Cfeline single-chain antibody targeting feline calicivirus as well as preparation method and application of feline single-chain antibody

Abstract

The invention discloses a feline single-chain antibody targeting feline calicivirus as well as a preparation method and application of the feline single-chain antibody, and belongs to the technical field of biology. The single-chain antibody includes a complementary determining region (CDR) of a heavy chain variable region (VH) and a light chain variable region (VL) having a specific amino acid sequence. A phage display technology is utilized for the first time, an FCV VP1-CDE antigen is prepared through prokaryotic expression, after a cat is immunized, a cat source scFv phage display library is constructed, five specific single-chain antibodies are obtained through multiple rounds of panning and identification, the neutralizing titer of one specific single-chain antibody reaches 1: 64, and the specific single-chain antibodies have remarkable FCV neutralizing activity. The single-chain antibody is high in specificity, low in immunogenicity and easy and convenient to prepare, can be used for preparing feline calicivirus diagnostic reagents or therapeutic drugs, solves the problem that in the prior art, FCV lacks special-effect diagnosis and treatment means, and provides new molecular materials and technical supports for prevention and control of FCV infection.

Description

Technical Field

[0001] This invention belongs to the fields of biotechnology and veterinary medicine, specifically relating to a feline calicivirus (FCV) single-chain variable fragment (scFv) antibody, its preparation method, and its application in the diagnosis and treatment of FCV. Background Technology

[0002] Feline calicivirus (FCV) infection is one of the most common infectious upper respiratory tract diseases in cats, causing a variety of symptoms ranging from mild localized infection to systemic disease. FCV infection often manifests as upper respiratory symptoms such as sneezing, runny nose, and coughing. Highly virulent strains (such as VS-FCV) can induce severe illnesses such as high fever, skin edema, and necrosis of internal organs, with a mortality rate as high as 50%-79%. Currently, there are no specific drugs for FCV, posing a serious threat to the health of felines.

[0003] FCV is a lipid-free RNA virus, approximately 30-40 nm in diameter, with a 7.7 kb linear single-stranded genome containing three open reading frames (ORFs). ORF2 encodes the major FCV capsid protein, which interacts with host cell receptors and influences viral invasion. ORF2 is translated into a 75 kDa capsid protein precursor. This precursor protein is cleaved by viral proteases to remove a 14 kDa leader protein, yielding the mature capsid protein VP1, approximately 62 kDa. The VP1 protein can be divided into six functional regions (A-F). Regions C, D, and E (CDE regions) have been confirmed as the major neutralizing epitopes of FCV, playing a crucial role in viral immune escape and regulation of the host immune response, making them ideal antigen targets for preparing FCV-specific antibodies.

[0004] Single-chain variable fragments (scFvs) are formed by linking the antibody heavy chain variable region (VH) and light chain variable region (VL) via a flexible linker (Gly4Ser)3. They possess advantages such as low molecular weight, strong penetration, good specificity, low immunogenicity, and complete antigen-binding activity. scFvs contain four backbone regions (FRs) and three complementarity-determining regions (CDRs), with the CDRs being the core regions for antigen recognition and binding, determining the antibody's specificity. Commonly used scFv display systems include phage display, ribosome display, and yeast surface display. Among these, phage display antibody technology can construct high-capacity antibody libraries. Through multiple rounds of "adsorption-elution-enrichment" processes, specific antibodies targeting antigens can be screened. It is characterized by its simplicity, efficiency, and low cost, making it a mature and widely used method for constructing scFv immune libraries. M13 phage is the most commonly used vector in this technology. Its characteristics include not lysing host bacteria and being highly resistant to factors such as high temperature, pH, denaturants, ultraviolet light, and proteolytic enzymes.

[0005] In recent years, phage display technology has been successfully applied to the construction of various antibody libraries. For example, a New Zealand white rabbit-derived scFv antibody library can effectively inhibit rabies virus infection, and a chicken-derived scFv antibody library is used to combat Helicobacter pylori. However, there are currently no reports on screening FCV-specific scFvs based on phage display antibody technology. Traditional murine monoclonal antibodies are heterologous antibodies, which are prone to producing anti-antibodies during continuous immunization and have a large molecular weight, making them unsuitable for in vitro expression and practical applications. Therefore, the preparation of efficient, safe, and highly specific feline FCV single-chain antibodies is of great significance for the diagnosis and treatment of FCV infection and can provide molecular materials and technical support for the development of related innovative products. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of the prior art and provide a feline single-chain antibody targeting feline calicivirus with high specificity, good antigen-binding activity and virus neutralizing ability, as well as its preparation method and application.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: This invention provides a feline single-chain antibody scFv-3A5 targeting feline calicivirus. The single-chain antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL). The amino acid sequences of the complementarity-determining regions CDR1, CDR2, and CDR3 of the heavy chain variable region are shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively. The amino acid sequences of the complementarity-determining regions CDR1, CDR2, and CDR3 of the light chain variable region are shown in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively.

