High-affinity single-chain neutralizing antibody targeting porcine epidemic diarrhea virus S1 protein and application of high-affinity single-chain neutralizing antibody

Abstract

The invention discloses a high-affinity single-chain neutralizing antibody targeting porcine epidemic diarrhea virus S1 protein and application thereof, and relates to the technical field of biology. The single-chain neutralizing antibody comprises a heavy chain variable region and a light chain variable region which are connected by a connecting peptide, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO. 1; the amino acid sequence of the light chain variable region is as shown in SEQ ID NO. 2. Functional verification experiments show that the single-chain neutralizing antibody can effectively block infection of PEDV virus on cells and effectively inhibit virus replication and diffusion. ELISA detection proves that the binding activity of the monoclonal antibody and PEDV S1 protein is superior to that of a traditional monoclonal antibody, and SPR technology determination proves that the antigen recognition sensitivity of the monoclonal antibody is extremely high. The single-chain neutralizing antibody not only can be used for research and development of anti-PEDV infection drugs, but also can be used for constructing a high-sensitivity PEDV diagnosis system, and a brand new technical scheme is provided for precise prevention and control of PEDV.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a high-affinity single-chain neutralizing antibody targeting S1 protein of porcine epidemic diarrhea virus and application thereof. BACKGROUND

[0002] Porcine epidemic diarrhea (PED) is an acute enteric disease caused by porcine epidemic diarrhea virus (PEDV), with watery diarrhea, vomiting, dehydration, and anorexia as the main clinical features. PEDV can infect pigs of all ages, and the clinical symptoms vary depending on the virulence, immune status, and age of the animal. The mortality rate for newborn piglets under 3 days of age can be as high as 100%, and the mortality rate decreases with increasing age.

[0003] PEDV belongs to the single-stranded positive-sense RNA virus with a capsid, and belongs to the alpha-coronavirus subgenus. The virus particles are round or oval in shape, with a layer of rod-shaped spicules on the surface, and the diameter is about 80-120 nm. The genome is about 28Kd long, containing a 5' cap structure (encoding 10 amino acids), a 3' poly A tail structure, and 7 open reading frames (ORF). The spicule protein (S) is mainly responsible for the interaction with specific host cell receptors and mediates the membrane fusion of virus entry into host cells, and plays a crucial role in inducing host neutralizing antibodies.

[0004] According to the differences in S gene sequence, PEDV can be divided into G1 group (classical strain, such as CV777) and G2 group (variant strain), and the G2 group is further divided into G2a, G2b, G2c, G2d, etc. The current main epidemic strain is G2a subtype, which has strong virulence, fast transmission, and can cause 100% mortality in piglets.

[0005] Currently, vaccination is still the main means of preventing and controlling PEDV, and there are various types on the market, including classical strain live vaccine, variant strain live vaccine, inactivated vaccine, and attenuated vaccine. However, due to the continuous and rapid variation of the virus, the antigen matching degree between the existing vaccines, especially those based on early strains, and the current epidemic strains has decreased, resulting in a significant decrease in cross-protection effect. In addition to limited protection, there are other inherent limitations of vaccine immunization: first, immune protection requires a certain induction period, which cannot provide immediate passive immunity for newborn piglets; second, live vaccines, especially attenuated vaccines, may have potential safety risks such as virulence reversion. Therefore, in the face of the continuous emergence of new variants, the protection effect of traditional vaccines is not complete or unstable, increasing the difficulty of prevention and control.

[0006] Neutralizing antibodies play a crucial role in preventing and treating viral infections. They prevent viral entry into cells for replication and spread by recognizing and binding to specific proteins on the surface of the virus, preventing the virus from binding to host cell receptors. This mechanism not only reduces viral infection, but also significantly reduces disease symptoms caused by infection. However, although monoclonal antibodies have high specificity, they have large molecular weights, are difficult to diffuse, and have high expression costs, and need to be modified to have diverse functions and properties. Therefore, there is an urgent need in the art for a new PEDV prevention and control means that can overcome the above-mentioned defects.

