Monoclonal antibody of receptor binding region of feline infectious peritonitis virus s protein and application thereof

Abstract

This invention relates to the field of biotechnology and aims to provide a monoclonal antibody against the receptor-binding region of the feline infectious peritonitis virus (FIP) S protein and its applications. The amino acid sequence of the heavy chain variable region of this monoclonal antibody is shown in SEQ ID NO.1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.2. The reactivity of the antibody (MAb) was identified using Western blot and IFA methods, and the antigenic epitope recognized by MAb was also identified. This antigenic epitope is conserved in type I feline coronaviruses, indicating that this monoclonal antibody can recognize multiple type I FCoV strains, exhibiting broad-spectrum recognition, and is not incidentally targeting a single strain. The antigenic epitope shows no cross-reactivity with type II FCoVs, indicating high specificity and its ability to differentiate between type I and type II infections, thus possessing diagnostic value.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a monoclonal antibody against the receptor-binding region of the feline infectious peritonitis virus (FIP) S protein and its applications. Background Technology

[0002] Feline coronavirus (FCoV) belongs to the order Nidovirales, family Coronavirinae, and genus Alphacoronavirus. It is a single-stranded positive-sense RNA virus. The FCoV genome is approximately 30 kb in length and includes 11 open reading frames encoding four major structural proteins: spike protein S, membrane protein M, envelope protein E, and nucleocapsid protein N, as well as seven non-structural proteins. The S protein is a type I transmembrane protein primarily involved in viral invasion; its short C-terminal portion is inside the viral envelope, while the remaining portion is external. This protein can be divided into two domains: the N-terminal (S1) domain contains a receptor-binding domain (RBD) responsible for receptor binding, and the C-terminal (S2) domain contains a fusion peptide that mediates fusion with the target cell membrane. The subunits of the S protein are activated by hydrolysis, which mediates the binding of the RBD to the host cell, allowing the viral fusion peptide to bind to the host cell membrane and form a "hairpin trimer" structure. This allows the virus to invade through the fusion pore. The S protein of FCoV plays an important role in viral invasion, replication, and the host's immune response.

[0003] Based on their pathogenicity, FCoV is classified into two biotypes: feline infectious peritonitis virus (FIPV) and feline enteric coronavirus (FECV). FECV is highly prevalent in cat populations and highly contagious, but these infections are mostly asymptomatic or cause only mild and transient diarrhea. FIPV, on the other hand, causes fatal feline infectious peritonitis (FIP) and can spread between domestic and wild cats worldwide. Based on amino acid differences in the S gene, FCoV can be divided into two serotypes, type I and type II, both of which are present in both the FECV and FIPV biotypes. In natural infections, FCoV-I is more prevalent than FCoV-II (accounting for approximately 80%-95%) and dominates in FIP cases in cats; FCoV-ІІ strains replicate well in vitro, while FCoV-І strains replicate more poorly in vitro. Therefore, most FCoV research in the past decade has been based on the FCoV-ІІ type, with less research on the widely prevalent FCoV-І type.

[0004] Feline coronavirus (FCoV) infects almost all feline populations globally and is one of the leading pathogens causing infectious diseases in felines. Currently, effective diagnostic methods and treatments for FCoV remain scarce, primarily due to the virus's high mutation rate and antibody-dependent enhancement (ADE) effect. ADE can cause vaccine-induced antibodies to actually promote viral invasion of cells, thus worsening the disease, significantly hindering the development of traditional vaccines. There are currently no registered specific treatments for FCoV. Therefore, improving the accuracy, sensitivity, and specificity of early FCoV diagnosis, and creating conditions for early intervention and treatment, is of paramount importance for the clinical management of this disease. Summary of the Invention

[0005] The purpose of this invention is to provide a monoclonal antibody against the receptor-binding region of the feline infectious peritonitis virus (FIP) S protein and its application.

[0006] The present invention relates to a monoclonal antibody against the receptor-binding region of the feline infectious peritonitis virus S protein. The amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO.1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.2.

[0007] The amino acid sequence of CDR1 in the heavy chain variable region is shown as positions 31-35 in SEQ ID NO.1.