[0008] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:7, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:8. This antibody has been experimentally confirmed to have significant FCV virus neutralizing activity.

[0009] The present invention also provides another feline single-chain antibody targeting feline calicivirus, scFv-2H5, wherein the single-chain antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL), the amino acid sequences of the complementarity-determining regions CDR1, CDR2 and CDR3 of the heavy chain variable region are shown in SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:11, respectively; the amino acid sequences of the complementarity-determining regions CDR1, CDR2 and CDR3 of the light chain variable region are shown in SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:14, respectively.

[0010] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:15, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:16. This antibody exhibits FCV-specific binding activity and a certain degree of FCV virus neutralizing activity.

[0011] The exemplary amino acid sequences of the antibody complementarity-determining region (CDR) and variable region (VH / VL) involved in this invention are shown below. SEQ ID NO:1-8 correspond to scFv-3A5 with neutralizing activity; SEQ ID NO:9-16 correspond to another strain, scFv-2H5, with binding activity.

[0012] SEQ ID NO:1 (scFv-3A5 VH CDR1): TFSNYPMT SEQ ID NO:2 (scFv-3A5 VH CDR2): DISSTGLRTYYADSVKG SEQ ID NO:3 (scFv-3A5 VH CDR3): PSRDYWGHGA SEQ ID NO:4 (scFv-3A5 VL CDR1):TGVGSYVD SEQ ID NO:5 (scFv-3A5 VL CDR2): GNSYRPSW SEQ ID NO:6 (scFv-3A5 VL CDR3): VSWYSSGSAL SEQ ID NO:7 (scFv-3A5 VH): DVQLVESGGNLVKPGGSLRLTCVASGFTFSNYPMTWVRQAPGKGLQWVADISSTGLRTYYADSVKGRFTVSRDNAKNTLYLEMNKLKTEDTATYYCAKPSRDYWGHGALVTVSSASPTAPSVF SEQ ID NO:8 (scFv-3A5 VL): QSGLNQPSSVSGALGQRVTISCTGVGSYVDWYQQIPGMAPKTIIYGNSYRPSWVPDRFSGSKSGSTGTLTITGLHAEDEADYYCVSWYSTGSALFGGGTHLTVLSQPK SEQ ID NO:9 (scFv-2H5 VH CDR1): NFDTYTMV SEQ ID NO:10 (scFv-2H5 VH CDR2):TISERGGKTFYADSVKG SEQ ID NO:11 (scFv-2H5 VH CDR3): GRNNCIGNCPRFDVWGRGV SEQ ID NO:12 (scFv-2H5 VL CDR1): TGVGNYVD SEQ ID NO:13 (scFv-2H5 VL CDR2): GNTYRPS SEQ ID NO:14 (scFv-2H5 VL CDR3): SSWDSRGRALCFRRRDQ SEQ ID NO:15 (scFv-2H5 VH): DVQLVESGGDLVKPGGSLKLTCVASGFNFDTYTMVWVRQAPGKGLQWVATISERGGKTFYADSVKGRVTISRDNAKNTLLLQMNTLQTDDTGTYYCVRGRNNCIGNCPRFDVWGRGVLVTVSSASPTAPSVF SEQ ID NO:16 (scFv-2H5 VL): QSGLNQPSSVSGALGQRVTISCTGVGNYVDWYQQIPGMAPKTVIYGNTYRPSGSLIDSPAPSLAAQAPWPSLGLQAADEADYYCSSWDSRGRALCFRRRDQVTVLGQPK。

[0013] This invention provides a method for preparing the above-mentioned feline single-chain antibody targeting feline calicivirus, comprising the following steps: (1) Cats were immunized with the CDE region (VP1-CDE) of the feline calicivirus VP1 protein as an immunogen, and peripheral blood lymphocytes were collected from the immunized cats. (2) The antibody heavy chain variable region (VH) gene and light chain variable region (Vλ) gene are amplified from the lymphocytes and linked by a linker peptide sequence to form a single-chain antibody (scFv) gene; (3) The scFv gene was cloned into a phage display vector to construct a cat-derived scFv phage display library; (4) Using VP1-CDE protein as the target, the phage display library is subjected to multiple rounds of screening to obtain phage clones that specifically bind to VP1-CDE protein; the multiple rounds of screening are 3 rounds of solid-phase “adsorption-elution-enrichment” screening; (5) Obtain the scFv gene from the positive phage clone, express and purify it to obtain the cat-derived single-chain antibody.

