Cathepsin D epitope (CDE) specific chicken single-chain antibody against feline calicivirus VP1 and preparation and application thereof

Abstract

The application discloses a kind of targeting cat calicivirus VP1-CDE protein chicken source single-chain antibody and its preparation and application, belong to biotechnology field.The single-chain antibody is selected from X2B2, X2E1 Or X2G5, its heavy chain variable region and light chain variable region amino acid sequence are respectively as shown in SEQ ID NO:1-6.The application is prokaryotic expression and purified VP1-CDE protein as antigen immunization chicken, amplifies VH And VL Gene and is spliced into scFv gene by (Gly4Ser) 3 linker, constructs library capacity not less than 5.25×10 7 CFU / mL phage display library, specific single-chain antibody clone is obtained by solid-phase panning, and its binding activity is verified by Western blot, ELISA, indirect immunofluorescence test after eukaryotic expression and purification.The results show that the three single-chain antibodies can specifically bind VP1-CDE protein and FCV virus particles, and there is no cross reaction, which provides key experimental materials for basic research of cat calicivirus, can be used for developing FCV rapid diagnostic reagent or kit, also provides candidate molecules for the development of FCV infection treatment drugs, and has important theoretical significance and application value.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to a chicken-derived single-chain antibody against feline calicivirus VP1-CDE protein, its preparation method, and its application. Background Technology

[0002] Feline calicivirus (FCV) is a single-stranded positive-sense RNA virus belonging to the genus Varicellavirus in the family Caliciviridae. It is one of the main pathogens causing respiratory diseases in felines. FCV is highly contagious and can infect almost all felines; it has also been isolated from dogs. In recent years, with the continuous evolution and mutation of FCV, some strains have become highly virulent systemic feline caliciviruses (VS-FCV) with efficient replication characteristics. Compared with traditional FCV strains, VS-FCV has significantly enhanced pathogenicity and exhibits diverse clinical symptoms. In addition to typical skin ulcers and oral ulcers, severe infections can lead to necrosis of multiple organs, including the pancreas, liver, and spleen, resulting in a significantly increased mortality rate in affected cats and posing a severe challenge to the prevention and control of FCV.

[0003] The full-length FCV genome is 7.7 kb and contains three open reading frames (OFRs). OFR2 encodes the viral structural protein precursor, which matures after proteolytic hydrolysis to form the major capsid protein VP1. The VP1 protein consists of six functional regions (A-F). Region C has a high amino acid sequence variation rate and is closely related to viral antigenic drift and genetic diversity. Region E is a functional adhesion region that specifically recognizes and binds to receptor adhesion molecules on the host cell surface; this region also contains the core site for FCV neutralizing antibodies. Previous studies have confirmed that regions C, D, and E of the VP1 protein together constitute the major neutralizing epitope region. This region is not only a key target for inducing a protective immune response in the host but also plays a central regulatory role in FCV evading host immune surveillance and achieving persistent infection.

[0004] Single-chain variable fragments (scFvs) are composed of the variable region of the heavy chain (VH) and the variable region of the light chain (VL) linked together by flexible linker peptides. Due to their relatively small molecular weight, they possess excellent tissue penetration and diffusion capabilities, showing broad application prospects in disease diagnosis, targeted therapy, and biosensing. Compared to traditional murine and human antibodies, avian antibodies, due to their unique antibody gene structure, require only one pair of primers each for amplifying the VH and VL genes. Therefore, constructing chicken-derived single-chain antibody libraries has the advantage of simpler technical operation and readily generates highly specific and high-affinity antibody responses against heterologous mammalian antigens. Since Yamanaka et al. first successfully constructed a chicken-derived single-chain antibody library and screened for specific antibodies using phage display technology in 1996, research on chicken-derived single-chain antibodies has gradually become a hot topic in the field of genetically engineered antibodies. However, no chicken-derived single-chain antibodies targeting the FCV VP1-CDE protein have been reported to date, and the lack of specific antibody tools for rapid diagnosis and treatment of FCV limits the development of FCV prevention and control technologies.