[0008] The amino acid sequence of CDR2 in the heavy chain variable region is shown as positions 50-66 in SEQ ID NO.1.

[0009] The amino acid sequence of CDR3 in the heavy chain variable region is shown as positions 99-104 in SEQ ID NO.1;

[0010] The amino acid sequence of CDR1 in the light chain variable region is shown as positions 24-34 in SEQ ID NO.2;

[0011] The amino acid sequence of CDR2 in the light chain variable region is shown as positions 50-56 in SEQ ID NO.2;

[0012] The amino acid sequence of CDR3 in the light chain variable region is shown as positions 89-96 in SEQ ID NO.2.

[0013] Furthermore, the nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO.3, and the nucleotide sequence encoding the light chain variable region of the monoclonal antibody is shown in SEQ ID NO.4.

[0014] Furthermore, the antigenic epitope of the feline infectious peritonitis virus (FIP) S protein, which can specifically bind to the aforementioned monoclonal antibody targeting the receptor-binding region of the FIP S protein, is described above. 39 DGSIFVNGYKYF 50 .

[0015] The present invention provides a nucleic acid capable of encoding the above-mentioned antigenic epitopes, wherein the nucleic acid is a gene sequence capable of encoding an antigenic epitope of the feline infectious peritonitis virus S protein.

[0016] The present invention also provides the use of the above-mentioned monoclonal antibody against the receptor-binding region of the feline infectious peritonitis virus (FIP) S protein in the preparation of FIP detection kits and / or test strips.

[0017] A feline infectious peritonitis virus (FIP) detection kit comprising a monoclonal antibody against the receptor-binding region of the FIP S protein.

[0018] The beneficial effects of this invention are:

[0019] FCoV is serologically classified into type I and type II. This invention selected the HLJ / HRB / 2016 / 10 strain (type I) with biotype FIPV. The base sequence of its S protein RBD region was optimized according to E. coli codons, and the protein was expressed and purified using a prokaryotic system. After immunizing mice, an S-RBD protein monoclonal antibody was prepared. Western blot and IFA methods were used to identify the reactivity of the MAb and to identify the antigenic epitope recognized by the MAb. This antigenic epitope is conserved in type I feline coronaviruses, indicating that the monoclonal antibody can recognize multiple type I FCoV strains, exhibiting broad-spectrum recognition, and is not incidentally targeting a single strain. This antigenic epitope showed no cross-reactivity with type II, indicating high specificity. It can be used to differentiate between type I and type II infections and has diagnostic value.

[0020] The preparation of monoclonal antibodies against the S protein of FCoV type I will help to further understand the immunological characteristics of FCoV and will also have direct application value for the development of FCoV diagnostic methods and vaccine strategies. Attached Figure Description

[0021] Figure 1 SDS-PAGE results for expression and purification of recombinant protein HLJ-S-RBD; 1~2: Supernatant and precipitate of uninduced recombinant bacterial pCold TF-HLJ-S-RBD / BL21(DE3) lysate; 3~4: Supernatant and precipitate of induced recombinant bacterial pCold TF-HLJ-S-RBD / BL21(DE3) lysate; M: Protein molecular weight standard;

[0022] Figure 2Western blot results for expression and purification of recombinant protein HLJ-S-RBD; 9: purified rHLJ-S-RBD protein; M: protein molecular weight standard;

[0023] Figure 3 IFA detection of serum antibody titers in mice immunized with rHLJ-S-RBD protein;

[0024] Figure 4 The results of Western blot identification of MAb reactivity;

[0025] Figure 5 The results of IFA identification of MAb reactivity;

[0026] Figure 6 The results of the identification of the HLJ-2D7 MAb antigenic epitope;

[0027] Figure 7 The sequence alignment results of the S protein of FCoV-1 and FCoV-2 coronaviruses;

[0028] Figure 8 The results of Western blot identification of the reactivity of MAb HLJ-2D7 with FCoV-1 S1 protein;

[0029] Figure 9 The results of IFA identification of the reactivity of MAb HLJ-2D7 with FCoV-I type S1 protein. Detailed Implementation

[0030] The embodiments of the present invention will be described in detail below. The following embodiments are implemented based on the technical solution of the present invention, and detailed implementation schemes and specific operation processes are given. However, the protection scope of the present invention is not limited to the following embodiments.