[0007] Single-chain variable fragment (ScFv) is a kind of recombinant protein formed by connecting the heavy chain variable region (VH) and the light chain variable region (VL) through a flexible peptide linker. Its unique structure has many unique advantages: first, the molecular weight is small, ScFv shows stronger tissue penetration ability and lower immunogenicity; and it lacks the constant region (Fc segment), which is highly plastic and can bind to various molecules to mediate diverse killing effects. These characteristics make ScFv an ideal building block that can be further developed into targeted biological agents such as immunotoxins, bispecific antibodies, CAR-T cells, etc. However, in the current technical field, there is still a key technical gap in the research of single-chain antibodies against porcine epidemic diarrhea virus (PEDV), and there is no related report on single-chain antibodies against porcine epidemic diarrhea virus (PEDV) with high neutralizing activity. In view of this, the core purpose of the present application is to fill this technical gap and provide single-chain antibodies against porcine epidemic diarrhea virus (PEDV) with high neutralizing activity to meet the application needs in the related field. SUMMARY

[0008] The purpose of the present application is to provide a high-affinity single-chain neutralizing antibody targeting porcine epidemic diarrhea virus S1 protein and its application to solve the problems existing in the prior art. The single-chain neutralizing antibody can effectively neutralize porcine epidemic diarrhea virus and block porcine epidemic diarrhea virus infection.

[0009] To achieve the above-mentioned purpose, the present application provides the following solutions: The present application provides a high-affinity single-chain neutralizing antibody targeting porcine epidemic diarrhea virus S1 protein, comprising a heavy chain variable region and a light chain variable region connected by a connecting peptide. The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO. 1; the amino acid sequence of the light chain variable region is shown in SEQ ID NO. 2.

[0010] Further, the amino acid sequence of the high-affinity single-chain neutralizing antibody is shown in SEQ ID NO. 5.

[0011] The application also provides a gene encoding the high-affinity single-chain neutralizing antibody.

[0012] The application also provides a recombinant expression vector comprising the gene.

[0013] The application also provides a recombinant host cell comprising the recombinant expression vector.

[0014] The application also provides use of the gene, the recombinant expression vector or the recombinant host cell in preparation of the high-affinity single-chain neutralizing antibody.

[0015] The application also provides use of the high-affinity single-chain neutralizing antibody in preparation of a medicine for resisting porcine epidemic diarrhea virus.

[0016] The application also provides a medicine for resisting porcine epidemic diarrhea virus, wherein the active ingredient comprises the high-affinity single-chain neutralizing antibody.

[0017] The application also provides use of the high-affinity single-chain neutralizing antibody in preparation of a detection kit for porcine epidemic diarrhea virus.

[0018] The application also provides a detection kit for porcine epidemic diarrhea virus, comprising the high-affinity single-chain neutralizing antibody.

[0019] The application discloses the following technical effects: The application successfully develops a single-chain neutralizing antibody targeting porcine epidemic diarrhea virus (PEDV), wherein the antibody connects a heavy chain variable region and a light chain variable region through a flexible connecting peptide to form a complete structure. Function verification experiments show that the neutralizing potency of the single-chain neutralizing antibody to PEDV is 4.38 mu g / mL, the single-chain neutralizing antibody can block the infection of Vero cells by PEDV in a concentration-dependent manner, and effectively inhibits the replication and spread of the virus. ELISA detection proves that the binding activity of the single-chain neutralizing antibody to PEDV S1 protein is better than that of a traditional monoclonal antibody, and the affinity constant KD of the single-chain neutralizing antibody to S antigen is as low as 0.0467 nM, and the antigen recognition sensitivity is extremely high. In addition, the single-chain neutralizing antibody has small molecular weight, strong tissue penetration and low immunogenicity, can be efficiently prepared through a eukaryotic expression system, can be used for research and development of an anti-PEDV infection medicine, and can build a high-sensitivity PEDV diagnosis system, and provides a new technical scheme for accurate prevention and control of PEDV, and has great industrialization value and clinical application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0020] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed in the embodiments will be briefly introduced below. Obviously, the drawings in the following description only constitute some embodiments of the present application, and for those skilled in the art, other drawings can also be obtained from these drawings without creative effort.