[0005] This invention uses prokaryotically expressed VP1-CDE protein as an immunogen to inoculate SPF chickens. The amplified chicken-derived antibody VH and VL genes are spliced ​​into an scFv gene using overlap PCR. An scFv phage display library targeting FCV VP1-CDE protein is constructed. The reactivity and virus neutralization capacity of each single-chain antibody with VP1-CDE protein are determined using Western blot, ELISA, indirect immunofluorescence, and virus neutralization assays. This provides core technical support and a solid material foundation for the subsequent development of single-chain antibodies and rapid test strips for FCV treatment. Summary of the Invention

[0006] The purpose of this invention is to provide a chicken-derived single-chain antibody that can specifically bind to the feline calicivirus VP1-CDE protein, its preparation method, and its application in FCV detection.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: This invention provides a chicken-derived single-chain antibody against feline calicivirus VP1-CDE protein, wherein the single-chain antibody is selected from X2B2, X2E1, or X2G5; wherein: The amino acid sequence of the heavy chain variable region of X2B2 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 amino acid sequence of the heavy chain variable region of X2E1 is shown in SEQ ID NO:3, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:4. The amino acid sequence of the heavy chain variable region of X2G5 is shown in SEQ ID NO:5, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:6.

[0008] X2B2 heavy chain variable region amino acid sequence (SEQ ID NO:1): AVTLDESGGGLQTPGGALSLVCKASGFDFSSYAMYWVRQAPGKGLEYVAGISNNGGTGYGSAVKGRATISRDNGQSTVGLQLNNLRAEDTGIYYCTRSTSIDTWGHGTEVIVSP X2B2 light chain variable region amino acid sequence (SEQ ID NO:2): ALTQPASVSANPGETVKITCSGGGSGYNYGWFQQKSPGSAPVTVIYWNDKRPSDIPSRFSGALSGSTATLTITGVQAEDEAVYYCGSWDGSNMFGAGTTLTVLRTEPKSCYKTHTCPPCPAPELLGGPSVFPLPPKTQDTLMISRTPESHAWWWT X2E1 heavy chain variable region amino acid sequence (SEQ ID NO:3): AVTLDESGGGLQTPGRALSLVCKASGFTFSSYNMGWVRQAPGKGLEFVAGIDNTGRYTDYGSAVKGRATISRDNGQSTVRLQLNNLRAEDTATYFCTRSPGGYSCVGGWCTPYFTGDIDAWGHGTEVIVSS X2E1 light chain variable region amino acid sequence (SEQ ID NO:4): ALTQPSSVSANPGETVKITCSGGSSYYGWHQQKSPGSAPVTLIYDNTNRPSNIPSRFSGSKSGSTATLTITGVQADDEAVYYCGNEDSNGAAIFGAGTTLTVLRTEPKSCYKTHTCPPCPAPELLGGPSVFLFPPKPKTPLMISRTPESTCVVWT X2G5 heavy chain variable region amino acid sequence (SEQ ID NO:5): AVTLDESGGGLQTPGGALSLVCKASGFTFSSFYMGWVRQAPGKGLEFVGGITSDGRYTEYGPAVKGRATISRDNGQSTLRLQLNNLRAEDTGTYYCTKCAYRDDCAGDDIDAWGRGTEVIVSS X2G5 light chain variable region amino acid sequence (SEQ ID NO:6): ALTQPSSVSANPGETVKITCSGDDRWYGWYQQKSPGSAPVSVIYANTKKPSGIPSRFSGSLSGSTNTLTITGVQVEDEAVYFCGGYDGSNMFGAGTTLTVLRTEPKSCYKTHTCPPCPAPELLGGPSVFPLPPKTQDTLMISRTPESHAWWWT The method for preparing the above-mentioned chicken-derived single-chain antibody targeting the feline calicivirus VP1-CDE protein includes the following steps: (1) Chickens were immunized with prokaryotically expressed and purified feline calicivirus VP1-CDE protein as an antigen; (2) Total RNA was extracted from peripheral blood lymphocytes of immunized chickens, and cDNA was synthesized by reverse transcription. The VH gene and VL gene were amplified, and the VH gene and VL gene were spliced ​​together by the linker peptide (Gly4Ser)3 through overlap extension PCR to obtain single-chain antibody genes. (3) The single-chain antibody gene is cloned into a phage display vector to construct a chicken-derived single-chain antibody phage display library; the library size of the phage display library is not less than 5.25 × 10⁻⁶. 7 CFU / mL; (4) Using VP1-CDE protein as the target antigen, the phage display library is subjected to solid-phase panning to screen for single-chain antibody clones that can specifically bind to VP1-CDE protein; (5) The single-chain antibody gene obtained by screening is cloned into a eukaryotic expression vector, transfected into host cells for expression, and purified to obtain the chicken-derived single-chain antibody.

