Monoclonal antibody against feline calicivirus VP1 protein and its antigenic epitope peptide and its application

Abstract

The present invention discloses a monoclonal antibody against the VP1 protein of feline calicivirus. The mAb against the VP1 protein was prepared by immunizing BALB / c mice with the whole virus of SH14 strain FCV as an immunogen by intraperitoneal injection. The specific monoclonal antibody 3A3C1 strain was successfully obtained by ELISA screening, which can specifically recognize and bind to FCV SH14 strain, F9 strain and GZ22 strain. The present invention also discloses the linear antigen epitope of the 3A3C1 strain mAb. By truncating and expressing the VP1 protein and performing detailed Western blot analysis, it was finally determined that the linear antigen epitope of the 3A3C1 strain mAb is located at the VP1 protein. 426 PSRLTPAGDYAITSG 440 In addition, the present invention also discloses a nucleic acid encoding the 3A3C1 strain variable region mAb, and the use of the monoclonal antibody in the preparation of an FCV detection kit. The 3A3C1 strain mAb prepared by the present invention is not only an important tool for understanding the immunological characteristics of FCV, but also has potential application value in the development of FCV diagnostic methods and vaccine strategies.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical antibodies, and in particular to a monoclonal antibody against feline calicivirus VP1 protein and an antigenic epitope peptide recognized by the antibody and an application thereof. Background Art

[0002] Feline calicivirus (FCV) is an important pathogen that causes infectious diseases in cats. Infection with FCV can lead to clinical symptoms such as oral ulcers, upper respiratory tract diseases and chronic gastroenteritis. FCV belongs to the genus Vesivirus of the family Calicivirdae. It has no envelope and its genome is a single-stranded positive-strand RNA. The virus is widely prevalent worldwide and poses a serious threat to the health of domestic cats and other felines. Especially in kittens under one year old, the morbidity and mortality of FCV infection are high, with a mortality rate of up to 67% (Radford AD, Coyne KP, Dawson S. et al. Feline calicivirus [J]. Vet Res. 2007, 38 (2): 319-335.).

[0003] The capsid protein VP1 of FCV is a key molecule for studying its pathogenicity and developing diagnostic tools. The VP1 protein is the main structural protein of the virus and the main site of interaction between the virus and host cells. Studies have shown that the VP1 protein of FCV has an important influence on the biological characteristics of the virus. The VP1 protein consists of six regions from A to F, each of which has its own unique functions and characteristics. Region A is located at the amino terminus of the protein and is a highly conserved region. During the maturation of the capsid protein, region A is usually cleaved. Region B contains myristylated glycine and ATP / GTP binding sites and is part of the core structure of the virus. Region C is a short hypervariable sequence that is related to the variability of the virus. Region D is another highly conserved region. Region E is the binding site of neutralizing monoclonal antibodies (mAbs) and is another hypervariable region. Region F is located at the highly conserved carboxyl terminus of the capsid protein and is the binding site of non-neutralizing mAbs. Therefore, the preparation of monoclonal antibodies (mAbs) against the VP1 protein is not only crucial for studying the biological characteristics of FCV, but also has direct application value in the development of diagnostic tools and vaccine strategies.

[0004] The previous literature (Cheng Chenxi, Liu Jiasen, Hu Xiaoliang, et al. Preparation of monoclonal antibodies against feline calicivirus VP1 protein and identification of its antigenic epitope [J]. Chinese Journal of Preventive Veterinary Medicine, 2017, 39(12): 1017-1021.) disclosed the preparation of monoclonal antibodies against feline calicivirus VP1 protein and identification of its antigenic epitope, and obtained two hybridoma cells E2F1 and E2F6 that stably secreted anti-FCV VP1-E MAbs. Both antibodies were of IgG1 subtype and had a κ light chain. Both antibodies could specifically recognize FCV-2280, and the recognized VP1-E sequences were 467 YICGSLQRAWG 477 This antigen epitope is relatively conserved in FCV VP1 protein. This MAb lays the foundation for the establishment of a highly specific and sensitive FCV detection method. However, its antigenic conserved sequence 467 YICGSLQRAWG 477 Located close to the inner surface of the nucleocapsid protein, MAb antibodies are easily interfered with during their reaction with antigens. Summary of the invention