[0021] Figure 1 Agarose gel electrophoresis diagram of total RNA; Figure 2 Agarose gel electrophoresis diagram of heavy chain (VH), light chain (VL) and single chain antibody (ScFv); wherein, M: DNA marker DL5000; Figure 3 Western Blot detection diagram of single chain antibody expression; Figure 4 Determination result diagram of single chain antibody and PEDV neutralization experiment; Figure 5 ELISA detection result diagram of single chain antibody and antigen binding activity; Figure 6 SPR detection result diagram of single chain antibody and S antigen affinity. DETAILED DESCRIPTION

[0022] The various exemplary embodiments of the present application will now be described in detail, which should not be considered as limiting the present application, but should be understood as a more detailed description of certain aspects, characteristics and embodiments of the present application.

[0023] It should be understood that the terms described in the present application are only for describing the specific embodiments, and are not used to limit the present application. In addition, for the numerical range in the present application, it should be understood that each intermediate value between the upper limit and the lower limit of the range is also specifically disclosed. Each smaller range between any stated value or intermediate value in the range, and any other stated value or intermediate value in the range is also included in the present application. The upper limit and the lower limit of these smaller ranges can be independently included or excluded from the range.

[0024] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present application pertains. Although preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials in connection with which the documents are concerned. In the event of conflict between the content of the specification and any incorporated document, the content of the specification controls.

[0025] Many modifications and variations of the present application described in the specification are possible without departing from the scope or spirit of the application. Other implementations of the application will be apparent to those skilled in the art from consideration of the specification and practice of the application disclosed herein. The specification and examples are illustrative only.

[0026] As used herein, the terms "comprise", "comprising", "include", "including", "have", "having" and the like are open-ended terms that are intended to mean including, but not limited to.

[0027] Example 1 Obtaining of Anti- Porcine epidemic diarrhea virus single-chain antibody encoding gene The hybridoma cell strain secreting anti-porcine epidemic diarrhea virus monoclonal antibody was prepared by immunizing mice with porcine epidemic diarrhea virus S1 protein. The hybridoma cells in the logarithmic growth phase were collected, and the RNA in the cells was extracted by using the RNA extraction kit of QIAGEN company, which has high cell lysis and RNA separation capacity. The product has three obvious bands, 28S, 18S and 5S respectively Figure 1 ), which correspond to different subunits of ribosomal RNA respectively. The bands are clear and no diffusion appears, which indicates that the RNA sample is of good quality, non-degraded and complete, providing a high-quality template for subsequent gene amplification. Then, reverse transcription was performed using the Prime seript TMRT-PCR Kit reagent kit of TAKARA company to obtain cDNA.

[0028] The cDNA product was amplified by using KOD plus high-fidelity enzyme and mouse-derived heavy chain variable region (VH) and light chain variable region (VL) primers in the Ig-Primer Sets kit of Novagen company to obtain VH and VL genes, which are about 357 bp and 321 bp in size respectively, consistent with the actual gene fragment base size, and meet the expected results Figure 2 The left), and then the PCR product was recovered by using the gel recovery kit to recover the target fragment.

[0029] The PCR reaction system was as follows: 10x buffer 5 μL, Mg2SO4 3 μL, 10 mM dNTP 5 μL, upstream primer 1.5 μL, downstream primer 1.5 μL, template DNA 5-100 ng, KOD plus DNA Poymerase 1 μL, and nuclease-free water was added to 50 μL.

