimer tag and at the 3¢ end. Xho I. Restriction site. Specific primer information is shown in Table 1, and PCR amplification system and conditions are shown in Table 2.

[0017] Table 1 Amplification Primers

[0018] Table 2 PCR reaction system and procedure

[0019] use EcoR I and Xho The pCAGGS vector was double-digested with restriction endonucleases at 37°C for 20 min. After confirming the correct band size using agarose gel electrophoresis, the target fragment was recovered. Figure 1 As shown, sample well 1 contains the pCAGGS-FIPV-S plasmid digested with two enzymes. The results show two clear bands at 4474 bp and 2991 bp, and the fragment size after double enzyme digestion is as expected. Sample well 2 contains... EcoR Single digestion of the pCAGGS-FIPV-S plasmid with restriction endonuclease I showed a clear band at 7465 bp, as expected. Sample well 3 contained the empty pCAGGS vector.

[0020] The recovered linear plasmid pCAGGS was homologously recombinated with the amplified target fragment FIPV S gene using homologous recombinase. The optimized in vitro ligation reaction program was set to be maintained at 50℃ for 15 minutes (specific parameters are detailed in Table 3). After the recombinant plasmid vector was introduced into DH5α competent cells, it was evenly spread on the surface of LB solid medium containing ampicillin (100 mg / mL) and transferred to a 37℃ incubator for 12 h of inverted culture. Single colonies with smooth edges were randomly selected and inoculated into LB liquid medium containing the same antibiotic concentration for amplification. After screening for single colonies by polymerase chain reaction (PCR), plasmids were extracted using a plasmid micro-extraction kit. EcoR I and Xho After double enzyme digestion verification, the pCAGGS-FIPV-S recombinant plasmid sample was sent to a sequencing institution for bidirectional sequencing. Sequence alignment analysis was performed using DNAman bioinformatics software. The PCR-identified positive monoclonal colonies were inoculated at a ratio of 1:100 into 100 mL of LB liquid medium containing ampicillin (working concentration 100 mg / mL), and placed in a 37°C constant temperature shaking incubator. The culture was carried out at 220 rpm for 16 h, and the supernatant was collected for plasmid extraction.

[0021] Table 3 DNA Ligation System

[0022] (2) Resuscitation and passage culture of HEK293F cells Preheat the water bath to 40°C. Use tweezers to remove the frozen HEK293F cells from the liquid nitrogen container, place them vertically inside a disposable PE glove, and put them in the water bath. Be careful to keep the liquid level below the mouth of the cryovial to prevent contamination.

[0023] After thawing for 2 minutes, open the cryovial in a clean bench and transfer the cell cryopreservation solution to a 15 mL centrifuge tube. Add 9 mL of fresh DMEM medium to dilute the cryopreservation solution and centrifuge at 800 rpm for 8 min. Carefully discard the supernatant, gently resuspend the cells in 1 mL of culture medium, and then transfer them to a 100 mL cell culture flask. Add 19 mL of cell culture medium, seal the flask with aluminum foil, and incubate at 37°C, 5% CO2, and 150 rpm in a shaker. Observe the cell status and count the cells every 24 h.

[0024] After counting, when the cell density reached 2.5 × 10⁻⁶ 6When the cell density and condition reach the required level (cells / mL), observe the cell state. Once the desired cell density and condition are met, cell passage can be performed. For cell passage, add 20 mL of cell suspension to a 250 mL culture flask and supplement with 40 mL of cell culture medium. Observe the cell state and count the cells every 24 hours. If cells show poor condition or low density, discard them and resuscitate the cells.

[0025] (3) Expression of FIPV S recombinant protein When HEK293F cells reached the third passage and their density and condition were normal, 800 mL of cells were used for transient transfection. Two 15 mL centrifuge tubes were used. One tube contained 800 mg of pCAGGS-FIPV-S plasmid diluted to 10 mL with 150 mM NaCl, and the other contained 800 mg of PEI transfection reagent diluted to 10 mL with 150 mM NaCl. Both tubes were incubated for 5 min. Then, the PEI reagent was slowly added to the centrifuge tube containing the plasmid. After incubating for 10 min, the PEI reagent was added dropwise to the cell culture flask. The flask was shaken to prevent over-transfection of cells and plasmid in certain areas. After all the plasmid was added, the flask mouth was sterilized with an alcohol lamp and sealed with aluminum foil. The flask was then incubated in a 37°C shaker. Add 28 mL of feed solution at 24 h, 72 h, and 120 h post-transfection. After adding the feed solution to the cells, loosen the bottle opening to allow CO2 produced by cell metabolism to escape. Close the CO2 valve of the cell culture incubator to prevent pH changes in the cell culture medium due to CO2 accumulation, which could inhibit the expression of exogenous proteins. Observe the cell status daily and collect samples on days 7-10 post-transfection.

[0026] (4) Purification of FIPV S recombinant protein 1) Sample purification pretreatment Aliquot 800 mL of cell culture medium into four 225 mL centrifuge flasks, balance them, and place them in a large-capacity ultracentrifuge. Centrifuge at 5000 rpm for 1 h, collect the supernatant, filter twice through a 0.45 mm filter membrane, then replace with a 0.22 mm filter membrane and filter twice more until the cell supernatant is clear and free of impurities. The cell supernatant contains the target protein, which is easily degraded after sample collection; therefore, the purification process must be carried out at a low temperature throughout.

[0027] 2) Nickel column purification of proteins ① Column pretreatment: A peristaltic pump system was used. First, the delivery tubing was deionized with ultrapure water, and then connected to the TED nickel column. Five column volumes (CV) of ultrapure water rinsing and equilibration buffer soaking were performed sequentially at a flow rate of 2 mL / min.