[0014] This invention provides the application of the above-mentioned feline single-chain antibody targeting feline calicivirus in the preparation of reagents or kits for detecting or diagnosing feline calicivirus infection.

[0015] This invention provides the use of the above-mentioned feline single-chain antibody targeting feline calicivirus in the preparation of a medicament for the prevention or treatment of feline calicivirus infection.

[0016] The present invention provides a kit for detecting or diagnosing feline calicivirus infection, characterized in that it contains a feline single-chain antibody targeting feline calicivirus.

[0017] The beneficial effects of this invention are: 1. This invention is the first to successfully prepare a feline single-chain antibody against the VP1-CDE region of the key FCV antigen using phage display technology, overcoming the shortcomings of traditional mouse monoclonal antibodies, which have strong immunogenicity and complex preparation.

[0018] 2. The feline single-chain antibody (such as scFv-3A5) obtained in this invention has excellent FCV antigen binding activity and significant virus neutralization ability, with a neutralization titer of up to 1:64 and a minimum neutralizing concentration of 1.172 µg / mL, showing great potential as a therapeutic antibody.

[0019] 3. The antibody provided by this invention is a feline antibody, which has lower immunogenicity than traditional mouse antibodies and is more suitable for long-term or repeated treatment of cats.

[0020] 4. The single-chain antibody provided by this invention has a small molecular weight and simple structure, making it easy to perform genetic engineering and large-scale production, thus providing core molecular material for the development of novel FCV diagnostic reagents and therapeutic drugs. Attached Figure Description

[0021] Figure 1 Figure 1 shows the prokaryotic expression, purification, and Western blot identification results of recombinant VP1-CDE protein. M. Protein molecular weight standard; A. 1. Induced bacterial cells; 2. Supernatant after sonication; 3. Precipitate after sonication; 4. Flow-through solution after binding; 5. VP1-CDE protein after elution; B. 1. Negative control; 2. VP1-CDE protein.

[0022] Figure 2 The image shows the serum antibody titer results of cats immunized three times with VP1-CDE protein.

[0023] Figure 3 Electrophoresis images of feline VH and Vλ gene amplification and scFv gene splicing results. M. DNA standard DL5000; A. 1-3. VH amplified fragment; B. 1-3. Vλ amplified fragment; C. 1-3. scFv amplified fragment.

[0024] Figure 4 PCR identification and sequence diversity analysis of the scFv phage display library. M. DNA standard DL2000; A. PCR identification of 24 colonies (1-24); 25. Negative control; B. Amino acid sequence phylogenetic analysis.

[0025] Figure 5 Figure A shows the solid-phase panning enrichment results and phage ELISA screening results of the scFv phage library. A. Solid-phase panning results of the scFv phage library (10...) n (Refers to the dilution factor of the bacterial culture); B.phage ELISA results.

[0026] Figure 6 Electrophoresis image for PCR identification of positive scFv phage clones. M. DNA standard DL 5000; PCR amplification of 1-30 scFv phage clones.

[0027] Figure 7 SDS-PAGE analysis of five recombinant scFv strains after eukaryotic expression and purification. M. Protein molecular weight standard; 1. scFv-3A5; 2. scFv-3B8; 3. scFv-3H5; 4. scFv-2H5; 5. scFv-2I5.

[0028] Figure 8The image shows the Western blot identification results of five recombinant scFv strains. M: Protein molecular weight standard; AE: scFv-3A5, scFv-3B8, scFv-3H5, scFv-2H5, and scFv-2I5, respectively, based on Western blot analysis. 1. VP1-CDE protein; 2. FPV VP2 protein.

[0029] Figure 9 The image shows the indirect ELISA detection results of five recombinant scFv strains.

[0030] Figure 10 The image shows the indirect immunofluorescence (IFA) detection results of five recombinant scFv strains. A. scFv-3A5; B. scFv-3B8; C. scFv-3H5; D. scFv-2H5; E. scFv-2I5; F. F81 Cell Control. Detailed Implementation

[0031] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. The following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods not specifically described in the embodiments are generally performed under conventional conditions or as recommended by the manufacturer.