[0031] Example 1: Construction and Identification of Recombinant Plasmids

[0032] The S-RBD gene sequence of FIPV HLJ / HRB / 2016 / 10 (KY566209.1) was selected. After codon optimization, the gene sequence was sent to Cysens (Harbin) Biotechnology Co., Ltd. for gene synthesis. Primers were designed using Premier 5.0 software to amplify the full-length S-RBD gene. The amplification primers used are shown in Table 1. Cysens (Harbin) Biotechnology Co., Ltd. synthesized the S-RBD gene into the pET-30a plasmid to obtain the pET-30a-HLJ-S-RBD plasmid. Using the pET-30a-HLJ-S-RBD plasmid as a template, the above primers were used, with Sac I and Xho I selected as restriction sites, to amplify the viral S-RBD gene by PCR and clone it into the pCold TF vector to obtain the recombinant plasmid pCold TF-HLJ-S-RBD. Samples that were correctly identified by PCR were sent to Harbin Ruibo Biotechnology Co., Ltd. for sequencing. Sequencing results showed that the sequence was correct, indicating that the prokaryotic expression plasmid pCold TF-HLJ-S-RBD was constructed correctly.

[0033] Nanjing GenScript Biotechnology Co., Ltd. was commissioned to synthesize the HLJ-S1, Felix-S1 and Black-S1 genes into the pSecTag2 plasmid, and an Igκ leader sequence was added before each gene to obtain the pSecTag2-HLJ-S1, pSecTag2-Felix-S1 and pSecTag2-Black-S1 plasmids.

[0034] The HLJ / HRB / 2016 / 10 (KY566209.1) mentioned above has been published in the article Li C, Liu Q, Kong F, Guo D, Zhai J, Su M, Sun D. Circulation and genetic diversity of feline coronavirus type I and II from clinically healthy and FIP-suspected cats in China. Transbound Emerg Dis. 2019; 66(2): 763-775.

[0035] The HLJ-S1 gene described is derived from HLJ / HRB / 2016 / 10 (KY566209.1) and has been published in the article Li C, Liu Q, Kong F, Guo D, Zhai J, Su M, Sun D. Circulation and genetic diversity of feline coronavirus type I and II from clinically healthy and FIP-suspectedcats in China. Transbound Emerg Dis. 2019; 66(2): 763-775.

[0036] The Felix-S1 gene described is derived from Felix (MG893511.1) and has been published in the article Ehmann R, Kristen-Burmann C, Bank-Wolf B, et al. Reverse genetics for type I feline coronavirus field isolate to study the molecular pathogenesis of feline infectious peritonitis. mBio. 2018;9(4):e01422-18. Published 2018 Jul 31.

[0037] The Black-S1 gene described is derived from Black (EU186072.1) and has been published in the article Tekes G, Hofmann-Lehmann R, Stallkamp I, Thiel V, Thiel HJ. Genome organization and reversegenetic analysis of a type I feline coronavirus. J Virol. 2008;82(4):1851-1859.

[0038] Table 1. Amplification primers used to construct recombinant plasmids.

[0039]

[0040] Example 2: Induced expression, purification and identification of recombinant protein

[0041] The recombinant plasmid pCold TF-HLJ-S-RBD was transformed into BL21(DE3) competent cells and transferred to ampicillin-resistant LB medium. The cells were then cultured at 37 ℃ and 180 r / min until the bacterial OD value was reached. 600 When the pH value was 0.5–0.6, IPTG was added to a final concentration of 0.1 mmol / L, and expression was induced at 16 °C for 16 h. Bacterial cells were collected, the precipitate was resuspended in PBS, sonicated, and the supernatant and precipitate were collected separately to prepare protein samples. The recombinant protein was purified using a Ni column purification system and then analyzed by SDS-PAGE. Figure 1 As shown, SDS-PAGE analysis revealed target bands at approximately 84 kDa and 82 kDa in the supernatant and precipitate of the recombinant bacteria, respectively. Western blot analysis of the purified protein using Anti-6×His tag® antibody (1:5000) showed a single target band after purification (e.g., [image of target band]). Figure 2 The above results indicate that the rHLJ-S-RBD protein was expressed in prokaryotic cells, and the purification effect was good.