[0028] ② Sample loading: After pretreatment by filtering the cell culture supernatant that has been treated with low temperature through a 0.22 mm microporous membrane, the sample loading operation is carried out by controlling the pumping rate in the range of 1-2 mL / min, and the column breakthrough liquid is monitored and collected simultaneously.

[0029] ③ Target protein elution procedure: First, wash the A and B liquid path systems with 50 mL of ultrapure water and equilibration buffer at a flow rate of 10 mL / min to eliminate residual ethanol and interference from foreign proteins. Then, establish a linear concentration gradient between pump A (equilibration buffer) and pump B (imidazole gradient buffer), initially setting the B phase to 0% and equilibrating at a flow rate of 2 mL / min until the UV absorption baseline stabilizes. As the imidazole concentration gradient increases from 0.025 M to 0.5 M, monitor the 280 nm absorbance value in real time. Collect the eluted fraction at the appearance of the characteristic absorption peak and transfer it to a 100 kDa ultrafiltration concentrator. After replacement with PBS buffer, perform SDS-PAGE purity analysis. The final product is concentrated by centrifugation, aliquoted into 1.5 mL cryovials, rapidly frozen in liquid nitrogen, and then stored in an ultra-low temperature freezer at -80℃.

[0030] ④ Maintenance of the chromatography column and protein purification instrument: Chemical cleaning was performed sequentially using 10 CV ultrapure water (5 mL / min), 10 CV 1M sodium hydroxide (2.5 mL / min), and 10 CV 30% isopropanol (2.5 mL / min). Finally, the nickel column was regenerated using a 20% ethanol solution. After disassembling the nickel column, the A and B liquid path systems were cleaned with 1M NaOH and ultrapure water at a flow rate of 10 mL / min, respectively.

[0031] 3) StrepTrap XT affinity chromatography procedure ① Equilibrate the StrepTrap XT column: After rinsing with 0.22 mm filtered ultrapure water for 5 CV, switch to equilibration buffer and complete the 5 CV column equilibration at a flow rate of 2 mL / min to ensure low residual ethanol content in the medium.

[0032] ② Sample loading: Load the nickel column elution product onto the StrepTrap XT column at a controlled flow rate of 1-2 mL / min, simultaneously collect the breakthrough fraction and record the parameters.

[0033] ③ Elution of the target protein: Rinse the A and B channels of the protein purifier with equilibration buffer at a flow rate of 10 mL / min. After the UV absorbance value stabilizes, set the B channel to 100% to elute the target protein. Collect the sample immediately when the UV absorbance value shows a significant jump. After ultrafiltration concentration, replace the sample with phosphate buffer.

[0034] ④ Maintenance of the chromatography column and protein purification instrument: Rinse with 10 column volumes of ultrapure water at a flow rate of 5 mL / min. Rinse with 10 column volumes of 1M NaOH and 30% isopropanol at a flow rate of 2.5 mL / min. Regenerate the StrepTrap XT column by rinsing with regeneration buffer for 10 CVs. Finally, store the medium with 20% ethanol. Sterilize the liquid chromatography system with 50 mL of 1M NaOH and ultrapure water respectively. Add the eluent purified by the StrepTrap XT column to an ultrafiltration tube, concentrate and replace the medium with PBS buffer, collect the protein, aliquot it into 1.5 mL EP tubes, add 10 mL of 5×Loading Buffer to 50 mL of the eluent, boil in a 100℃ water bath for 5 min, then in an ice bath for 10 min. Stain the protein bands using Coomassie Brilliant Blue on SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Purify the protein using a TED Ni chromatography column based on its affinity. Figure 2 As shown in Figure A, the peak value was 240 mAu. After recovering the eluent, it was purified a second time using a StrepTrap XT column for affinity purification. Figure 2 As shown in Figure B, the peak value was 55 mAu. After collecting the eluent, it was immediately centrifuged at low temperature using a 100 kDa concentration tube, concentrated, and stored in PBS buffer. FIPV S protein is a trimeric protein; the monomeric protein size is approximately 160 kDa, and glycosylation modification using a eukaryotic expression system can reach 180-200 kDa. The size of the target band was identified by SDS-PAGE gel electrophoresis, as shown... Figure 2 As shown in C, the main bands of FIPV S protein without DTT and with DTT are both around 200 kDa. However, the main band of FIPV S protein without DTT has a light band above it (about 500 kDa), suggesting that some FIPV S protein exists in the form of a trimer.

[0035] (ii) Random library nanobody gene amplification Using the synthesized FR1-FR3 gene as a template, the FR1-FR3 region of the nanobody was amplified using primers VHH-F and VHH-R1, with an Sfi I restriction site added to the 5¢ end. The reaction system and conditions for both amplification processes are shown in Table 4. After gel electrophoresis, the size of the PCR1 band was identified, and the PCR1 product was recovered and purified using a DNA purification kit. Using the PCR1 product from the first amplification as a template, amplification was performed using primers VHH-F and VHH-R2, with the CDR3 region and an Sfi I restriction site added to the 3¢ end. The reaction system and conditions for the second amplification are shown in Table 5. The resulting PCR2 product is the complete nanobody gene, and the size of the PCR2 band was identified using a 2% agarose gel. The PCR1 product from the first amplification is shown in Table 5. Figure 3As shown in (a), the target band size is 300 bp. Using the PCR1 product as a template, a PCR2 reaction was performed, and a 420 bp band was recovered after gel electrophoresis, as shown in (a). Figure 3 As shown in (b).