[0032] The main reagents and instruments used in this invention are as follows: Main reagents: pET-28a-VP1-CDE plasmid, pComb3XSS phage vector, pFUSE-Fc eukaryotic expression vector, and F81 cat kidney cells were preserved by the Public Health Research Group of the College of Veterinary Medicine, Gansu Agricultural University; two male cats were provided by the Lanzhou Institute of Animal Husbandry and Veterinary Medicine, Chinese Academy of Agricultural Sciences. E. coli BL21(DE3) competent cells, Qingke (Beijing) Biotechnology Co., Ltd.; E. coli. SS320 competent cells, E. coli NEB5α F' competent cells and M13KO7 helper phages were purchased from NEB Corporation. Trizol reagent, 2 × Rapid Taq Master Mix, gel DNA extraction kit, and reverse transcription kit were purchased from Novizan (Nanjing) Biotechnology Co., Ltd. Ni affinity chromatography resin and Protein A affinity chromatography column were purchased from Sangon Biotech (Shanghai) Co., Ltd. HRP-labeled anti-M13 antibody and HRP-labeled IgG-Fc antibody were purchased from Sino Biologicals. CY3 conjugated anti-human IgG was purchased from Sigma-Aldrich.

[0033] Main instruments: Ultrasonic disruptor (Ningbo Xinzhi Biotechnology Co., Ltd.); BIO-RAD-2000 gel imaging system; protein electrophoresis apparatus (Bio-Rad, USA); Spectra Max i3x multi-functional enzyme-linked immunosorbent assay (ELISA) system (MolecularDevlces, USA); gradient PCR instrument (Eppendorf GmbH, Germany); low-temperature centrifuge (HITACHI, Japan); constant temperature incubator shaker (Shanghai Nanrong Experimental Equipment Co., Ltd.).

[0034] Example 1: Expression, purification, and animal immunization of recombinant VP1-CDE protein 1. Prokaryotic induction and purification of recombinant pET-28a-VP1-CDE The pET-28a-VP1-CDE plasmid, preserved in the laboratory, was transformed into E. coli BL21(DE3) competent cells using a heat shock transformation method. The transformed cells were plated on LB solid medium containing kanamycin (50 µg / mL) and incubated overnight in an inverted incubator at 37 ℃. Single colonies were picked and inoculated into LB liquid medium containing kanamycin (50 µg / mL) and incubated overnight at 37 ℃ and 200 r / min using a shaker. The overnight culture of single colonies was then transferred at a 1:100 volume ratio to 300 mL of kanamycin-containing medium. In (50µg / mL) LB liquid culture medium, wait for OD 600 When the pH reached 0.6-0.8, isopropyl-β-D-thiogalactoside (IPTG) was added to a final concentration of 0.5 mmol / L, and expression was induced overnight in a shaker at 16℃ and 180 r / min. After induction, the bacterial culture was collected, centrifuged at 4℃ and 8000 r / min for 10 min, the supernatant was discarded, the cells were resuspended in PBS buffer, and sonicated on ice for 30 min. The lysed culture was then centrifuged at 4℃ and 8000 r / min for 15 min to separate the supernatant from the precipitate. The recombinant protein in the supernatant was purified by Ni affinity chromatography. The purified sample was analyzed by SDS-PAGE electrophoresis and Western blot to identify the molecular size and specificity of the protein and to determine the protein concentration. The recombinant VP1-CDE protein was obtained and stored at -80℃.

[0035] Soluble expression was induced in the pET-28a-VP1-CDE recombinant plasmid. The protein supernatant was purified using Ni affinity chromatography and analyzed by SDS-PAGE and Western blot. Results showed a single band around 18 kDa, consistent with the theoretical size of the VP1-CDE protein. Figure 1 ).

[0036] 2. Animal Immunology and Serological Assay Purified VP1-CDE protein was used as an antigen to inoculate cats. For the first immunization, it was fully emulsified with Freund's complete adjuvant at a 1:1 ratio and injected subcutaneously at multiple sites on the back. For the second and third immunizations, it was mixed with Freund's incomplete adjuvant at a 1:1 ratio and administered once every two weeks. After the third immunization, cat serum was collected from the hind limb veins. The purified VP1-CDE protein was used as the coating antigen at a concentration of 100 ng / well. The serum antibody titer was detected by indirect ELISA. When the serum titer was higher than 1:64000 (P / N>2.5), anticoagulated whole blood was collected from the cats for subsequent construction of a phage display antibody library.

[0037] Purified VP1-CDE protein was used as an antigen to immunize cats. Using an indirect ELISA method, the antibody titer against VP1-CDE in the cat serum after the third immunization reached 1:256,000, meeting the criteria for phage display library construction. Figure 2 Example 2: Construction of a VP1-CDE-specific feline single-chain antibody phage immunotherapy library. 1. Primer design According to the literature report by Lu Z et al., feline antibody gene sequences were searched in GenBank (GenBank numbers: KY795030-KY795318). Relatively conserved sequences were identified by comparing the variable regions (VH) and (Vλ) of the feline IgG heavy chain and light chain, and VH and Vλ amplification primers were designed using SnapGene software (Table 1). All primers were synthesized by Wuhan Jinkairui Biotechnology Co., Ltd.