[0005] The purpose of the present invention is to provide a monoclonal antibody against the VP1 protein of feline calicivirus and the antigenic epitope peptide and application thereof; specifically, to provide a monoclonal antibody against the FCV VP1 protein and to identify its antigenic epitope. The present invention uses the SH14 strain of FCV as an immunogen, immunizes BALB / c mice with the whole virus, and then successfully prepares a monoclonal antibody specific for the VP1 protein by an enzyme-linked immunosorbent assay (ELISA) screening method. Recombinant antibodies are then constructed and their biological activity is verified. The work of the present invention lays the foundation for a deep understanding of the immunological characteristics of FCV and the development of new diagnostic and treatment methods. In addition, the monoclonal antibodies obtained by the present invention provide an important tool for further FCV research, especially in detection methods and vaccine development.

[0006] In this application, the 3A3C1 hybridoma cell line was reverse transcribed by 5'RACE method, and its light and heavy chain variable region sequences were cloned and sequenced. The obtained sequence was analyzed based on the IMGT database to determine the V (variable region), D (diversity region) and J (joining region) regions of the sequence, and the sequencing results were compared with the reference sequence using CodonCode Aligner, DtaiBio Tools, DNAMAN and Snapgene software to detect mutations, insertions and deletions, and finally obtain the complete variable region sequence information. Subsequently, the cat and mouse IgG constant region sequences were synthesized into the pcDNA 3.4 eukaryotic expression vector, and the light and heavy chain variable region sequences obtained by sequencing were cloned upstream of their expression frames to construct the light and heavy chain expression plasmids of the recombinant antibodies. Subsequently, the mouse and cat light and heavy chain expression vectors were co-transfected into the Expi-CHO expression system, and after expression, the recombinant antibodies were confirmed to have the same specific reactivity as the mouse antibodies by Western blot.

[0007] The objective of the present invention is achieved through the following technical solutions:

[0008] <First aspect>

[0009] The present invention provides a monoclonal antibody against feline calicivirus VP1 protein, wherein the monoclonal antibody comprises an antibody heavy chain and an antibody light chain.

[0010] The variable region of the antibody heavy chain includes CDR1 consisting of the amino acid sequence shown in SEQ ID NO.1, CDR2 consisting of the amino acid sequence shown in SEQ ID NO.2, and CDR3 consisting of the amino acid sequence shown in SEQ ID NO.3;

[0011] The variable region of the antibody light chain includes CDR1 consisting of the amino acid sequence shown in SEQ ID NO.4, CDR2 having the amino acid sequence of DTS, and CDR3 consisting of the amino acid sequence of SEQ ID NO.5.

[0012] In the present invention, the monoclonal antibody of feline calicivirus includes a heavy chain and a light chain, each heavy chain includes a heavy chain variable region (VH) and a heavy chain constant region (CH). Each light chain includes a light chain variable region (VL) and a light chain constant region (CL). The VH region and the VL region can be further divided into complementary determining regions (CDRs), and a framework region (FR) is inserted between each two complementary determining regions. Each VH and VL includes three CDRs and four FRs, and the order of connection from the amino terminus to the carboxyl terminus is: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0013] As one embodiment of the present invention, the monoclonal antibody comprises the heavy chain variable region amino acid sequence as shown in SEQ ID NO.16 or a homologous sequence thereof; and / or,

[0014] It comprises the light chain variable region amino acid sequence shown in SEQ ID NO.17 or a homologous sequence thereof.

[0015] The homologous sequences are sequences having at least 90%, optionally at least 95%, preferably at least 97%, more preferably at least 98%, and most preferably at least 99% sequence identity.

[0016] As an embodiment of the present invention, the antibody heavy chain further comprises FR1 consisting of the amino acid sequence shown in SEQ ID NO.6, FR2 consisting of the amino acid sequence shown in SEQ ID NO.7, FR3 consisting of the amino acid sequence shown in SEQ ID NO.8, FR4 consisting of the amino acid sequence shown in SEQ ID NO.9, and / or,

[0017] The antibody light chain also includes FR1 consisting of the amino acid sequence shown in SEQ ID NO.10, FR2 consisting of the amino acid sequence shown in SEQ ID NO.11, FR3 consisting of the amino acid sequence shown in SEQ ID NO.12, and FR4 consisting of the amino acid sequence shown in SEQ ID NO.13.