[0036] Table 4 PCR reaction system and conditions

[0037] Table 5 PCR reaction system and conditions

[0038] (III) Purification of Nanobody Fragments Purification of DNA fragments amplified by overlapping PCR using ethanol precipitation: An equal volume of pre-chilled anhydrous ethanol was added to the PCR2 product, followed by 1 / 10 volume of 3M NaAC (pH 5.2). The mixture was then inverted and thoroughly mixed. The product was placed in a -20°C freezer and allowed to stand for 30 min to precipitate the overlapping PCR product. After centrifugation at 4°C for 10 min, the precipitate was collected. Ethanol was added and mixed, followed by another 10 min centrifugation. The precipitate was collected in a 1.5 mL centrifuge tube. After a brief centrifugation, the remaining ethanol was removed using a 100 mL pipette. The tube was then dried at room temperature for 10-15 min, dissolved in an appropriate volume of water, and mixed using a pipette. The mixture was then subjected to gel electrophoresis for identification. A gel fragment the size of the target band was collected, and the PCR2 product was recovered and purified using a DNA recovery kit. Ethanol precipitation removes impurities such as salt ions and proteins from the PCR2 product, yielding high-purity nanobody genes, such as... Figure 3 - (c) is shown.

[0039] (iv) Construction of phage random peptide libraries (1) Enzymatic digestion of nanobody fragments and phage vectors: The nanobody fragments and phage vector pComb3XSS were digested with restriction endonuclease Sfi I. The digestion system and conditions are shown in Table 6. After digestion, the results were identified by agarose gel electrophoresis. Figure 4 As shown, the phage vector pComb3XSS is 4992 bp in length and has two Sfi I restriction sites. After Sfi I digestion, two bands of 1664 bp and 3319 bp were obtained, as shown in the figure. Figure 4 As shown in (a), the 3319 bp linearized pComb3XSS vector was recovered using a DNA recovery kit. After amplification via overlap PCR, Sfi I restriction sites were added to both ends of the nanobody fragment. Digestion of the nanobody gene with Sfi I restriction endonuclease showed no significant change in band size. Agarose gel electrophoresis confirmed the band size to be approximately 400 bp. Figure 4 As shown in (a).

[0040] (2) Ligation and Transformation: The linearized phage vector generated after enzyme digestion was ligated with nanobody fragments at a ratio of 1:2 under the action of T4 ligase. The ligation system and conditions are shown in Table 7. TG1 competent cells were electroporated into the gene transfer instrument: TG1 competent cells were taken out of the -80℃ freezer 15 min in advance, and the 0.2 cm electroporation cuvette was placed on ice for pre-cooling. The tube wall was gently tapped. After the TG1 competent cells thawed, the ligation product was taken out and added to the TG1 competent cells. After mixing, the mixture was added to the electroporation cuvette, gently tapped, and labeled. 1 mL of SOC medium was pipetted into a 15 mL centrifuge tube for later use. The parameters of the gene transfer instrument were set to EC2, 1.8 KV, and 4.9 s interval. After placing the electroporation cuvette into the gene transfer instrument, the start button was clicked. After the gene transfer was completed, 1 mL of SOC medium was quickly added to the 15 mL centrifuge tube. Incubate at 37°C and 180 rpm for 40 min on a shaker. Add 10 mL of Amp-resistant LB liquid medium and incubate for another 1 h. Add 50 mL of helper phage M13K07 and incubate for 30 min. Transfer the liquid from the centrifuge tube to 300 mL of Amp and Kana-resistant LB medium and incubate for another 4 h. Add 2 M IPTG at a ratio of 1:3000 and incubate overnight at 30°C and 180 rpm on a shaker. Centrifuge the overnight culture at 7000 G and 4°C for 10 min and collect the supernatant. Add 1 / 5 volume of PEG8000 to the supernatant and incubate at 4°C for 3-4 h to allow the phage to settle. Centrifuge the settled phage at 16000 G and 4°C for 20 min using a high-speed refrigerated centrifuge. Carefully discard the supernatant; the resulting precipitate is the amplified phage. The amplified phage was thoroughly resuspended in 20 mL of PBS solution, dissolved, and centrifuged repeatedly at 7000 G, 4 °C for 10 min. The supernatant was collected, and 1 / 5 volume of PEG8000 was added. The mixture was then incubated on ice for 3–4 h to allow the phage to precipitate. After precipitation, the mixture was centrifuged at 12000 G, 4 °C for 10 min, the supernatant was discarded, and the precipitate was resuspended in 10 mL of 50% glycerol PBS. After thorough mixing, the precipitate was aliquoted and stored at -80 °C.

[0041] Table 6 Enzyme digestion reaction system and conditions

[0042] Table 7 Connection Reaction System and Conditions

[0043] (v) Determination of the library size of phage libraries Add 10 mL of phage library to 990 mL of PBS. Take eight 1.5 mL centrifuge tubes and add 180 mL of PBS to each tube. Take 20 mL of the phage library diluted 100 times and add it to the first centrifuge tube. Perform serial dilutions of 10-fold to the eighth well. Use the four highest dilutions to perform SS320 cell infection experiments. Infect SS320 cells with serially diluted phage for 10 min. After infection, take 100 mL of each phage and spread it on Amp-resistant and Kana-resistant LB plates. After incubating overnight at 37°C, count the single colonies on the Amp-resistant and Kana-resistant LB plates. The colonies growing on the Amp-resistant plates represent the total number of successfully constructed phages and helper phages, while the number of helper phages can be obtained by counting the colonies on the Kana-resistant plates. The number of single colonies on the Amp-resistant plate minus the number of single colonies on the Kana-resistant plate yields the number of successfully constructed phage random peptide libraries, which is then used to calculate the library size. Single colonies were randomly picked from Amp-resistant LB plates after phage infection of SS320 bacteria. Seventeen single colonies were identified by PCR using upstream and downstream primers for SS320 bacteria. The complete nanobody sequence size is approximately 750 bp. Figure 5 As shown, the positive rate was 100%, and the capacity of the phage random peptide library constructed in this experiment reached 7 × 10⁻⁶. 11 The PFU / mL level meets the capacity requirements of non-immune libraries and can be used for subsequent panning experiments for specific antibodies.