[0038] 2. scFv gene ligation Fresh, anticoagulated whole blood was collected from cats, and peripheral blood lymphocytes were isolated. Total RNA was extracted from the peripheral blood lymphocytes using the conventional Trizol method, and the purity and concentration of RNA were determined using a micro-ultraviolet spectrophotometer. The total RNA was then reverse transcribed into cDNA. Using the cDNA as a template, the VH and Vλ genes were amplified using specific primers (sequences shown in Table 1), and the target fragments were recovered. Overlap extension PCR was performed using the recovered and purified VH and Vλ gene fragments as templates. The VH and Vλ genes were ligated together using the (Gly4Ser)3Linker (amino acid sequence GGGGSGGGGSGGGGS) to form the scFv gene. The scFv gene was then ligated into the pComb3XSS phage vector using homologous recombination.

[0039] Total RNA was extracted from peripheral blood lymphocytes and reverse transcribed into cDNA. VH and Vλ gene fragments were then amplified using specific primers, successfully producing a VH gene of approximately 400 bp and a Vλ gene of approximately 350 bp. Figure 3 A and Figure 3B), consistent with the expected length. Using the gel recovery products of VH and Vλ as templates, overlap extension PCR amplification was performed, successfully obtaining an amplified band of the scFv gene of approximately 750 bp. Figure 3 C).

[0040] 3. Preparation of phage-displaying scFv libraries The scFv-pComb3XSS ligation product was electroporated into SS320 competent cells. The electroporated bacterial culture was then transferred to 30 mL of antibiotic-free 2YT medium and cultured at 37 ℃ with shaking at 200 r / min for 1 h. 100 µL of the culture was then diluted 10⁻⁶ times. 3 -10 5 The phage library volume was calculated by counting the number of single colonies on the plate using a dropper plate. The formula for calculating the volume is: Volume = Number of single colonies × Dilution factor × Total volume of mixed bacterial solution. The remaining bacterial solution was transferred to 300 mL of 2YT medium containing Amp-Kan antibiotic and incubated at 37°C and 200 rpm. -1 Incubate overnight with shaking. The next day, collect the bacterial culture and incubate at 4 °C and 8000 r·min. -1 Centrifuge for 10 min and collect the supernatant. Add 1 / 4 volume of PEG8000 / NaCl solution to the supernatant, incubate on ice for 30 min, then centrifuge again and discard the supernatant. Resuspend the precipitate in 1 mL of PBS buffer to obtain the primary phage library of single-chain antibodies. Randomly select 24 single colonies, amplify by PCR to identify the number of positive clones, and send the PCR amplification products of the positive clones to a sequencing company for sequencing analysis.

[0041] The scFv gene fragment was cloned into the linearized pComb3XSS phage vector, and a phage display library was constructed after electroporation. The bacterial culture was diluted and plated. The results showed that the volume of the antibody library reached 2.1 × 10⁻⁶. 8 CFU / mL. Twenty-four single colonies were randomly selected for PCR identification, and all amplified bands approximately 750 bp in size. Figure 4 A), the vector ligation efficiency is 100%, and a phylogenetic tree of the sequences was drawn using MEGA software. The 24 sequences are all unique. Figure 4 B). The above data indicate that the phage display library constructed in this study has good diversity and can be used for subsequent enrichment and screening experiments.

[0042] Example 3: Screening and identification of VP1-CDE-specific scFv 1. Phage display scFv library solid-phase panning VP1-CDE protein (2 µg / well) was used to coat the cells overnight at 4 °C. Four antigen-coated wells and four negative control wells were prepared. After coating, the liquid in the wells was discarded, and the cells were blocked at 37 °C for 2 h with 5% skim milk powder. The cells were washed three times with PBST. 100 µL of primary phage library was added to each well, and the cells were incubated at 37 °C with shaking for 2 h. After incubation, the cells were washed 10 times with PBST to remove non-specifically bound phages. 100 µL of 0.1 mol / L HCl elution buffer was added to each well, and the cells were incubated at room temperature for 5 min. Then, 1 / 4 volume of 1 mol / L Tris-HCl was quickly added to neutralize the elution buffer. The neutralized elution buffer was then transferred to 1 mL of NEB5α F' bacterial culture and incubated at 37 °C. After incubating at 200 rpm for 1 h with shaking, 2 µL of M13KO7 helper phage was added, and the culture was continued at 37 ℃ with shaking at 200 rpm for another 1 h. After the culture was completed, the bacterial culture was transferred to 35 mL of 2YT liquid medium containing Amp-Kan antibiotic for expansion. The above screening procedure was repeated three times. The number of phages eluted from the antigen-coated wells and the number of phages eluted from the control wells were compared by drop plate analysis to determine whether specific scFvs against the antigen had been enriched.