[0018] As an embodiment of the present invention, the antibody heavy chain further includes a signal peptide sequence as shown in SEQ ID NO.14, and / or the antibody light chain further includes a signal peptide sequence as shown in SEQ ID NO.15.

[0019] As one embodiment of the present invention, the monoclonal antibody is prepared by animal immunization using the whole virus of SH14 strain FCV as an immunogen.

[0020] <Second Aspect>

[0021] The present invention provides a nucleic acid encoding the heavy chain of the monoclonal antibody as described in any one of the above items, and / or encoding the light chain of the monoclonal antibody as described in any one of the above items.

[0022] As one embodiment of the present invention, the nucleic acid comprises nucleotide sequences encoding CDR1, CDR2, and CDR3 of the heavy chain variable region, as shown in SEQ ID NO. 18, 19, and 20, respectively, and / or,

[0023] Nucleotide sequences for encoding CDR1, CDR2, and CDR3 of the light chain variable region, wherein the nucleotide sequence encoding the CDR1 is as shown in SEQ ID NO.21, the nucleotide sequence encoding the CDR2 is GACACATCC, and the nucleotide sequence encoding the CDR3 is as shown in SEQ ID NO.22.

[0024] As an embodiment of the present invention, the nucleic acid further comprises nucleotide sequences for encoding FR1, FR2, FR3, and FR4 of the heavy chain variable region, as shown in SEQ ID NO. 23, 24, 25, and 26, respectively; and / or,

[0025] The nucleotide sequences encoding FR1, FR2, FR3 and FR4 of the light chain variable region are shown in SEQ ID NOs. 27, 28, 29 and 30, respectively.

[0026] <Third Aspect>

[0027] The present invention provides an antigenic epitope peptide of feline calicivirus VP1 protein, wherein the antigenic epitope peptide specifically binds to the above-mentioned monoclonal antibody.

[0028] As one embodiment of the present invention, the amino acid sequence of the antigenic epitope peptide is 426 PSRLTPAGDYAITSG 440 , as shown in SEQ ID NO.47.

[0029] <Fourth Aspect>

[0030] The present invention provides a biological material containing the nucleic acid, wherein the biological material is one or more of a kit, an expression vector and a eukaryotic or prokaryotic host cell.

[0031] <Fifth Aspect>

[0032] The present invention also provides a use of any of the above-mentioned monoclonal antibodies in FCV detection.

[0033] As an embodiment of the present invention, it includes the use of the monoclonal antibody in the preparation of an FCV detection kit.

[0034] Compared with the prior art, the present invention has the following beneficial effects:

[0035] 1) The antigen epitope obtained is extremely conservative and appears for the first time in China. It can detect all feline caliciviruses, greatly reducing the possibility of missed detection;

[0036] 2) The preparation of recombinant antibodies reduces the cost of secondary production and reduces the variability of antibodies between batches;

[0037] 3) Currently, there are no reports of relevant recombinant antibodies against FCV. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Other features, objects and advantages of the present invention will become more apparent from the detailed description of non-limiting embodiments made with reference to the following drawings:

[0039] Figure 1 The Western blot diagram of the monoclonal antibody in Example 2 reacting with VP1 proteins of different strains; wherein, Figure 1 A is the WB image of mAb and five FCV strains. Figure 1 B is the WB graph of mAb and SH14 strain FCV at different reaction times. Figure 1 C is the WB graph of mAb and F9 strain FCV at different reaction times. Figure 1 D is the WB graph of mAb and GZ22 strain FCV at different reaction times;

[0040] Figure 2 This is the IFA identification result of mAb ascites and different FCV strains in Example 3;

[0041] Figure 3 The figure is a segmented pattern diagram and a Western blot analysis diagram of the antigen epitope identification of the 3A3C1 monoclonal antibody in Example 3; wherein, Figure 3 A is a schematic diagram of dividing the VP1 protein into two fragments: 126-426aa (B, C, D regions) and 426-668aa (E, F regions). Figure 3 B is a WB image for identification of the above two fragments and the full length of VP1; Figure 3 C is Figure 3 Schematic diagram of the positive region 426-668aa (E and F regions) identified in B divided into three segments P1, P2, and P3. Figure 3 D is the WB image for identification of the above three fragments; Figure 3 E is for Figure 3 Schematic diagram of the positive region 426-486aa identified in D divided into three segments P4, P5, and P6. Figure 3 F is a WB image for identification of the above three fragments;