[0044] Example 2: Screening and Identification of FIPV S-Specific Antibodies (I) Screening and enrichment of FIPV S-specific phages The ELISA plate was coated with FIPV S protein, and the phage random library, natural library, and human single-chain antibody library were diluted with PBS to 1×10⁻⁶. 12 Add pfu / mL to an ELISA plate and incubate at room temperature in a microplate shaker for 2 h. Wash the plate 9 times with 0.1% PBST, elute with 0.2 M Gly-HCl (pH 2.2), incubate at room temperature for 15 min, aspirate the eluent, and quickly neutralize with 1 M Tris-HCl (pH 9.1). Culture XL1-blue to the logarithmic growth phase, and take 5 mL of OD... 600nmXL1-blue culture medium with a pH of 0.6 was neutralized with the above-mentioned neutralization solution and incubated at 37°C for 30 min. The culture was then transferred to LB (Amp+) liquid medium and incubated at 37°C and 220 rpm for 1 h. Helper phage M13K07 with a multiplicity of infection (MOI) of 20:1 was added, and the culture was incubated at 37°C for 30 min, followed by shaking incubation at 37°C and 220 rpm for 30 min. Then, 60 mL of LB liquid medium (containing 100 mg / mL Amp, 50 mg / mL Kana, and 50 mg / mL Tet) was added, and the culture was incubated overnight at 37°C and 220 rpm. The supernatant was collected after centrifugation at 5000 G for 30 min at 4°C, precipitated with 0.8 mol / L IPTG, centrifuged at 8000 G for 20 min at 4°C, and PEG8000 was added to the supernatant. The culture was then incubated overnight at 4°C to precipitate the phage. The phage display library obtained after the first round of panning was collected by centrifugation at 4℃ and 8000 G for 20 min, and used for the next round of screening. The panning process was repeated three times to enrich phage monoclonals expressing anti-FIPV nanobodies. The phage titer was determined by the number of clones formed by serial dilutions of SS320. 100 monoclonal colonies were randomly picked from each of the nano-natural antibody, nano-random antibody, and single-chain antibody LB plates, and PCR identification was performed using the picked single colonies as templates. The results are as follows: Figure 6 The results showed that the amplified product of the nanobody was around 700 bp, while the single-chain antibody was approximately 1000 bp, consistent with the target fragment size, indicating a 100% colony positivity rate. (II) Construction, expression and reactiveness detection of recombinant expression plasmids Antibody strains exhibiting high binding affinity in ELISA tests were selected for nucleic acid sequencing. Antibodies with CDR3 region sequence duplication were excluded by BLAST alignment. Specific primers were designed for the light / heavy chains of the selected single-chain antibodies, as well as for PCR amplification of the natural and random libraries. After identifying the target band, the antibody fragments were recovered from the gel. The purified antibody fragments, human pCAGGS-H and pCAGGS-K vectors were digested with enzymes, ligated using T4 ligase, and transformed into DH5α competent cells. Transformed single colonies were picked for PCR, enzyme digestion, and sequencing identification. After amplification culture, the successfully constructed recombinant plasmid was extracted using an endotoxin-free plasmid extraction kit. The results are as follows: Figure 7 The results showed that the light chain amplification fragment of the single-chain antibody was approximately 320 bp, the heavy chain amplification fragment was approximately 380 bp, and the amplification fragment of the nanobody was 450 bp, all of which were consistent with the expected amplification fragment sizes.

[0045] Mix the light / heavy chain recombinant plasmid (or 5 μg nanobody recombinant plasmid) in a 2:3 ratio in a 1.5 mL EP tube. In another 1.5 mL EP tube, add 15 μL of PEI transfection reagent. Incubate in a clean bench for 5 min each, then mix and incubate again for 10 min. Remove the original cell culture medium. Gently rinse each well twice with 1 mL of culture medium. Add the plasmid-PEI mixture to HEK293T cells and incubate in a sterile cell culture incubator for 6 h. After incubation, carefully remove the cell supernatant. Gently rinse each well twice with 1 mL of culture medium. Add 2 mL of DMEM medium containing 2% FBS and incubate in a cell culture incubator for 48 h. Collect the HEK293T cell supernatant.

[0046] (1) Western Blot detection: 1) Prepare separating gels of 6% and 15% concentrations in the lower layer, and uniformly prepare a 5% stacking gel in the upper layer to complete the gel preparation process.

[0047] 2) Take 20 mL of HEK293T cell supernatant and mix it with 5 mL of protein loading buffer. Boil at 100℃ for 5 min to complete the denaturation treatment. Immediately place it on ice to cool for 10 min before loading the sample.

[0048] 3) Fill the electrophoresis tank with 1× electrophoresis buffer, set the initial voltage to 80 V, and after the sample migrates to the separating gel area, adjust the voltage to 200 V until electrophoresis is complete.

[0049] 4) The PVDF membrane was pretreated with methanol solution for 5 min before use. After gel formation and electrophoresis, the gel was placed on a transfer clamp for transfer. The transfer times for single-chain antibody and nanobody samples were different, and the transfer parameters were set to 200 mA constant current for 100 min and 200 mA constant current for 45 min, respectively.