[0042] Example 3: Detection of antibody titer in mouse serum and preparation of MAb

[0043] The prepared protein was mixed with 50 μg of Bio-Drone 3w adjuvant at a 1:1 ratio with each mouse and immunized 6-8 week old BALB / c female mice via intramuscular injection in the hind leg. A booster immunization was performed 14 days later. Seven days after the third immunization, blood was collected, and the eukaryotic expression plasmid pSecTag2-HLJ-S1 was transfected into HEK293T cells. Detection was performed 48 hours later. Serially diluted (1:500, 1:1000, 1:2000) mouse serum immunized with rHLJ-S-RBD protein was used as the primary antibody, and Alexa Fluor® 488-labeled goat anti-mouse IgG (H+L) (1:400) was used as the secondary antibody. IFA was used to detect the antibody titer in mouse serum to determine if the mouse serum reached the titer required for monoclonal antibody preparation. After confirming the fusion titer was reached, a booster immunization was performed 3 days before cell fusion by intraperitoneal injection of 50 μg / mouse of the recombinant protein without adjuvant. After immunization, the spleens of immunized mice were harvested, and mouse spleen cells were fused with SP2 / 0 myeloma cells using a fusion agent (50% PEG1450). After fusion, the cells were transferred to 96-well cell culture plates and selectively cultured in HAT medium. The supernatant (stock solution) of the hybridoma cells to be tested was used as the primary antibody, and Alexa Fluor® 488-labeled goat anti-mouse IgG (H+L) (1:400) was used as the secondary antibody. IFA was used to screen for positive hybridoma cell lines. The antibody-positive hybridoma cells were subcloned three times to screen for cell lines that could stably secrete HLJ-S-RBD MAb. These cells were then expanded and cryopreserved.

[0044] The results showed that after three immunizations, the serum antibody titer of mice in the rHLJ-S-RBD protein immunization group reached 1:2000 (e.g., Figure 3 After confirming a serum antibody titer of 1:2000 in mice, the mice were boosted with immunoglobulin. Three days later, mouse spleen cells were fused with mouse myeloma cells SP2 / 0. Following IFA selection and three subcloning processes, a hybridoma cell line, MAbHLJ-2D7, was obtained. The MAb subclass identification kit showed that MAb HLJ-2D7 was of the IgG1 subtype with a κ light chain.

[0045] The amino acid sequence of the heavy chain variable region of a monoclonal antibody:

[0046] EVKMVESGGGLVKPGGSLKLSCATSGFAFSNYDMSWVRQTPEKRLEWVATISSGGSHTYYPDSVKGRFTISRDNARNTLYLQMSSLRSDDAALYYCARRGPFDFWGQGTSLTVSS

[0047] The amino acid sequence of CDR1 in the heavy chain variable region is: NYDMS

[0048] The amino acid sequence of CDR2 in the heavy chain variable region is: TISSGGSHTYYPDSVKG

[0049] The amino acid sequence of CDR3 in the heavy chain variable region is: RGPFDF

[0050] The amino acid sequence of the light chain variable region of a monoclonal antibody:

[0051] DIQMTQSPSSLSASLGDKVIITCKASQDINKYIAWYQHKPGKGPRLLIHFTSTVQPGIPSRFSGSGSGRDYSFSIRNLEPEDFATYYCLQYDYFYTFGGGTKLEIK

[0052] The amino acid sequence of CDR1 in the light chain variable region is: KASQDINKYIA

[0053] The amino acid sequence of CDR2 in the light chain variable region is: FTSTVQP

[0054] The amino acid sequence of CDR3 in the light chain variable region is: LQYDYFYT