[0043] Antibody libraries were screened based on antigen-antibody specific binding, and the scFv phage library underwent three rounds of "adsorption-elution-enrichment," as determined by plate drop results. Figure 5 A) Enrichment was observed in both the second and third rounds. The number of phages eluted from the VP1-CDE protein-coated wells was significantly higher than that eluted from the control wells, indicating effective enrichment of specific antibodies.

[0044] 2. Screening positive clones using phage-ELISA Coat wells with 100 ng / well of VP1-CDE protein at 4°C overnight. After coating, discard the liquid in the wells, add 5% skim milk blocking buffer, and block at room temperature for 2 h. Serially dilute the phage library obtained from the third round of screening and plate it. Randomly select 92 single clones, and set up 4 negative controls. Incubate overnight at 37°C with shaking at 200 rpm. The next day, centrifuge at 4°C and 4000 rpm for 20 min, collect the supernatant (primary antibody for single-clone phages), add the supernatant to the coated wells, incubate at 37°C for 1 h, add HRP-labeled anti-M13 antibody (1:8000 dilution) as the enzyme-labeled secondary antibody, and incubate at 37°C for 1 h. OD values ​​are then read. 450 Value, positive OD 450 / Negative OD 450A P / N ratio ≥ 2 was used to determine positive clones. Positive clones with higher P / N values ​​were selected for subsequent identification and eukaryotic expression experiments. Using a P / N ratio (OD450 of positive wells / OD450 of negative wells) ≥ 2 as the criterion, 30 positive clones were selected. Figure 5 B).

[0045] 3. Positive clone sequence analysis: PCR identification was performed on 30 positive scFv phage clones with high phage ELISA binding activity, and a total of 26 target bands were successfully detected. Figure 6 Fifteen PCR-positive clones were randomly selected for sequencing and sequence alignment. Using DNAMAN software, amino acid sequence alignment revealed five clones with different amino acid sequences, named 3A5, 3B8, 3H5, 2H5, and 2I5. In contrast, the VH sequence of the five positive scFv clones was highly conserved, which is closely related to primer design. The Vλ region, however, exhibited rich amino acid diversity, with the main differences concentrated in the CDR3 region. The CDR3 region of the five scFvVλ clones varied by approximately 10-13 amino acids, while the FR region was relatively conserved.

[0046] Example 4: Eukaryotic expression, purification, and activity identification of scFv 1. Eukaryotic expression, purification, and Western blot identification of scFv The scFv genes of five positive clones were ligated into the pFUSE-Fc eukaryotic expression vector to construct recombinant pFUSE-Fc-scFv plasmids. The recombinant plasmids were transiently transfected into Expi293F suspension cells. Five to seven days after transfection, the cell culture medium was centrifuged at 4°C and 8000 r / min to collect the supernatant. The supernatant was filtered through a 0.22 μm filter, and the antibody was purified using a Protein A affinity chromatography column. The eluted protein was then analyzed by SDS-PAGE electrophoresis to determine the scFv molecule size and purity. Subsequently, the protein specificity was further identified by Western blot. VP1-CDE protein was subjected to SDS-PAGE electrophoresis, with feline parvovirus VP2 protein set as a negative control. After electrophoresis, the membrane was transferred and blocked with 5% skim milk powder at room temperature for 1 h. The purified recombinant scFv (1:500 dilution) was added as the detection primary antibody, and HRP-labeled anti-human IgG-Fc (1:8000 dilution) was added as the enzyme-labeled secondary antibody. The membrane was incubated at 37 °C for 1 h. After washing with PBST to remove unbound secondary antibody, colorimetric analysis was performed.

[0047] Five scFv strains were ligated into the pFUSE-Fc eukaryotic expression vector, transfected, and expressed. After 5 days, the supernatant was collected by centrifugation for purification. The eluted protein was analyzed by SDS-PAGE, and a single protein band appeared around 56 kDa, which was consistent with the theoretical value of the protein. Figure 7 The VP1-CDE protein was subjected to SDS-PAGE electrophoresis, with feline parvovirus VP2 protein as a negative control. After transfer, five purified scFv strains were incubated with primary antibody and HRP-labeled IgG-Fc as secondary antibody. Western blot analysis showed that all five scFv strains specifically reacted with the VP1-CDE protein, while the negative control did not react. The target protein band was visible around 18 kDa, and the protein purification results were as expected. Figure 8 ).