[0050] 5) After the transfer is completed, the blot membrane is immersed in 5% skim milk blocking solution and shaken for 1 h in a shaker at 60 rpm. Then it is washed three times with TBST buffer, each time shaking for 5 min.

[0051] 6) Prepare goat anti-human HRP antibody containing 5% skim milk (dilution ratio 1:5000). Incubate the blot membrane in the antibody for 2 h, and repeat the washing process.

[0052] 7) Under light-protected conditions, mix substrate A and substrate B in equal volume ratio, drop them evenly onto the membrane surface, and immediately perform the development operation.

[0053] The results are as follows Figure 8The results showed that the nanobody band without DTT addition expressed at approximately 100 kDa. Figure 8 (a) The human single-chain antibody has two distinct bands at 100 kDa and 150 kDa, consistent with the light and heavy chain size of single-chain antibodies. Figure 8 (b)). Therefore, a total of 9 antibodies that could be expressed normally were obtained, of which 7 were nanobodies, namely 2S-29, 2S-31, 2S-39, 2S-42, 3S-3, 3S-22 and 3S-56, and 2 were single-chain antibodies, namely 3Fc-70 and 3Fc-128.

[0054] (2) Indirect ELISA detection: 1) Antigen coating: Dilute the eukaryotically purified FIPV S protein with coating buffer to ensure that the amount of antigen coated in each well of the ELISA plate is 100 ng / 100 μL. Coating conditions can be selected as 4°C overnight or 37°C incubation for 2 h.

[0055] 2) Washing and blocking: Discard the coating solution and wash three times with 200 μL of PBST washing buffer, each time for 5 min. After washing, add 200 μL of 5% BSA blocking buffer to each well. Incubate at 37°C for 1 h.

[0056] 3) After removing the blocking solution, wash the microplate three times with PBST buffer (200 μL per well), add 100 μL of the cell supernatant to be tested as the primary antibody to each well, and incubate at 37°C for 60 min.

[0057] 4) After removing the cell supernatant and washing 3 times, add 100 μL of HRP-labeled goat anti-human secondary antibody (dilution ratio 1:10000) to each well and incubate at room temperature for 60 min. After incubation, wash 3 times with 200 mL PBST per well.

[0058] 5) Add 100 μL of TMB colorimetric solution to each well. After color development for 10 min, add 50 µL of stop solution to terminate the reaction and read the OD. 450nm Absorbance value.

[0059] The results are as follows Figure 9 The results showed that the two nanobodies, 3S-3 and 3S-22, exhibited significant binding advantages, while in the recombinant single-chain antibody group, the binding activity of the eukaryotic antibodies 3Fc-70 and 3Fc-128 was reduced compared to prokaryotic expression.

[0060] (III) Eukaryotic purification of recombinant antibodies HEK293F suspension cells were revived and cultured at 37℃ and 5% CO2 until the cell density reached 2.5 × 10⁻⁶ cells / year. 6When cells / mL and in good condition, transiently co-transfect the pCAGGS-VH-Fc / pCAGGS-Vκ-Fc recombinant plasmid (or 100 μg nanobody recombinant plasmid) at a ratio of 3:2. After 7 days of transfection, the antibody is purified by affinity chromatography using Protein A column. (1) Sample pretreatment: 100 mL of cell culture medium was aliquoted into centrifuge bottles, balanced, and placed in large-capacity ultracentrifuge tubes. The tubes were centrifuged at 5,000 rpm for 1 h. The supernatant was collected and filtered twice with a 0.45 mm filter membrane, followed by two more filtrations with a 0.22 mm filter membrane, until the cell supernatant was clear and free of impurities. After sample collection, the cell supernatant contained the target protein, which is easily degraded. Low temperature was maintained throughout the process.

[0061] (2) Column equilibration: Rinse the pump tubing with 0.22 mm filtered ultrapure water, connect the Protein A column, and rinse sequentially at a flow rate of 2 mL / min: 5 CV ultrapure water to remove ethanol and contaminating proteins and 5 CV equilibration buffer until the baseline is stable.

[0062] (3) Sample loading: Mix cell supernatant with equilibration solution at a ratio of 1:1, load the sample at 1-2 mL / min, collect the flow-through solution simultaneously and monitor UV280.

[0063] (4) Elution: Rinse the AB flow path with 10 mL / min of ultrapure water and equilibration solution. Set pump A (equilibration solution) to 2 mL / min and pump B (elution solution) to 0% until the baseline is stable. Then switch to 100% B solution (elution solution) for one-step elution. Collect the antibody from the sample outlet and immediately add 1 M Tris-HCl (pH 9.0) for neutralization.

[0064] (5) Maintenance of Protein A affinity chromatography column and protein purification instrument: First, rinse the column with elution buffer. After the UV absorbance value stabilizes, rinse with 1M acetic acid at a flow rate of 2 mL / min for 5 column volumes, followed by rinsing with 0.1M NaOH solution for 5 column volumes. After completing the above operations, equilibrate the chromatography column with 5 column volumes of equilibration buffer, then rinse with ultrapure water for 10 column volumes. Finally, seal the column with 20% ethanol solution. After disassembling the chromatography apparatus, adjust the flow rate to 10 mL / min and pump 50 mL of 1M NaOH and ultrapure water into the instrument through pipelines A and B, respectively. Finally, seal the pipelines with 20% ethanol.