[0042] Figure 4 The diagram is a schematic diagram of the FCV VP1 protein structure and mAb antigen epitope structure in Example 3; wherein, Figure 4 A is a top view of the monomer structure of VP1 protein. Figure 4 B is a top view of the VP1 protein trimer; Figure 4 C is a plan view of the VP1 protein trimer;

[0043] Figure 5 The sequence logo is generated using Weblogo for the 426-440aa antigenic epitope of the VP1 protein of 185 strains of FCV in Example 3;

[0044] Figure 6 The predicted signal peptides of the light and heavy chain variable regions of the monoclonal antibody in Example 4;

[0045] Figure 7 is a schematic diagram of the amino acid sequence of the monoclonal antibody variable region obtained in Example 4;

[0046] Figure 8 The figure is an agarose gel electrophoresis diagram of the variable region DNA sequence amplified in Example 4; wherein, band M is a marker; Lane 1-2 is a heavy chain, Lane 3 is a heavy chain positive control, Lane 4-5 is a light chain, and Lane 6 is a light chain positive control. DETAILED DESCRIPTION

[0047] The present invention is described in detail below in conjunction with specific embodiments. The following embodiments will help those skilled in the art to further understand the present invention, but are not intended to limit the present invention in any form. It should be noted that, for those of ordinary skill in the art, several variations and improvements may be made without departing from the concept of the present invention. These all belong to the protection scope of the present invention.

[0048] The cell lines and virus strains used in the present invention include cat kidney cell line (CRFK), human embryonic kidney cell line (293T), and FCV strains SJ14, XA20, SH14, F9 and GZ22, truncated VP1 protein (330-668aa) and its expression plasmid, all of which are preserved by the companion animal biosafety risk warning and prevention and control technology team of Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences. Among them, FCV strain SH14 has been published in document 1 (Huimin Guo, Qiuhong Miao, Jie Zhu, Zhibiao Yang, Guangxiang Liu Isolation and molecular characterization of a virulent systemic felinecalicivirus isolated in China [J] Infect Genet Evol. 2018 Nov: 65: 425-429. doi: 10.1016 / j.meegid.2018.08.29); F9 has been published in document 2 (Carter MJ, Milton ID, Turner PC, et al. Identification and sequence determination of the capsid protein gene of feline calicivirus [J]. Arch Virol, 1992, 122 (3-4): 223-35.); SJ14 is a strain isolated by our laboratory, the full length of which has been sequenced, Genbank accession number: PP061537; XA20 is a strain isolated by our laboratory, the full length of which has been sequenced, Genbank accession number: OR791865; GZ22 is a strain isolated by our laboratory, the full length of which has been sequenced, Genbank accession number: OQ814185. Truncated VP1 protein (330-668aa) and its expression plasmid were obtained by conventional methods.

[0049] The plasmid pCMV-3*Flag-14 used in the present invention was purchased from Thermofisher Company in the United States; pCold-SUMO was purchased from Takara Company in Japan.

[0050] The reagents and materials used in the present invention include complete Freund's adjuvant, incomplete Freund's adjuvant, PEG SOLUTION 50%, horseradish peroxidase-labeled goat anti-mouse IgG (IgG-HRP), goat anti-mouse IgG-FITC, and anti-Histag mAb purchased from Sigma, infrared fluorescent-labeled goat anti-mouse IgG (IgG-RBITC), and goat anti-cat IgG (H+L)-HRP purchased from QIAGEN, protein marker purchased from Sevier, and IgG antibody subclass identification kit purchased from Beyotime. The experimental animals are 6-8 week-old female SPF BALB / c mice purchased from Shanghai Jiesijie Experimental Animal Co., Ltd.

[0051] Example 1. Preparation of hybridoma cells

[0052] 1.1 Virus purification

[0053] The FCV strain SH14 was purified using a sucrose gradient centrifugation purification method, and after purification, β-propiolactone was added to a final concentration of 0.025% to inactivate the virus as an immunogen.