[0030] The PCR reaction conditions were as follows: 94℃ 2 min; 94℃ 30 s, 55℃ 30 s, 68℃ 2 min, 30 cycles; 72℃ 7 min, 16℃ ∞.

[0031] The recovered VH and VL genes were ligated using linker peptide coding sequences to construct the ScFv gene: VL, linker peptide, and VH fragments were sequentially ligated using overlap extension PCR (SOE-PCR), with the linker peptide being (Gly4Ser)3. The primers used included a VL 5' end sequence in the upstream primer, a VH 3' end sequence in the downstream primer, and overlapping sequences between the VL 3' end and the linker peptide 5' end, and between the linker peptide and the VH 5' end, respectively. The size of the spliced ​​single-chain antibody gene was approximately 723 bp. Figure 2 (Right) The corresponding bands can be clearly seen, indicating that the splicing reaction was successful and a single-chain antibody fragment of the expected size was obtained.

[0032] Sequencing analysis of the obtained ScFv gene revealed the following: the amino acid sequence of its heavy chain variable region is shown in SEQ ID NO. 1, and the nucleotide sequence of its encoding gene (VH gene) is shown in SEQ ID NO. 3; the amino acid sequence of its light chain variable region is shown in SEQ ID NO. 2, and the nucleotide sequence of its encoding gene (VL gene) is shown in SEQ ID NO. 4. These results demonstrate that this invention successfully obtained a single-chain antibody encoding gene against porcine epidemic diarrhea virus with a clearly derived and defined sequence, providing a reliable genetic basis for subsequent recombinant expression, purification, and functional verification of the single-chain antibody.

[0033] SEQ ID NO. 1: EVQLQQSGPELVKPGASVKISCKASGYTFTDYYMTWVKQSHGKSLEWIGHIYPNNGGTSYNQKFKGKATLTVDKSSSTAYMEVRSLTSEDSAVYYCARLGILGWYFDVWGAGTTVTVSS.

[0034] SEQ ID NO. 2: DIQMTQSPASSLSASEGETVTITCRASENIYSYLVWYQQKQGKSPQLLVYNAKTLAEGVPSRFSGSGSGTQFSLKINSLQPEDFGSYYCQHHYGIPFTFGSGTKLEIK.

[0035] SEQ ID NO.3: GAGGTCCAGCTGCAACAGTCTGGACCTGAGCTGGTGAAGCCTGGGGCTTCAGTGAAGATATCCTGTAAGGCTTCTGGATACACGTTCACTGACTACTACATGACCTGGGTGAAGCAGAGCCATGGAAAGAGCCTTGAGTGGATTGGACATATTTATCCTAACAATGGTGGTACTAGCTACAACCAGAAATTCAAGGGCAAGGCCACATTGACTGTAGACAAGTCCTCCAGCACAGCCTACATGGAGGTCCGCAGCCTGACATCTGAGGACTCTGCAGTCTATTACTGTGCAAGATTAGGGATACTGGGCTGGTACTTCGATGTCTGGGGCGCAGGGACCACGGTCACCGTCTCCTCA。

[0036] SEQ ID NO.4: GACATTCAGATGACGCAGTCTCCAGCCTCCCTATCTGCATCTGAGGGGGAGACTGTCACCATCACATGTCGAGCAAGTGAGAATATTTACAGTTATTTAGTATGGTATCAACAGAAACAGGGAAAATCTCCTCAGCTCCTGGTCTATAATGCAAAAACCTTAGCAGAAGGTGTGCCATCAAGGTTCAGTGGCAGTGGATCAGGCACACAGTTTTCTCTGAAGATCAACAGCCTGCAGCCTGAAGATTTTGGGAGTTATTACTGTCAACATCATTATGGTATTCCATTCACGTTCGGCTCGGGGACAAAGTTGGAAATAAAA。