[0065] The purified antibodies were identified using SDS-PAGE gel electrophoresis. The results are as follows: Figure 10 The results show that the nanobody bands without DTT are around 100 kDa, while the full-length human antibody is around 250 kDa. Figure 10(a)). The main bands of the nanobodies with added DTT were all around 35 kDa, while the recombinant full-length human antibody showed a relatively single target protein band at approximately 55 kDa. Figure 10 (b) The size was consistent with the prediction, indicating that the expression and purification of these 9 antibodies were effective.

[0066] (iv) Indirect immunofluorescence assay (IFA) of antibodies HEK293T cells were seeded into 24-well culture plates. After cell adhesion reached 70%–80%, the cells were transfected with the pCAGGS-FIPV-S recombinant plasmid. Thirty h after transfection, the cells were fixed with 4% paraformaldehyde. Nanobodies and single-chain antibodies (1:300) were used as primary antibodies. Mouse anti-His IgG (1:300) was used as the primary antibody in the positive control wells, and PBS buffer was used as the primary antibody in the negative control wells. Goat anti-human IgG FITC (1:300) was used as the secondary antibody in the experimental and negative control groups, while goat anti-mouse IgG FITC (1:300) was used as the secondary antibody in the positive control group. IFA was used to identify the specific binding of the antibodies to the FIPV-S protein. Results are as follows: Figure 11 The results showed that green fluorescence appeared in cells incubated with 5 nanobodies and 2 full-length human antibodies as primary antibodies, consistent with the positive control. The 5 nanobodies were 2S-29, 2S-39, 2S-42, 3S-3 and 3S-56, and the 2 full-length human antibodies were 3Fc-70 and 3Fc-128. The cells incubated with the other two nanobodies as primary antibodies and the negative control did not show green fluorescence. This indicates that 7 out of the 9 antibodies can specifically bind to the FIPV S protein.

[0067] Example 3: Establishment of a FIPV double antibody sandwich ELISA detection method (a) Horseradish peroxidase (HRP) labeled antibody This experiment used the HRP antibody labeling kit manufactured by Mekwand, and HRP was labeled onto full-length antibodies and nanobodies according to the instructions.

[0068] (1) Take the antibody to be labeled out of the -80℃ freezer, place it on ice to thaw, mix well and measure the antibody concentration. Adjust the concentration with PBS buffer to dilute the antibody concentration to about 1 mg / mL to facilitate subsequent steps. (2) Add 1 mg of antibody to the tube that has activated HRP, mix well, add 100 mL of labeling buffer, and incubate on a shaker in the dark for 3 h. (3) Add the reaction enhancer to the labeling tube at a volume ratio of 1:10, vortex mix, and react at room temperature in the dark for 15 min; (4) Add 100 mL of storage solution to stop the reaction. Incubate at room temperature in the dark for 15 min. Then transfer to an ultrafiltration tube and replace the free HRP with PBS buffer. Add an equal volume of glycerol to the enzyme-labeled antibody and mix well. Store at -20℃.

[0069] (5) Add an equal volume of glycerol to the enzyme-labeled antibody, mix well, and store at -20℃.

[0070] (II) Determination of enzyme-labeled antibody titer (1) Using FIPV S protein as the coating antigen, the antigen was diluted to a final concentration of 1 mg / mL with coating buffer. 100 mL of diluted antigen was added to each well of a 96-well microplate and the plate was placed at 4°C for 16 h of solid-phase coating.

[0071] (2) Take 5 HRP-labeled nanobodies and serially dilute the enzyme-labeled antibodies. The dilution gradient is set as follows: 1:200, 1:400, 1:800, 1:1600, 1:3200, 1:6400, 1:12800, 1:25600, 1:51200, 1:102400; (3) After discarding the coating solution, wash the wells with phosphate-buffered saline (PBST) containing 0.1% Tween-20 at a volume of 150 mL / well, repeating 3 times. After the last wash, pat dry any remaining liquid on absorbent paper. Add the diluted enzyme-labeled antibody to the corresponding wells and incubate at 37°C for 30 min. Repeat the washing steps 3 times after incubation.

[0072] (4) After washing the plate three times, pat it dry. Add 100 mL of colorimetric reagent to each well and develop the color for 5 min in the dark. After the color development is complete, add 50 mL of stop solution to terminate the reaction and read the OD. 450nm Absorbance. Results are as follows: Figure 12 The results showed that among the five enzyme-labeled antibodies, the 2S-29 antibody had the highest titer, followed by the 2S-39 antibody, while the 3S-3 antibody had the lowest titer.

[0073] (iii) Antibody pairing test (1) Seven human nanobodies and two single-chain antibodies were used as capture antibodies, diluted with PBS buffer to 2 mg / mL, coated with 100 mL / well of ELISA plates, and incubated overnight at 4 °C. (2) Wash the overnight coated microplate three times with 0.1% PBST solution, invert the microplate onto absorbent paper, discard the liquid in the wells, add 200 mL of 5% BSA to each well, and block at 37℃ for 1 h. (3) After sealing, wash each well three times with 200 mL PBST, add 100 mL FIPV S protein, and incubate at 37 ℃ for 1 h; (4) After washing with PBST 3 times, add 0.5 mg / mL HRP enzyme-labeled antibody as secondary antibody (100 mL / well) and incubate at 37 °C in the dark for 1 h.

[0074] (5) Wash the microplate three times with 0.1% PBST solution, pat the liquid in the wells dry, add 100 mL of TMB chromogenic solution to each well, and develop the color at room temperature in the dark for 10 min. After development, add stop solution (50 mL / well) to stop the color development.