[0054] 1.2 Immunization of mice

[0055] During the first immunization, the purified SH14 was mixed with an equal amount of complete Freund's adjuvant, and each mouse was intraperitoneally injected with 100 μg of virus (a total of 200 μl of the mixture). After 2 weeks and 4 weeks, the same amount of FCV whole virus and incomplete Freund's adjuvant were used for booster immunization. One week after the booster immunization, blood was collected from the eyeballs, and the antibody titer against FCV VP1 (330-668aa) protein in the serum was determined by indirect ELISA. The full-length sequence of FCV VP1 protein was derived from Genebank id: KT000003.

[0056] The monoclonal antibody was prepared by immunizing mice with the whole virus and then using the truncated VP1 protein as the coating antigen for screening. The main reasons for not using the complete VP1 protein or 126-668aa are: (1) In previous experiments, it was found that the expression of the full-length VP1 protein was very difficult and almost impossible to express; (2) The A region (Leader Capsid) of the VP1 protein (1-125aa) was removed by the viral protease during FCV infection. In the actual detection process, the monoclonal antibody obtained against this segment has no use value; (3) In the process of reading previous literature, it was found that there are very few antigenic epitopes on 126aa-329aa. 330-668aa is the longest length of VP1 protein that can be stably expressed, which was gradually screened out in the previous experiments. It can also avoid the false positive errors that may occur in ELISA detection and be confirmed by secondary verification. Therefore, the truncated region of 330-668aa of the FCV VP1 protein was selected as the coating antigen.

[0057] 1.3 Cell fusion

[0058] The mice with the highest titer of serum antibody against truncated VP1 (330-668aa) protein were selected for cell fusion experiment. After boosting immunity, mouse spleen cells were collected and mixed with SP2 / 0 myeloma cells at a ratio of 5:1, and cell fusion was performed using PEG1000 as a fusion promoter.

[0059] 1.4 Screening and cloning of positive hybridoma cells

[0060] After 14 days of culture, positive hybridoma clones that can continuously produce antibodies specific to FCV VP1 (330-668aa) protein were screened by ELISA technology. Positive clones were cultured by limiting dilution until a cell line that stably secreted monoclonal antibodies specific to VP1 protein (330-668aa) was obtained.

[0061] Through three clone purifications, a hybridoma cell line that can stably secrete mAb against VP1 truncated protein (330-668aa) was obtained, named 3A3C1. After subtype identification, the mAb belongs to IgG1 type, and the light chain is κ chain.

[0062] 1.5 Ascites preparation and purification

[0063] BALB / c female mice were intraperitoneally injected with 0.5 mL of liquid paraffin, and one week later, approximately 2 × 10 6 Monoclonal cell line in logarithmic growth phase. When the abdomen of the mouse is obviously swollen, ascites is extracted, centrifuged at 10000r / min for 10min, and the supernatant is collected. The antibody is purified by ammonium sulfate precipitation method.

[0064] Example 2: Identification of monoclonal antibodies

[0065] 2.1 Subtype identification

[0066] The monoclonal antibodies extracted from ascites were subtyped by ELISA using a commercial immunoglobulin subtyping kit to determine the subtype of the immunoglobulin.

[0067] 2.2 Indirect immunofluorescence (IFA) identification

[0068] Feline kidney cell lines (CRFK) infected with FCV SH14, GZ22, and F9 strains were used as target cells, ascites was used as primary antibody, FITC-labeled goat anti-mouse IgG was used as secondary antibody, and specific binding was observed using a fluorescence microscope.

[0069] IFA results show that ( Figure 2), 3A3C1 mAb was used as the primary antibody to react with CRFK cells infected with F9, SH14, and GZ22, respectively, and a specific green fluorescence signal appeared, while there was no fluorescence in the negative control group.

[0070] 2.3 Western blot analysis

[0071] FCV SH14, F9, GZ22, SJ14, and XA14 strains were used to infect CRFK cells (cat kidney cells), and the cells were collected at 16 and 24 hours, lysed with Ripa lysis buffer, and subjected to SDS-PAGE and membrane transfer. Ascites was used as the primary antibody and goat anti-mouse IgG-RBITC was used as the secondary antibody. The reactivity of the antibody with the VP1 protein of different FCV strains was detected by Western blot analysis.

[0072] Western blot results showed that mAb 3A3C1 could specifically bind to the VP1 proteins of five FCV strains: SJ14, XA20, SH14, F9, and GZ22 ( Figure 1 A), selected representative F9 (classic vaccine strain), SH14 (type I epidemic strain), and GZ22 (type II epidemic strain) for secondary WB verification, and the binding strength increased with the extension of virus infection time ( Figure 1 B. Figure 1 C. Figure 1 D).