[0037] Example 2 Expression and Purification of Single-Chain Antibody Based on the unique light chain (VL) and heavy chain (VH) sequences obtained, VL and VH are connected by a flexible Linker (Gly4Ser)3 (in the direction of VL-Linker-VH) and 6×His tags are added. Homologous arms matching the GSV0 vector were introduced at both ends of the gene. The GSV0 vector was digested with restriction endonucleases, and the linearized fragment was recovered. The reaction system was NEB CloneExpressII One Step Cloning Kit (catalog number C112): 50 ng linearized GSV0 vector, 150 ng ScFv gene, 4 μL 5×CE II Buffer, 2 μL Exnase II, and enzyme-free water to a final volume of 20 μL. The reaction was carried out at 37°C for 30 min, and terminated on ice. The recombinant product was electroporated into BL21 competent cells, and after recovery at 37°C and 220 rpm for 1 h, the cells were plated on LB plates containing 50 μg / mL kanamycin. Single clones were picked, cultured, and the recombinant plasmid was extracted. The correctness of the ScFv sequence was verified by sequencing using universal primers for the GSV0 vector. For clones with correct sequencing, plasmids were extracted in large quantities using the Omega plasmid extraction kit, the concentration was determined, and the plasmids were stored at -20°C for later use.

[0038] First, ExpiCHO cells were revived and cultured at 37°C, 8% CO2, and 125 rpm to maintain a cell density of 2 × 10⁶ cells / year. 6 cells / mL; adjust cell density to 3×10⁶ cells / mL 1 day before transfection. 6 cells / mL; In a 1 L culture system, 2 mg of ScFv expression plasmid was mixed with 6 mL of ExpiFectamine. TM CHO Reagent was used with 5 mL of OptiPRO. TM SFM was diluted, incubated at room temperature for 5 min, mixed, and incubated at room temperature for 20 min to form a DNA-liposome complex; the complex was then added to 1 L of Expi CHO cell suspension (density 5 × 10⁻⁶). 6 (cells / mL), cultured at 37℃, 8% CO2, and 125 rpm; 20 h after transfection, ExpiFectamine was added. TM CHOEnhancer and Expi CHO TM Feed, continue to cultivate for 7 days.

[0039] After 7 days of transfection culture, the cells were centrifuged at 5000×g for 15 min at 4℃, and the supernatant was collected and filtered through a 0.22 μm filter membrane to remove impurities. ProteinIso, manufactured by Beijing TransGen Biotech Co., Ltd., was used. ® The Ni-NTA Resin protein purification kit was used to purify the cell supernatant after centrifugation and harvest single-chain antibodies. The procedure is as follows: a) Column equilibrium: Take an appropriate amount of Ni 2+ -NTA, equilibrate with binding buffer, ready for use; b) Sample loading and binding: The collected supernatant is combined with pre-equilibrated Ni 2+ -NTA affinity chromatography medium was gently incubated overnight at 4°C to allow the histidine-tagged protein to fully bind to the medium. The next day, the mixture was loaded onto the chromatography column to specifically immobilize the target protein within the column; c) Washing: Rinse the chromatography column with washing buffer to remove non-specifically bound proteins; d) Elution: Elute using elution buffer and collect the eluent; e) Concentration: Combine the eluents containing the target protein and concentrate them using an ultrafiltration tube with a molecular weight cutoff of 10 kDa; f) Protein concentration was determined using the BCA protein quantification kit and aliquoted into 1.5 mL centrifuge tubes (100-500 μL per tube) and stored at -80℃.