[0075] (6) Preheat the multi-functional microplate reader, use PBS buffer as a negative control, record the absorbance value at A450, and compare the ratio of absorbance of the experimental group to the negative control (P / N value) to determine the optimal antibody pairing. Results are as follows: Figure 13 The results show that when 2S-39 is used as the detection antibody and 2S-29 is used as the capture antibody, the OD... 450nm The values ​​are the highest. Based on this, 2S-29 was selected as the best capture antibody and 2S-39 as the best detection antibody. These two nanobodies can effectively recognize and bind to the FIPV S protein with high affinity.

[0076] (iv) Optimization of reaction conditions (1) Determination of the optimal working concentration of antibodies for capturing and detecting antibodies The optimal coating concentration of the capture antibody and the working concentration of the detection antibody were determined by matrix titration.

[0077] 1) Dilute the capture antibody 2S-29 to concentrations of 10 mg / mL, 8 mg / mL, 6 mg / mL, 5 mg / mL, 4 mg / mL, 3 mg / mL, 2 mg / mL, and 1 mg / mL, add 100 mL to each well, and coat the well overnight at 4°C; 2) Add 150 mL of PBST buffer to each well, let stand for 2 min, wash the plate three times, add 100 mL of 5% BSA solution to each well, and block at 37℃ for 1 h. 3) After sealing, wash each well three times with 200 mL PBST, add 100 mL FIPV S protein, and incubate at 37 ℃ for 1 h. Use PBS buffer as a negative control. 4) After completing the antigen incubation, wash each well three times with 200 mL PBST. 2S-39 serially dilute the enzyme-labeled antibody with dilution gradients of 1:800, 1:1600, 1:3200, and 1:6400. Add 100 mL of enzyme-labeled antibody dilution solution to each well and incubate at 37°C for 1 h. 5) After color development and termination, read the OD using a microplate reader. 450nm Value. Result as follows Figure 14 The results showed that the optimal concentration of the enzyme-labeled antibody was 6 μg / mL, the optimal ratio of the detection antibody was 1:1600, and the P / N value was 10.76.

[0078] (2) Optimization of sealing fluid Based on the determined optimal working concentrations of the capture antibody and the detection antibody, different concentrations of BSA solution and skim milk powder were added to the ELISA plate to optimize the type and concentration of blocking buffer. Since the optimal concentration of the capture antibody was 6 mg / mL and the dilution ratio of the detection antibody was 1:1600, this study used six blocking buffers: 1%, 2%, and 5% bovine serum albumin (BSA) solution and skim milk powder. Blocking was performed at 37℃ for 1 h, and the optimal blocking time was determined based on the P / N ratio. The results are as follows: Figure 15 The results showed that the optimal type and concentration of the blocking solution was 5% BSA, and the optimal blocking time was 1 h. Therefore, this experiment selected 5% BSA as the blocking solution and incubated the reaction at 37 ℃ for 1 h.

[0079] (3) Optimize the incubation time of the antigen protein. This experiment determined the optimal antigen incubation time based on the optimized concentrations of capture and detection antibodies, as well as the type and concentration of blocking buffer. Incubation was performed at 37°C for 15 min, 30 min, 45 min, 60 min, and 75 min, respectively. After color development, the P / N ratio was calculated to obtain the optimal antigen incubation time. Results are as follows: Figure 16 The results showed that the antigen incubation time was 1 hour when the P / N value was highest. Therefore, the optimal incubation time for the antigen protein was ultimately determined to be 1 hour.

[0080] (4) Optimize the incubation time for antibody detection To determine the optimal reaction time for the detection antibody, the effects of different incubation times on the detection results were compared under constant conditions of antibody working concentration, blocking solution type and concentration, and antigen protein incubation time. The specific procedure was as follows: the incubation time of the detection antibody was set to five time gradients: 15, 30, 45, 60, and 75 minutes. After the reaction was terminated, the optimal antigen incubation time was determined by calculating the P / N value. The results are as follows. Figure 17 The results showed that the optimal incubation time for the enzyme-labeled antibody was 1 hour when the P / N value was highest. Therefore, this experiment ultimately determined the optimal time for antibody detection to be 1 hour.

[0081] (5) Sensitivity detection Based on the optimal conditions determined in the above experiments (the optimal reaction concentration of capture nanobody 2S-29 is 6 mg / mL, the dilution ratio of detection antibody 2S-39 is 1:1600, the blocking solution conditions are 5% BSA blocking for 1 h, and the optimal incubation time for antigen protein and detection antibody is 1 h), FIPV S protein was serially diluted to concentrations of 2 mg / mL, 1 mg / mL, 0.5 mg / mL, 0.25 mg / mL, 0.125 mg / mL, 0.062 mg / mL, 0.031 mg / mL, 0.015 mg / mL, 0.008 mg / mL, 0.004 mg / mL, and 0.002 mg / mL, for a total of 11 dilutions, and the absorbance values ​​were measured for each. Table 5-8 shows that, using concentration (ng / mL) as the independent variable (x) and OD450 absorbance as the dependent variable (y), linear regression analysis revealed a good linear relationship between FIPV S protein concentrations in the range of 2 ng / mL to 1 μg / mL. The obtained linear regression equation was: y = 0.5655x - 0.3665, with an R² value of 0.9932. Therefore, under optimal reaction conditions, the limit of detection for this method reaches 4 ng / mL. Figure 18 ).

[0082] Table 8. Results of sensitivity analysis of the ELISA method

[0083] (6) Specific detection Based on previous experimental results, three antigen proteins—TGEV, PDCoV, and ASFV—were selected and simultaneously incubated with the FIPV S protein under the same conditions. OD was then quantitatively detected using an enzyme-linked immunosorbent assay (ELISA) reader. 450nm The absorbance values ​​were measured, and the P / N ratio was calculated to analyze the specificity of the constructed double-antibody sandwich ELISA detection method. The results are as follows: Figure 19 The results showed that when FIPV S protein was used as the antigen, a P / N value greater than 2 was considered positive, while when TGEV, PDCoV and ASFV proteins were used as antigens, a P / N value less than 2 was considered negative. Therefore, the double-antibody sandwich ELISA method constructed in this experiment has good specificity.