[0055] Example 4: Identification of MAb subclasses

[0056] (a) Western blot analysis of MAb reactivity

[0057] HEK293T cells were transfected with the eukaryotic expression plasmid pSecTag2-HLJ-S1. Cells were collected 48 h later, with untransfected 293T cells serving as a control. Cells were collected after 24 h, discarding the supernatant. The cells were lysed using 1% Triton X-100, centrifuged, and the supernatant was collected. The supernatant secreted by the prepared MAb (stock solution) was used as the primary antibody, and FITC-labeled goat anti-mouse IgG (H+L) (1:10000) was used as the secondary antibody. Western blot analysis was performed to identify the reactivity of the monoclonal antibody.

[0058] Western blot results showed that cells transfected with the plasmid exhibited a specific band at approximately 130 kDa, while the negative control did not show this band (e.g., ...). Figure 4 This indicates that MAb HLJ-2D7 can be used for Western blot identification of rHLJ-S1 expression in eukaryotic cells.

[0059] (ii) IFA identification of MAb reactivity

[0060] After transfecting HEK293T cells with pSecTag2-HLJ-S1 for 48 h, the cells were fixed with 4% paraformaldehyde, permeabilized with 0.3% Triton X-100 for 15 min, and incubated overnight at 4 °C with MAb HLJ-2D7 secretion supernatant (stock solution) as the primary antibody. Untransfected 293T cells were used as a negative control. The cells were then incubated with Alexa Fluor® 488-labeled goat anti-mouse IgG (H+L) (1:400) as the secondary antibody for 1 h at room temperature in the dark. The results were then analyzed using an inverted fluorescence microscope. CRFK cells were infected with the DF2 strain and cultured at 37 °C for 24 h. Uninoculated CRFK cells were used as a negative control. Cells were fixed with 4% paraformaldehyde, permeabilized with 0.3% Triton X-100 for 15 min, and incubated with MAb HLJ-2D7 secretion supernatant (stock solution) as the primary antibody. Incubate overnight at 4°C, using Alexa FLuor® 488-labeled goat anti-mouse IgG (H+L) (1:400) as the secondary antibody, incubate at room temperature in the dark for 1 h, and detect using an inverted fluorescence microscope.

[0061] The results showed that MAb HLJ-2D7 exhibited specific green fluorescence in HEK293T cells transfected with pSecTag2-HLJ-S1; however, no specific green fluorescence was observed in CRFK cells infected with DF2 strain, and no fluorescence signal was observed in the negative control cells (e.g., Figure 5The above results indicate that MAb HLJ-2D7 can be used for IFA detection of rHLJ-S1 protein expressed in eukaryotic cells, but cannot detect DF2-S protein. This monoclonal antibody specifically recognizes FCoV-I S1 protein and has no cross-reactivity with FCoV-II S1 protein.

[0062] Example 5: Identification of antigenic epitopes of MAb

[0063] Using the HLJ-S-RBD gene as a template, approximately 420 bp fragments of the HLJ-S-RBD gene were amplified by PCR using primers for fragments E1 and E2 in Table 2, and cloned into the pCMV-HA vector to construct recombinant plasmids expressing the HLJ-S-RBD protein, which were then sequenced and identified. After transfecting HEK293T cells with the constructed recombinant plasmids for 48 hours, the protein was collected. Using MAb HLJ-2D7 (stock solution) as the primary antibody and FITC-labeled goat anti-mouse IgG (H+L) (1:10000) as the secondary antibody, Western blot was used to identify the recombinant protein reacting with MAb HLJ-2D7, and the antigenic epitopes recognized by MAb HLJ-2D7 were analyzed. Based on the results, the HLJ-S-RBD gene was further truncated and cloned into the pCMV-HA vector, which was then transfected into 293T cells. The same Western blot method was then used to further identify the minimal antigenic epitope recognized by MAb HLJ-2D7. The amplification primers used for HLJ-S-RBD truncated expression are shown in Table 2.

[0064] Table 2. Amplification primers used to identify the antigenic epitope recognized by MAb.