[0009] Preferably, in step (1), the immunization method is as follows: for the first immunization, VP1-CDE protein is emulsified with Freund's complete adjuvant and injected into the pectoral muscle at multiple points, with an immunization dose of 0.5 mg / bird; for the subsequent four booster immunizations, VP1-CDE protein is emulsified with Freund's incomplete adjuvant, with an immunization interval of 14 days and an immunization dose of 0.5 mg / bird each time; after immunization, the titer of chicken serum against VP1-CDE protein is ≥1:64000.

[0010] Preferably, in step (2), the primers used to amplify the VH gene are: VH-F: CTACAAATGCCTATGCATCCGCCGTGACGTTGGACG (SEQ ID NO:7); VH-R: CCACCCGACCCACCTCCGCCCGAGCCACCACCTCCGGAGGAGACGATGAC (SEQ ID NO: 8); The primers used to amplify the VL gene are: VL-F: GGCTCGGGCGGAGGTGGGTCGGGTGGAGGCGGTTCAGCRCTGACTCAGCCGKC (SEQ ID NO: 9); VL-R: AAACAACTTTCAACAGTGGATAGGACGGTCAGGGTT (SEQ ID NO: 10).

[0011] Preferably, in step (5), the eukaryotic expression vector is pFUSE-hIgG-Fc vector, and the host cell is human embryonic kidney Expi293F cell.

[0012] This invention provides a kit for detecting feline calicivirus, which contains the above-mentioned chicken-derived single-chain antibody.

[0013] This invention provides the application of the above-mentioned chicken-derived single-chain antibody in the preparation of diagnostic reagents or kits for detecting feline calicivirus.

[0014] The beneficial effects of this invention are as follows: This invention successfully constructed a chicken-derived single-chain antibody phage display library targeting the key FCV antigen VP1-CDE protein, and the constructed phage display library has a high volume (up to 5.25 × 10⁻⁶). 7 The samples showed good diversity (CFU / mL), and specific binding antibodies (X2B2, X2E1, X2G5) were screened from them. The three single-chain antibodies (X2B2, X2E1, X2G5) specifically bind to FCV VP1-CDE protein and viral particles, exhibiting no cross-immunoreactivity with feline parvovirus VP2 protein. They demonstrate high specificity and affinity, and can be used for rapid FCV diagnosis, providing core materials for the development of highly sensitive, low-cost diagnostic kits and test strips. Attached Figure Description

[0015] Figure 1 shows the construction results of pET-28a-VP1-CDE plasmid and the purification results of VP1-CDE protein; where A: PCR amplification results of VP1-CDE gene (M: DNA Marker, 1-4: VP1-CDE gene amplification target band); B: VP1-CDE protein purification results (M: standard protein Marker, 1: sample before induction, 2: sample after induction, 3: lysed supernatant, 4: precipitate after lysing, 5-7: VP1-CDE protein elution samples); C: Western blot validation results (M: standard protein Marker, 1: VP1-CDE protein, 2: FCV); Figure 2 shows the serum titer results of SPF chickens 14 days after five immunizations; Figure 3 shows the amplification results of VH and VL genes and the splicing results of scFv; where A: gel image of total RNA from chicken peripheral blood lymphocytes; B: VL gene amplification results (M: DNA Marker, 1-5: VL gene amplification bands); C: VH gene amplification results (M: DNA Marker, 1-5: VH gene amplification bands); D: scFv gene overlap PCR amplification bands. Figure 4 shows the construction and quality evaluation results of the scFv antibody library; where A: PCR identification of the ligation efficiency of the original antibody library; B: Antibody sequence phylogenetic tree diversity analysis; Figure 5 shows a comparison of the number of colonies selected from the scFv antibody library enrichment panning; the left side shows the results of the second round of panning; the right side shows the results of the third round of panning. Figure 6 shows the Phage-ELISA screening results; Figure 7 shows the amplification results of five specific scFv genes (M: DNA Marker, 1-2: X1A2, 3-4: X2B2, 5-6: X2C1, 7-8: X2E1, 9-10: X2G5). Figure 8 shows the eukaryotic expression and Western blot results of five scFv-Fc proteins; where A: eukaryotic expression and purification results of scFv-Fc (M: protein marker, 1: X1A2, 2: X2B2, 3: X2C1, 4: X2E1, 5: X2G5); BF: Western blot results of the five scFv-Fc proteins (M: protein marker, 1: VP1-CDE protein, 2: FPV VP2 protein). Figure 9 shows the results of ELISA evaluation of the reactivity of scFv-Fc and VP1-CDE proteins; Figure 10 shows the results of IFA detection of the binding ability of each scFv-Fc recombinant protein to FCV. Detailed Implementation

[0016] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments, but the scope of protection of the present invention is not limited thereto.