[0084] (7) Precision determination of FIPV S protein double antibody sandwich ELISA detection method Intra-assay repeatability testing: FIPV S protein was diluted at three concentration gradients of 30 ng / mL, 20 mg / mL, and 10 mg / mL. Six intra-assay repeatability tests were performed on the same batch of ELISA plates using these three concentrations. Results are as follows: Figure 20 The coefficients of variation were 7.19%, 4.43%, and 7.81%, respectively, all <10%.

[0085] Inter-batch repeatability testing: FIPV S protein was diluted at three concentration gradients of 30 ng / mL, 20 mg / mL, and 10 mg / mL. Absorbance was measured once daily for three consecutive days. Results are as follows: Figure 21 The coefficients of variation were 7.09%, 4.28%, and 7.64%, respectively. The experimental results indicate that the coefficients of variation were <10% for both intra-batch and inter-batch differences.

[0086] The above description is only for better explaining the embodiments of the present invention and is not intended to limit them. Any modifications or equivalent substitutions that do not depart from the spirit and scope of the present invention shall fall within the scope of the present invention.

Claims

1. A nanobody targeting feline infectious peritonitis virus, characterized in that, The nanobodies include 2S-29 antibody and 2S-39 antibody; The nanobody has a heavy chain variable region comprising three complementarity-determining regions HCDR1, HCDR2, and HCDR3 and four framework regions HFR1, HFR2, HFR3, and HFR4, arranged in the order HFR1, HCDR1, HFR2, HCDR2, HFR3, HCDR3, and HFR4. For the 2S-29 antibody: the amino acid sequence of HCDR1 is shown in SEQ ID NO:1.1; the amino acid sequence of HCDR2 is shown in SEQ ID NO:2.1; the amino acid sequence of HCDR3 is shown in SEQ ID NO:3.1; the amino acid sequence of HFR1 has at least 80% identity with the sequence shown in SEQ ID NO:4.1; the amino acid sequence of HFR2 has at least 80% identity with the sequence shown in SEQ ID NO:5.1; the amino acid sequence of HFR3 has at least 80% identity with the sequence shown in SEQ ID NO:6.1; the amino acid sequence of HFR4 has at least 80% identity with the sequence shown in SEQ ID NO:7.

1. For the 2S-39 antibody: the amino acid sequence of HCDR1 is shown in SEQ ID NO:1.2; the amino acid sequence of HCDR2 is shown in SEQ ID NO:2.2; the amino acid sequence of HCDR3 is shown in SEQ ID NO:3.2; the amino acid sequence of HFR1 has at least 80% identity with the sequence shown in SEQ ID NO:4.2; the amino acid sequence of HFR2 has at least 80% identity with the sequence shown in SEQ ID NO:5.2; the amino acid sequence of HFR3 has at least 80% identity with the sequence shown in SEQ ID NO:6.2; and the amino acid sequence of HFR4 has at least 80% identity with the sequence shown in SEQ ID NO:7.

2.

2. The nanobody against feline infectious peritonitis virus according to claim 1, characterized in that, The amino acid sequence of the 2S-29 antibody is shown in SEQ ID NO:8.1; the amino acid sequence of the 2S-39 antibody is shown in SEQ ID NO:8.

2.

3. The nanobody against feline infectious peritonitis virus according to claim 1, characterized in that, The nanobody described herein has a heavy chain constant region containing a sequence that has at least 70% identity with the sequence shown in SEQ ID NO:

9.

4. Any one of the following substances: (i) A polynucleotide molecule comprising a nucleotide sequence encoding the nanobody of any one of claims 1 to 2; (ii) Expression vectors, including the polynucleotide molecules in (i); (iii) Engineered bacteria, including the expression vectors in (ii); (iv) Host cells, including the expression vectors in (ii); (v) Products for detecting feline infectious peritonitis virus, comprising the nanobody as claimed in any one of claims 1 to 2 and / or the polynucleotide molecule in (i) and / or the expression vector in (ii) and / or the engineered bacteria in (iii) or the host cell in (iv); (vi) A pharmaceutical composition comprising the nanobody of any one of claims 1 to 2 and / or the polynucleotide molecule in (i) and / or the expression vector in (ii) and / or the engineered bacteria in (iii) and / or the host cell in (iv); (vii) A pharmaceutical formulation comprising the nanobody as claimed in any one of claims 1 to 2 and / or the polynucleotide molecule in (i) and / or the expression vector in (ii) and / or the engineered bacteria in (iii) and / or the host cell in (iv) and / or the pharmaceutical composition in (vi).

5. The use of the nanobody as described in any one of claims 1 to 2, or the polynucleotide molecule, expression vector, engineered bacteria, host cell, pharmaceutical composition, or pharmaceutical preparation as described in claim 3, in the preparation of a feline infectious peritonitis virus detection product.

6. A double-antibody sandwich ELISA method for detecting feline infectious peritonitis virus using the nanobody described in any one of claims 1 to 2, characterized in that, Includes the following steps: An enzyme-labeled 2S-29 antibody is prepared as a capture antibody, and an HRP-labeled 2S-39 antibody is prepared as a detection antibody. The sample to be tested is added to the capture antibody to allow it to bind, and then the detection antibody is added. After color development, the sample is ready. The double-antibody sandwich ELISA method is used for non-diagnostic detection.

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