[0048] 2. scFv indirect ELISA detection Coat microplates with 100 ng / well of VP1-CDE protein, and simultaneously coat PBS wells as a blank control, at 4°C overnight. Block with 5% skim milk powder at 37°C for 1 h, then wash three times with PBST. After adjusting the initial concentration of purified scFv antibody to a uniform level, serially dilute scFv with PBS buffer at ratios of 1:50, 1:100, 1:200, 1:400, 1:800, and 1:1600, and add to the corresponding wells, incubate at 37°C for 1 h, then wash three times with PBST. Add HRP-labeled anti-human IgG-Fc enzyme-labeled secondary antibody (1:8000 dilution), incubate at 37°C for 1 h, then wash three times with PBST. Add 100 µL of TMB chromogenic solution to each well and react in the dark for 10 min. Then add 50 µL of 2 M H2SO4 solution to stop the reaction and read the OD of each well. 450 value.

[0049] After serial dilution of recombinant scFv, its antigen-binding activity was detected by indirect ELISA. The results showed that the OD values ​​of the five scFv positive wells were [missing data]. 450 The value decreases with decreasing antigen coating concentration, and the corresponding OD value... 450 The values ​​were all significantly higher than those of the blank control wells (coated only with PBS), indicating that all five scFv strains possessed VP1-CDE antigen-binding activity. When the antibody dilution was 1:800 and 1:1600, the OD values ​​of scFv were significantly higher. 450 The values ​​were all relatively low, with scFv-3A5 showing the strongest binding reaction and excellent antigen-binding ability, while scFv-2I5 and scFv-3B8 showed the weakest binding reaction and relatively weak antigen-binding ability. Figure 9 ).

[0050] 3. scFv indirect immunofluorescence assay (IFA) 100 TCID doses 50 F81 cells containing FCV were cultured in 96-well cell culture plates for 3 h. The culture medium was discarded, and the cells were washed three times with PBS. 100 µL of 80% acetone solution, pre-cooled to -20 °C, was added to each well, and the cells were fixed at -20 °C for 25 min. After fixation, the acetone solution was discarded, and the cells were washed three times with PBST. The cells were blocked with 5% skim milk powder at 37 °C for 1 h, and washed three times with PBST. scFv was uniformly diluted to 0.2 mg / mL with PBS buffer and added to each well. The cells were incubated at 37 °C for 1 h, and washed three times with PBST. Cy3-conjugated anti-human IgG Fc antibody (1:500 dilution) was used as a secondary antibody and incubated at 37 °C in the dark for 1 h, followed by washing three times with PBST. 50 µL of Hoechst 33342 nuclear staining solution was added to each well, and the cells were stained at room temperature in the dark for 10 min before observation under a fluorescence microscope. A negative control without virus inoculation was also included.

[0051] To further verify whether recombinant scFv can specifically bind to FCV within cells, the reactivity of five scFv strains (3A5, 3B8, 3H5, 2H5, and 2I5) was detected by indirect immunofluorescence assay. The results showed that all five scFv strains specifically reacted with FCV-infected cells, further confirming that these five scFv strains possess FCV-specific reactivity. Figure 10 ).

[0052] 4. scFv virus neutralization test The initial concentrations of the five purified scFvs were first adjusted to 75 µg / mL, and then serially diluted 4-fold with DMEM medium. Each dilution of antibody was then mixed with an equal volume containing 100 TCID50. 50 The live FCV virus solution was thoroughly mixed and incubated at 37 °C for 1 h. The FCV-scFv mixture was then inoculated into F81 cat kidney cells, with four replicates per dilution. FCV-infected controls and blank cell controls were also included. Cells were cultured continuously at 37 °C in a 5% CO2 incubator for 48 h after inoculation, and the cytopathic effect was observed under an inverted microscope.

[0053] The neutralizing activity of five recombinant scFv feline calicivirus (FCV) strains was evaluated using a virus neutralization assay. Based on the cytopathic effect (CPE) results, scFv-3A5 showed a neutralizing titer of 1:64, demonstrating significant neutralizing activity against FCV, with a minimum neutralizing concentration (MINC) of 1.172 µg / mL. scFv-2H5 showed a neutralizing titer of 1:16, indicating some neutralizing ability, but with weak activity and a MINC of 4.688 µg / mL. The remaining three scFv strains showed no significant neutralizing activity against FCV (Table 2). This suggests that scFv-3A5 is a potential candidate molecule for developing into a therapeutic antibody against FCV.