[0017] The materials used in this invention are as follows: F81 cells, FCV strain, pET-28a-VP1-CDE plasmid, feline parvovirus VP2 protein, and FCV-positive serum were all preserved by the Veterinary Public Health Laboratory of the College of Veterinary Medicine, Gansu Agricultural University; pComb3XSS phage vector, NEB 5α F´ competent cells, E. coli SS320 competent cells, and M13 helper phage were purchased from NEB (UK); human embryonic kidney Expi293F cells were purchased from Pronosun Biotechnology Co., Ltd. (Wuhan); cDNA first-strand synthesis kit was purchased from Novizan (Nanjing); Ni affinity chromatography resin was purchased from Sangon Biotech (Shanghai); DMEM, 293F medium, and fetal bovine serum were purchased from Thermo Fisher Scientific (Shanghai); chicken peripheral blood lymphocyte isolation kit was purchased from Solarbio (Beijing); HRP-mouse anti-M13 antibody and HRP-goat anti-human IgG-Fc antibody were purchased from Sinopharm (Beijing); HRP-goat anti-chicken IgG... H&L antibody was purchased from Bio-Engineering Corporation (Beijing); Freund's complete adjuvant, Freund's incomplete adjuvant, and Cy3-conjugated human IgG-Fc antibody were purchased from Sigma-Aldrich (USA); Hochest 33342 was purchased from Beyotime International (Shanghai). Two SPF chickens were purchased from Vital River Laboratory Animal Technology Co., Ltd. (Beijing).

[0018] Example 1: Preparation and Identification of VP1-CDE Protein The recombinant plasmid pET-28a-VP1-CDE was transformed into Rosetta competent cells using the heat shock method. Single colonies were picked and inoculated into LB medium containing kanamycin for expansion culture. The OD of the bacterial culture was then measured. 600When the pH value reached 0.6-0.8, IPTG was added to a final concentration of 0.5 mM, and expression was induced at low temperature for 16 h at 16 ℃ and 200 r / min. After induction, the bacterial pellet was collected (8000 r / min, 4 ℃ for 30 min), and the pellet was lysed using ultrasonic disruption. The supernatant was collected (8000 r / min, 4 ℃ for 20 min), bound to Ni affinity chromatography resin for 2 h (4 ℃), and then washed with 50 mM and 100 mM washing buffer (20 mM Tris-HCl, 300 mM NaCl, 50 mM imidazole / 100 mM imidazole, pH=8.0) to remove impurities. Finally, the target protein was eluted with elution buffer (20 mM Tris-HCl, 300 mM NaCl, 500 mM imidazole, pH=8.0). The purified VP1-CDE protein was separated from FCV (positive control) by SDS-PAGE, and then transferred to a PVDF membrane by wet transfer. Feline FCV-positive serum diluted 1:2000 was used as the primary antibody, and HRP-conjugated goat anti-cat IgG (1:5000) was used as the secondary antibody. After blocking with 5% skim milk powder, antibody incubation, and color development, Western blot validation was performed.

[0019] PCR verification was performed on the pET-28a-VP1-CDE recombinant plasmid, and the results showed (according to agarose gel electrophoresis). Figure 1 A) The amplified target band was approximately 381 bp, consistent with the expected VP1-CDE gene fragment length, indicating correct construction of the recombinant plasmid. The pET-28a-VP1-CDE plasmid was transformed into Rosetta competent cells for prokaryotic induction expression; the cells were lysed using sonication, and the supernatant was collected and purified using Ni affinity chromatography. SDS-PAGE results showed ( Figure 1 B), a band of approximately 18 kDa appeared in the supernatant and purified eluent after cell lysis, consistent with the theoretical molecular weight of the VP1-CDE protein. Further Western blot validation results showed ( Figure 1 C) Clear and specific immune bands were observed in both the VP1-CDE protein sample lane and the FCV (positive control) lane, proving that the VP1-CDE protein was correctly expressed.