[0040] 10 µL of the purified protein was subjected to SDS-PAGE electrophoresis (80 V for 20 min, 120 V for 60 min). After electrophoresis, a PVDF membrane with an appropriate pore size was selected based on the protein molecular weight. The PVDF membrane was activated by soaking in methanol for 30 s and then equilibrated in transfer buffer. The membrane was placed using the sandwich method, and the transfer conditions were set to a constant current of 200 mA for 90 min. After transfer, the membrane was incubated at room temperature for 2 h with blocking buffer diluted with TBST containing 5% skim milk. After washing the membrane three times with TBST, a single-chain antibody primary antibody diluted 1:5000 with TBST buffer containing 3% bovine serum albumin (BSA) was added, and the membrane was incubated overnight at 4°C with shaking. The next day, the primary antibody was recovered, and the membrane was washed with TBST. Then, HRP-labeled goat anti-mouse IgG secondary antibody diluted 1:5000 with TBST buffer containing 5% skim milk was added, and the membrane was incubated at room temperature with shaking for 1 h. Finally, the membrane was washed with TBST and exposed using ECL chromogenic solution. Figure 3 ).

[0041] The amino acid sequence of the single-chain antibody prepared in this embodiment is shown in SEQ ID NO.5, wherein positions 1-107 are the light chain variable region, positions 108-122 are the linker peptide (Gly4Ser)3, and positions 123-241 are the heavy chain variable region; the nucleotide sequence of the encoding gene of the single-chain antibody is shown in SEQ ID NO.6.

[0042] SEQ ID NO.5:DIQMTQSPASLSASEGETVTITCRASENIYSYLVWYQQKQGKSPQLLVYNAKTLAEGVPSRFSGSGSGTQFSLKINSLQPEDFGSYYCQHHYGIPFTFGSGTKLEIKGGGGSGGGGSGGGGSEVQLQQSGPELVKPGASVKISCKASGYTFTDYYMTWVKQSHGKSLEWIGHIYPNNGGTSYNQKFKGKATLTVDKSSSTAYMEVRSLTSEDSAVYYCARLGILGWYFDVWGAGTTVTVSS。

[0043] SEQ ID NO.6:

[0044] Example 3 Functional Validation of Single-Chain Antibodies Using a virus-infected cell model (monkey kidney cells Vero), the neutralizing titer of the single-chain antibody prepared in Example 2 was determined according to the method of fixing the virus and diluting the antibody. The steps are as follows.

[0045] a) Seed Vero cells in 96-well plates. When the cell density is approximately 90%, dilute PEDV with MM medium containing 5 μg / mL trypsin to a concentration of 100 TCID. 50 The single-chain antibody was used in MM medium, starting at 40 μg / mL, and serially diluted 10 times to obtain 2-fold concentrations. The experiment was repeated three times.

[0046] b) Mix 50 μL of diluted virus with 50 μL of antibody dilution by vortexing and incubate at 37°C for 1 h.

[0047] c) The virus-single-chain antibody mixture was then added to Vero cells washed with PBS and incubated in a cell culture incubator for 2 h.

[0048] d) After co-incubation, discard the supernatant, wash with PBS, and add MM medium containing 5 μg / mL trypsin again for 72 h of further culture.

[0049] e) Collect samples and perform IFA experiments using PEDV N antibody as the primary antibody.

[0050] f) After washing twice with PBS, add 100 μL of 4% paraformaldehyde diluted in PBS, fix at room temperature for 15 min, wash the cells with PBS, add 100 μL of 0.5% Triton X-100 diluted in PBS, punch wells at room temperature for 10 min, and wash again with PBS.

[0051] g) After washing, add 100 μL of single-chain antibody diluted 1:1000 with PBS, incubate at room temperature for 1 h, then wash the cells. Add 500 μL of AlexFlour-488-labeled goat anti-mouse IgG secondary antibody (1:1000 dilution) in the dark, incubate at room temperature in the dark for 1 h, wash the cells, add 100 μL of DAPI (1:1000 dilution) in the dark, incubate at room temperature in the dark for 5 min, and then wash the cells.

[0052] h) Add 100 μL of PBS, and finally observe the green fluorescent protein in the cells using a fluorescence microscope, recording the number of fluorescent wells in each column. Calculate the neutralizing titer of the monoclonal antibody using the Reed-Muench method.