[0065]

[0066] The results showed that the antigenic epitope recognized by MAb HLJ-2D7 is located at aa1~aa141 of the S-RBD protein ( Figure 6 A). To further identify the antigenic epitopes recognized by this MAb, 15 overlapping coding gene fragments covering S-RBD aa1~aa141 were amplified by PCR and induced for expression, followed by Western blot identification. Figure 6 B). The results showed that the antigenic epitope recognized by MAb HLJ-2D7 was located at aa39~aa50 of S-RBD, and the corresponding amino acid sequence was... 39 DGSIFVNGYKYF 50 .

[0067] Example 6: Conservation analysis of the RBD region sequence of the FCoV-1 coronavirus S protein

[0068] The S protein sequences of FCoV-1 and FCoV-2 coronaviruses were located on the UniProt website. The S protein sequence FASTA format file was downloaded and imported into SnapGene software for multiple sequence alignment. Figure 7 As shown.

[0069] The results showed that this antigenic site 39 DGSIFVNGYKYF 50 This site is highly conserved in type I feline coronaviruses. It shows no sequence homology with the corresponding region of type II FCoV, but exhibits significant differences in amino acid composition and arrangement, no cross-reactivity, and strict type specificity.

[0070] Example 7: Identification of the reactivity of MAb HLJ-2D7 with different FCoV-1 S1 proteins

[0071] (a) Western blot identification

[0072] HEK293T cells were transfected with eukaryotic expression plasmids pSecTag2-HLJ-S1, pSecTag2-Felix-S1, and pSecTag2-Black-S1, respectively, with untransfected 293T cells serving as a control. Cells were collected after 48 h by discarding the supernatant. The cells were lysed with 1% Triton X-100 and the supernatant was collected by centrifugation. The reactivity of MAb HLJ-2D7 with FCoV-1 type S1 protein was assessed by Western blot using MAb HLJ-2D7 as the primary antibody and FITC-labeled goat anti-mouse IgG (H+L) (1:10000) as the secondary antibody.

[0073] like Figure 8 Western blot results showed that cells transfected with the plasmid exhibited a specific band at approximately 130 kDa, while the negative control did not show this band. This indicates that MAb HLJ-2D7 can be used for Western blot identification of the expression of rHLJ-S1, rFelix-S1, and rBlack-S1 in eukaryotic cells.

[0074] (II) IFA Identification

[0075] HEK293T cells were transfected with eukaryotic expression plasmids pSecTag2-HLJ-S1, pSecTag2-Felix-S1, and pSecTag2-Black-S1, respectively, with untransfected 293T cells serving as a control. After 48 h, the supernatant was discarded, and the cells were fixed with 4% paraformaldehyde, permeabilized with 0.3% Triton X-100 for 15 min, and incubated overnight at 4 °C with MAb HLJ-2D7 as the primary antibody. Then, the cells were incubated at room temperature in the dark for 1 h with AlexaFLuor® 488-labeled goat anti-mouse IgG (H+L) (1:400) as the secondary antibody, and detected using an inverted fluorescence microscope.

[0076] like Figure 9 The results showed that HLJ-2D7 MAb exhibited specific green fluorescence in transfected plasmid cells, while negative control cells showed no green fluorescence. These results indicate that MAb HLJ-2D7 can be used for IFA to identify the expression of rHLJ-S1, rFelix-S1, and rBlack-S1 in eukaryotic cells.

[0077] The S protein of FCoV is a key structural protein on the viral surface, playing a central role in viral infection and containing multiple neutralizing epitopes, making it a major target of the host's protective immune response. The S protein consists of two subunits, S1 and S2. The S1 subunit contains a receptor-binding domain (RBD), responsible for recognizing and adsorbing to host cell receptors; the S2 subunit mediates the fusion of the viral envelope and cell membrane. However, the natural S1 region suffers from complex glycosylation modifications and poor expression stability, limiting antibody development. Therefore, in-depth research into the immunodominant proteins and antibody response mechanisms of the FCoV S1 region is crucial for developing efficient diagnostic methods and novel prevention and control strategies.