[0073] 2.4 Neutralization activity assay

[0074] 200TCID 50 The virus was mixed with an equal volume of ascites at different dilutions (1:2, 1:4, 1:8, 1:16, 1:32, 1:64) and incubated at 37°C for 1 hour to promote virus-antibody binding. Each mixture was inoculated into a 96-well plate of CRFK cells (200 μL / well, 8 replicates), and an uninfected control group was set up. The cells were cultured at 37°C and 5% CO2 for 72 hours, and the cytopathic effect (CPE) was observed, based on which the monoclonal antibody (mAb) neutralization titer was calculated.

[0075] The results of the neutralization test showed that mAb 3A3C1 stock solution was also unable to neutralize the infection of the SH14 strain, indicating that the mAb had no neutralizing activity.

[0076] In summary, the mAb 3A3C1 prepared in the present invention can specifically identify cell samples infected with FCV strains of various genotypes and is suitable for the detection of FCV.

[0077] Example 3: Identification of antigenic epitopes

[0078] 3.1 VP1 protein region division and eukaryotic expression

[0079] The A region (1-125aa) of the FCV VP1 protein is removed during the infection process, so the research focuses on other regions: 126-426aa (B, C, D regions) and 426-668aa (E, F regions). The full-length sequence of the VP1 protein is divided into two main regions: VP1-126-426aa and VP1-426-668aa (e.g. Figure 3 A).

[0080] Eukaryotic expression plasmids pCMV-3Flag-14-VP1 (126-426aa), pCMV-3Flag-14-VP1 (426-668aa) and pCMV-3*Flag-14-VP1 full length were constructed and transfected into 293T cells respectively. Cells were lysed by Ripa, protein samples were extracted, and the positive area was preliminarily determined by Western blot. It was found that the 3A3C1 strain mAb only specifically bound to the 426-668aa fragment (such as Figure 3 B).

[0081] 3.2 Refinement of antigenic region and prokaryotic expression

[0082] According to the preliminary results of Western blot, three pairs of primers (as shown in P1-P3 in Table 1) were used to divide the positive region into four fragments and cloned into the pCold-SUMO prokaryotic expression vector to express the protein. Protein samples were collected and Western blot analysis was performed to further determine the antibody antigen epitope.

[0083] Specifically, according to the VP1-426-668aa sequence, it is divided into three small fragments: P1, P2 and P3 ( Figure 3 C). After the P1, P2 and P3 fragments were expressed in a prokaryotic expression system, they were identified by Western blot, which showed that the 3A3C1 strain mAb specifically reacted with the P1 fragment, thus narrowing the potential antigen epitopes to the range of 426-486aa ( Figure 3 D).

[0084] 3.3 Accurate identification of antigenic epitopes

[0085] Based on the Western blot results in 3.2, three pairs of primers were designed (as shown in P4-P6 in Table 1) to divide the positive region into four segments, which were cloned into the pCold-SUMO vector for prokaryotic expression. Protein samples were collected and analyzed by Western blot, and the antibody antigen epitope was finally limited to 15 amino acids.

[0086] Specifically, according to the VP1-426-486aa sequence, it is divided into three parts: P4, P5 and P6 ( Figure 3E). After prokaryotic expression of P4, P5 and P6 and Western blot analysis, the results showed that 3A3C1 mAb only reacted with the P4 fragment ( Figure 3 F), indicating that 3A3C1 mAb recognizes the VP1 protein sequence 426 PSRLTPAGDYAITSG 440 .

[0087] Table 1 Primer Design for Antigen Epitope Identification of Clone mAb

[0088]

[0089] The ChimeraX software was used to locate the position of the epitope on the VP1 protein trimer, and the results showed that the antigen epitope was located at the top of the VP1 trimer structure, that is, the surface of the virus particle ( Figure 4 ). A total of 185 FCV strains at home and abroad were selected for comparison of the amino acid sequences at the corresponding positions of VP1, and the Weblogo of 426-440aa was created through the Weblogo3 website. The graphic content shows that the epitope is highly conserved, and 431-435aa is extremely conserved ( Figure 5 ).