[0053] i) Use GraphPad Prism 6.0.1 to analyze the data and plot dose-response curves.

[0054] Single-chain antibodies significantly reduced PEDV infection in Vero cells, indicating that single-chain antibodies have a neutralizing effect on the virus, and that this neutralizing effect is concentration-dependent. Figure 4 The neutralizing titer of the single-chain antibody determined by this invention was 4.38 μg / mL.

[0055] ELISA plates were coated with 100 μL of PEDV S1 protein (1.0 μg / mL) per well. After coating overnight at 4°C, the liquid in the wells was discarded, and the plates were washed three times with PBST and patted dry. Then, 200 μL of PBST containing 5% skim milk blocking solution was added to each well, and the plates were incubated in a humidified chamber at 37°C for 1.5 h. The plates were washed three times with PBST and patted dry. Add 100 μL of single-chain antibody diluted with blocking buffer at different concentrations (5, 0.5, 0.1, 0.05, 0.01, 0.005, 0.001, and 0.0001 μg / mL, a total of 8 concentrations) to each well. Incubate at 37°C in a humidified chamber for 1 h. Wash three times with PBST, blot dry, and add 100 μL of HRP-goat anti-mouse IgG enzyme-labeled secondary antibody (1:5000 dilution) to each well. Incubate at 37°C in a humidified chamber for 1 h. Wash again, blot dry, add TMB chromogenic buffer, incubate at 37°C in the dark for 15 min, and then add stop solution. Finally, use a microplate reader to detect the OD of the ELISA plate. 450 Value. The binding titer of the single-chain antibody to the PEDV S1 protein was detected. The single-chain antibody showed high binding activity to the PEDV S1 protein ( Figure 5 ).

[0056] To assess the affinity of the single-chain antibody, SPR was used to evaluate its binding ability to the antigen. First, a CM5 chip was used to capture PEDV-S1 protein. Then, the single-chain antibody was serially diluted with 1 × HBS-EP buffer (75, 37.5, 18.75, 9.38, 4.69, 2.34, 1.17, 0.58 nM). The KD value was 0.0467 nM. Figure 6 This indicates that single-chain antibodies have a high affinity for antigens, therefore, using single-chain antibodies as a tool for virus detection is more sensitive.

[0057] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A high-affinity single-chain neutralizing antibody targeting the S1 protein of porcine epidemic diarrhea virus, characterized in that, This includes heavy chain variable regions and light chain variable regions linked by linker peptides; The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.1; the amino acid sequence of the light chain variable region is shown in SEQ ID NO.

2.

2. The single-chain neutralizing antibody according to claim 1, characterized in that, The amino acid sequence of the high-affinity single-chain neutralizing antibody is shown in SEQ ID NO.

5.

3. A gene encoding a high-affinity single-chain neutralizing antibody as described in any one of claims 1-3.

4. A recombinant expression vector, characterized in that, Includes the coding gene as described in claim 3.

5. A recombinant host cell, characterized in that, Includes the recombinant expression vector as described in claim 4.

6. The use of the encoding gene as described in claim 3, the recombinant expression vector as described in claim 4, or the recombinant host cell as described in claim 5 in the preparation of the high-affinity single-chain neutralizing antibody as described in claim 1 or 2.

7. The use of a high-affinity single-chain neutralizing antibody as described in claim 1 or 2 in the preparation of a medicament against porcine epidemic diarrhea virus.

8. A drug for treating porcine epidemic diarrhea virus, characterized in that, The active ingredient includes the high-affinity single-chain neutralizing antibody as described in claim 1 or 2.

9. The use of a high-affinity single-chain neutralizing antibody as described in claim 1 or 2 in the preparation of a detection kit for porcine epidemic diarrhea virus.

10. A detection kit for porcine epidemic diarrhea virus, characterized in that, Includes the high-affinity single-chain neutralizing antibody as described in claim 1 or 2.

Images