[0078] Example 8

[0079] The amino acid sequence of the heavy chain variable region of the feline infectious peritonitis virus S protein receptor binding region monoclonal antibody is shown in SEQ ID NO.1 of the sequence listing, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.2 of the sequence listing;

[0080] The amino acid sequence of CDR1 in the heavy chain variable region is shown as positions 31-35 in SEQ ID NO.1.

[0081] The amino acid sequence of CDR2 in the heavy chain variable region is shown as positions 50-66 in SEQ ID NO.1.

[0082] The amino acid sequence of CDR3 in the heavy chain variable region is shown as positions 99-104 in SEQ ID NO.1;

[0083] The amino acid sequence of CDR1 in the light chain variable region is shown as positions 24-34 in SEQ ID NO.2;

[0084] The amino acid sequence of CDR2 in the light chain variable region is shown as positions 50-56 in SEQ ID NO.2;

[0085] The amino acid sequence of CDR3 in the light chain variable region is shown as positions 89-96 in SEQ ID NO.2.

[0086] This embodiment utilizes the above-mentioned monoclonal antibody to prepare a feline infectious peritonitis virus detection kit and / or test strip.

[0087] Example 9

[0088] The feline infectious peritonitis virus (FIP) detection kit of this embodiment includes a monoclonal antibody against the receptor-binding region of the FIP S protein. The amino acid sequence of the heavy chain variable region of the FIP S protein receptor-binding region monoclonal antibody is shown in SEQ ID NO.1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.2.

[0089] The amino acid sequence of CDR1 in the heavy chain variable region is shown as positions 31-35 in SEQ ID NO.1.

[0090] The amino acid sequence of CDR2 in the heavy chain variable region is shown as positions 50-66 in SEQ ID NO.1.

[0091] The amino acid sequence of CDR3 in the heavy chain variable region is shown as positions 99-104 in SEQ ID NO.1;

[0092] The amino acid sequence of CDR1 in the light chain variable region is shown as positions 24-34 in SEQ ID NO.2;

[0093] The amino acid sequence of CDR2 in the light chain variable region is shown as positions 50-56 in SEQ ID NO.2;

[0094] The amino acid sequence of CDR3 in the light chain variable region is shown as positions 89-96 in SEQ ID NO.2.

[0095] This invention successfully expressed and purified the FCoV-1 type FIPV RBD protein using an Escherichia coli prokaryotic expression system, and obtained a hybridoma cell line capable of stably secreting the corresponding specific monoclonal antibody, and identified its antigenic epitopes. Experiments confirmed that the obtained MAb exhibited good reactivity. These biological materials and related research results provide a valuable experimental basis and reference for the subsequent development of rapid, simple, and accurate FCoV diagnostic reagents and for improving the early diagnosis of FCoV infection.

Claims

1. A monoclonal antibody against the receptor-binding domain of the feline infectious peritonitis virus (FIP), characterized in that, The amino acid sequence of the heavy chain variable region of this monoclonal antibody is shown in SEQ ID NO.1 of the sequence listing, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.2 of the sequence listing; The amino acid sequence of CDR1 in the heavy chain variable region is shown as positions 31-35 in SEQ ID NO.

1. The amino acid sequence of CDR2 in the heavy chain variable region is shown as positions 50-66 in SEQ ID NO.

1. The amino acid sequence of CDR3 in the heavy chain variable region is shown as positions 99-104 in SEQ ID NO.1; The amino acid sequence of CDR1 in the light chain variable region is shown as positions 24-34 in SEQ ID NO.2; The amino acid sequence of CDR2 in the light chain variable region is shown as positions 50-56 in SEQ ID NO.2; The amino acid sequence of CDR3 in the light chain variable region is shown as positions 89-96 in SEQ ID NO.

2.

2. The use of the feline infectious peritonitis virus (FIP) S protein receptor-binding domain monoclonal antibody as described in claim 1 in the preparation of FIP detection kits and / or test strips.

3. A feline infectious peritonitis virus (FIP) detection kit, comprising a monoclonal antibody against the FIP S protein receptor-binding domain as described in claim 1.

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