[0090] Example 4: Obtaining the sequence of the variable region of a monoclonal antibody

[0091] 4.1 Hybridoma Cell RNA Extraction and 5' RACE Reverse Transcription

[0092] First, collect about 8×10 7 Transfer hybridoma cells to a 1.5mL centrifuge tube, add 1mL Trizol reagent, mix thoroughly, and incubate at room temperature for 5 minutes. Add 0.2mL chloroform, shake for 15 seconds to fully mix the organic phase and the aqueous phase, and let stand for 2 minutes. Centrifuge at 12000×g for 15 minutes at 4℃, and carefully aspirate the upper colorless aqueous phase. Add an equal volume of isopropanol, mix gently, and let stand at room temperature for 10 minutes to form RNA precipitation. Centrifuge at 12000×g for 10 minutes at 4℃, and remove the supernatant. Add 1mL 75% ethanol to wash the RNA precipitate, centrifuge at 7500g for 5 minutes at 4℃, and aspirate the supernatant. After the RNA precipitate is air-dried, add an appropriate amount of DEPC water to dissolve the RNA, and incubate at 65℃ for 10-15 minutes to promote dissolution.

[0093] The cDNA was synthesized according to the instructions of 5'-RACE kit of Sangon Biotech Co., Ltd. and sent to Nanjing Mingyan Co., Ltd. for hybridoma sequencing.

[0094] 4.2 Monoclonal Antibody Heavy and Light Chain Variable Region Gene Amplification and Sequencing

[0095] According to the nucleotide sequence information obtained by sequencing analysis in 4.1, specific primer pairs MVH-F / MVH-R and MVL-F / MVL-R were designed and synthesized for the variable regions of the heavy chain and light chain, respectively (primer sequence information is shown in Table 2). The upstream primer is located in the FR1 region, while the downstream primer is located in the antibody C region. Using the cDNA prepared in step 4.1 as a template, a PCR amplification reaction was performed using a 2×Phanta MaxMaster Mix kit. The variable region genes in the cell line were amplified using polymerase chain reaction (PCR).

[0096] Prepare 1% agarose gel, mix the amplification reaction product with an appropriate amount of loading buffer and load it. After electrophoresis separation for 30 minutes, place the gel in a gel imaging system and observe the migration position of the band using ultraviolet transmission. Figure 8 As shown, the M band is a marker; Lane 1-2 is a heavy chain, Lane 3 is a heavy chain positive control (conventional mouse antibody heavy chain variable region), Lane 4-5 is a light chain, and Lane 6 is a light chain positive control (conventional mouse antibody light chain variable region). The target band can be clearly detected by gel electrophoresis, and its size is within the expected range according to the DNA molecular weight standard. The corresponding gel band was cut out and the purified DNA was recovered using a commercial kit (Agarose Gel Recovery Kit from Nanjing Novogene).

[0097] Table 2 Primer names and sequences

[0098]

[0099] The sequencing results were predicted by signal peptide ( Figure 6 ), IMGT database comparison and other bioinformatics analysis, label each gene segment, and obtain the amino acid sequence of the monoclonal antibody variable region, such as Figure 7 shown.

[0100] The VP1 capsid protein of feline calicivirus (FCV) is a key structural protein of the virus, and its complex structure plays a decisive role in the biological characteristics and immune response of the virus. The VP1 protein is mainly composed of two main domains: the shell (S) domain and the protrusion (P) domain. The S domain provides the basic framework of the virus particle and is the main part of the virus capsid, while the P domain is located on the outside of the virus particle and is directly exposed to the host immune system.

[0101] The P domain is subdivided into two subdomains, P1 and P2, of which the P2 subdomain is particularly important. It is located in the outermost layer of the virus particle and is the main interface for the interaction between the virus and the host cell receptor. It is also a key area for immune system recognition. The high variability of the P2 subdomain is one of the main mechanisms for FCV to evade the host immune system. Although FCV has only one serotype, the high variability of VP1 makes the cross-reactivity between FCV strains low, which greatly increases the probability of immune failure after the use of vaccines. The antigenic region of the VP1 protein is divided into six regions from A to F. Among them, the E region is located in the P2 subdomain, which is a key region for inducing immune responses and contains multiple immunogenic epitopes. These epitopes are key regions for the interaction between the virus and the host immune system and are crucial for the recognition and neutralization of the virus. Therefore, in-depth research on the E region, especially the precise identification and positioning of the immunogenic epitopes therein, is of great significance for understanding the immunological characteristics of FCV and developing effective diagnostic tools and vaccines.