[0054] In summary, this invention utilizes phage display technology to screen scFvs. The size of the antibody library is a key factor in selecting high-affinity antibodies; a larger library size facilitates the acquisition of high-affinity antibodies. Existing research indicates that an antibody library size of 10... 7 ~10 8 The CFU / mL concentration basically meets the screening requirements. The phage scFv library successfully constructed in this invention has a size of 2.1 × 10⁻⁶. 8 The library, with a concentration of CFU / mL and verified by amino acid sequence alignment, exhibited good diversity and its volume met the screening requirements. Solid-phase panning and phage ELISA played crucial roles in screening for specific VP1-CDE scFv clones. Solid-phase panning involves co-incubating the antibody library with immobilized antigen, eluting the bound phages by changing the pH, and then allowing the phages to multiply and amplify in E. coli. After multiple rounds of solid-phase panning, phage ELISA was used to assess the specific binding activity of the screened antibodies to identify positive clones. The phage ELISA results showed that 30 positive scFv clones with binding activity to the VP1-CDE protein were successfully screened.

[0055] This invention prepared the truncated FCV capsid protein VP1-CDE antigen via prokaryotic expression, which was then emulsified with Freund's adjuvant and used to immunize experimental cats. A phage display-specific single-chain antibody phage immunotherapy library was constructed. Positive clones were obtained after three rounds of adsorption-elution-enrichment screening, followed by sequencing and eukaryotic expression. The specificity, binding activity, and neutralizing capacity of the antibodies were detected by indirect ELISA, Western blot, immunofluorescence assay (IFA), and virus neutralization assay. The results showed that soluble recombinant VP1-CDE protein was successfully purified. After three immunizations of the experimental cats, the serum VP1-CDE-specific antibody titer reached 1:256000, meeting the requirements for library construction. The constructed feline anti-VP1-CDE library had a capacity of 2.1 × 10⁻⁶. 8 With a CFU / mL concentration and a 100% positivity rate, five VP1-CDE-specific single-chain antibodies were obtained via phage ELISA. These antibodies demonstrated good antigen-binding activity as confirmed by Western blot, ELISA, and IFA. Two of the antibodies exhibited significant virus-neutralizing activity as confirmed by neutralization assays. This study successfully obtained FCV-specific single-chain antibodies using phage display technology, providing molecular material for further exploration of the application of single-chain antibodies in the diagnosis and treatment of feline calicivirus.

Claims

1. A feline single-chain antibody targeting feline calicivirus, characterized in that, The antibody comprises a heavy chain variable region and a light chain variable region. The amino acid sequences of the complementarity-determining regions CDR1, CDR2, and CDR3 of the heavy chain variable region are shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively. The amino acid sequences of the complementarity-determining regions CDR1, CDR2, and CDR3 of the light chain variable region are shown in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively.

2. The single-chain antibody according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 7, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

8.

3. A feline single-chain antibody targeting feline calicivirus, characterized in that, The antibody comprises a heavy chain variable region and a light chain variable region. The amino acid sequences of the complementarity-determining regions CDR1, CDR2, and CDR3 of the heavy chain variable region are shown in SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11, respectively. The amino acid sequences of the complementarity-determining regions CDR1, CDR2, and CDR3 of the light chain variable region are shown in SEQ ID NO:12, SEQ ID NO:13, and SEQ ID NO:14, respectively.

4. The feline single-chain antibody targeting feline calicivirus according to claim 3, characterized in that, The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:15, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

16.

5. The method for preparing feline single-chain antibodies according to any one of claims 1-4, characterized in that, Includes the following steps: (1) Cats were immunized with the CDE region of the feline calicivirus VP1 protein as an immunogen, and peripheral blood lymphocytes were collected from the immunized cats. (2) Amplify the antibody heavy chain variable region gene and the light chain variable region gene from the lymphocytes, and connect them by linking peptide sequences to form a single-chain antibody gene; (3) The single-chain antibody gene was cloned into a phage display vector to construct a cat-derived scFv phage display library; (4) Using VP1-CDE protein as the target, the phage display library was screened in multiple rounds to obtain phage clones that specifically bind to VP1-CDE protein; (5) Obtain the single-chain antibody gene from the positive phage clone, express and purify it to obtain the cat-derived single-chain antibody.

6. The use of a feline single-chain antibody as described in any one of claims 1-4 in the preparation of a reagent or kit for detecting or diagnosing feline calicivirus infection.

7. The use of a feline single-chain antibody as described in any one of claims 1-4 in the preparation of a medicament for the prevention or treatment of feline calicivirus infection.

8. A kit for detecting or diagnosing feline calicivirus infection, characterized in that, It contains the feline single-chain antibody as described in any one of claims 1-4.

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