[0102] The conserved epitopes of the main antigen protein VP1 remain relatively stable in different FCV strains, and therefore can induce a wide range of immune responses. The effectiveness of FCV vaccines mainly depends on stimulating immune responses to highly conserved neutralizing epitopes, so using monoclonal antibodies to screen for conserved and neutralizing epitopes of FCV is crucial for developing vaccines that can effectively prevent viral infection and transmission.

[0103] In addition, the diversity of VP1 protein is also the main reason for the poor effect of FCV antigen detection products. The epitope targeted by the 3A3C1 strain mAb in this study is relatively conservative but has no neutralizing activity. However, this provides a potential new solution to overcome the current situation of insufficient effective FCV antigen reagents caused by the diversity of VP1 antigens.

[0104] In summary, the present invention successfully prepared the monoclonal antibody (mAb) 3A3C1 strain against FCV VP1 protein. The 3A3C1 strain mAb is of IgG1 type, and its light chain is a κ chain. It can specifically recognize and bind to multiple FCV strains, but has no neutralizing activity. The linear antigen epitope targeted by the 3A3C1 strain mAb is located in the VP1 protein. 426 PSRLTPAGDYAITSG 440 The (SEQ ID NO.47) region was compared with the amino acid sequences of the VP1 corresponding positions of 185 FCV strains at home and abroad. The results showed that the epitope was highly conserved and could be used as a candidate antibody for FCV universal antigen detection.

[0105] The above describes the specific embodiments of the present invention. It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art may make various modifications or variations within the scope of the claims, which do not affect the essence of the present invention.

Claims

1. A monoclonal antibody against feline calicivirus VP1 protein, characterized in that: The monoclonal antibody comprises an antibody heavy chain and an antibody light chain. The variable region of the antibody heavy chain includes CDR1 consisting of the amino acid sequence shown in SEQ ID NO.1, CDR2 consisting of the amino acid sequence shown in SEQ ID NO.2, and CDR3 consisting of the amino acid sequence shown in SEQ ID NO.3; The variable region of the antibody light chain includes CDR1 consisting of the amino acid sequence shown in SEQ ID NO.4, CDR2 having the amino acid sequence of DTS, and CDR3 consisting of the amino acid sequence of SEQ ID NO.

5.

2. The monoclonal antibody according to claim 1, characterized in that The monoclonal antibody comprises the heavy chain variable region amino acid sequence as shown in SEQ ID NO.16 or a homologous sequence thereof; and / or, It comprises the light chain variable region amino acid sequence shown in SEQ ID NO. 17 or a homologous sequence thereof.

3. A nucleic acid, characterized in that The nucleic acid encodes the heavy chain of the monoclonal antibody according to any one of claims 1 to 2; and the nucleic acid encodes the light chain of the monoclonal antibody according to any one of claims 1 to 2.

4. The nucleic acid according to claim 3, characterized in that comprising nucleotide sequences encoding CDR1, CDR2, and CDR3 of the heavy chain variable region, as shown in SEQ ID NOs. 18, 19, and 20, respectively, and Nucleotide sequences for encoding CDR1, CDR2, and CDR3 of the light chain variable region, wherein the nucleotide sequence encoding the CDR1 is as shown in SEQ ID NO.21, the nucleotide sequence encoding the CDR2 is GACACATCC, and the nucleotide sequence encoding the CDR3 is as shown in SEQ ID NO.

22.

5. An antigenic epitope peptide of feline calicivirus VP1 protein, characterized in that: The antigen epitope peptide specifically binds to the monoclonal antibody according to any one of claims 1 to 2; The amino acid sequence of the antigen epitope peptide is 426 PSRLTPAGDYAITSG 440 .

6. A biological material containing the nucleic acid according to claim 3 or 4, characterized in that: The biological material is one or more of an expression vector, a eukaryotic or prokaryotic host cell.

7. Use of the monoclonal antibody according to any one of claims 1 to 2 in the preparation of an FCV detection product.

8. The use according to claim 7, characterized in that: Including the use of monoclonal antibodies in the preparation of FCV detection kits.

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