[0020] Example 2 Animal Immunization and Antibody Titer Detection 0.5 mg of recombinant VP1-CDE protein was thoroughly mixed with an equal volume of Freund's complete adjuvant until a stable water-in-oil emulsion was formed (no diffusion was observed when added to distilled water). The primary immunization was performed via multiple intramuscular injections into the pectoral muscles of chickens. For booster immunizations, Freund's incomplete adjuvant was used, with four booster vaccinations administered at 14-day intervals. Each booster dose was 0.5 mg / bird. Blood was collected from the subwing vein before each immunization, and serum samples were separated for enzyme-linked immunosorbent assay (ELISA) to detect the specific antibody titer against VP1-CDE protein. HRP-conjugated goat anti-chicken IgG antibody (1:5000 dilution) was used as the secondary antibody, and the negative control serum was pre-immunization chicken serum (1:2000). The absorbance (OD) of each well was measured at 450 nm using an ELISA reader. 450nm According to serological test results, when the sample serum OD 450nm Values ​​and negative serum OD 450nm When the ratio of the values ​​(P / N value) is ≥2.1, it is interpreted as containing a specific antibody against the VP1-CDE protein.

[0021] Using VP1-CDE protein as an immunogen, experimental animals were immunized five times. Blood was collected from the subwing vein of chickens to separate serum, and serum titers were determined by indirect ELISA. The results showed ( Figure 2 The titers of the immune serum from the two experimental chickens were P / N > 2.1 at a dilution of 1:64000. 450nm Positive / OD 450nm The negative result indicates that the serum titer meets the standards for phage display library construction.

[0022] Example 3: scFv gene amplification and phage display library construction 1. Antibody variable region gene amplification and in vitro construction of scFv When the VP1-CDE specific antibody titer in chicken serum reached 1:64,000 or higher, peripheral blood was collected to isolate lymphocytes. Total RNA was extracted from lymphocytes according to the TriZol reagent instructions. After purity and integrity verification, first-strand cDNA synthesis was performed using the HiScrip® II qPCR kit. Referring to the primers shown in Table 1 (double-underlined regions are linker sequences), the VH and VL genes were amplified using the following amplification program: 95 ℃ for 3 min; 95 ℃ for 30 s, 62.5 ℃ for 30 s, 72 ℃ for 1 min, 22 cycles; 72 ℃ for 10 min. The amplified products were purified and recovered after 1% agarose gel electrophoresis. The purified VH and VL genes were mixed at a 1:1 molar ratio and spliced ​​into the scFv gene using overlap extension PCR with the (Gly4Ser)3linker (amino acid sequence GGGGSGGGGSGGGGS). The primers used were VH-F and VL-R (Table 1). The amplification program was: 95 ℃ for 3 min; 95 ℃ for 30 s, 60 ℃ for 30 s, 72 ℃ for 1 min, 20 cycles; 72 ℃ for 10 min. The final scFv gene fragment was purified using a gel extraction kit.

[0023] Total RNA was extracted from chicken peripheral blood lymphocytes. Figure 3 A) Total RNA samples from two chickens were mixed and used as a template for reverse transcription to synthesize cDNA, amplifying the VH and VL genes. Agarose gel electrophoresis results showed ( Figure 3 B, Figure 3 C) The amplified gene bands were approximately 420 bp and 360 bp in size, respectively, consistent with the expected lengths of the VH and VL target gene fragments. The VH and VL genes were spliced ​​using overlap extension PCR. Electrophoresis results showed a scFv-specific band of approximately 780 bp in the spliced ​​product, confirming the successful construction of the scFv gene splice. Figure 3 D).

[0024] 2. Construction and diversity identification of scFv antibody libraries The purified scFv target gene fragment was ligated into the pComb3X phage vector to construct a recombinant expression vector. The ligation mixture was electroporated into E. coli SS320 competent cells and incubated at 37 °C for 1 h. 10 µL of the bacterial culture was diluted 10-fold and plated onto LB agar plates containing ampicillin / kanamycin (Amp-Kan) antibiotics, and incubated overnight at 37 °C. Twenty single colonies were randomly selected, and plasmids were extracted. The correctness of the inserted fragment was verified by PCR. Positive recombinant plasmids were sequenced to assess sequence diversity, and the library size of the original antibody library was calculated based on the number of colonies on the plate. The remaining bacterial culture was transferred to 300 mL of 2YT medium and incubated at 37 °C on a shaker at 200 rpm for 16–18 h. After incubation, the bacterial culture was centrifuged at 4 °C and 8,000 r / min for 20 min, and the supernatant was collected. One-quarter volume of PEG8000 / NaCl solution was added to the supernatant and mixed thoroughly by inversion. The mixture was then incubated on ice for 1 h. Subsequently, the culture was centrifuged at 4 °C and 9,500 r / min for 15 min, the supernatant was discarded, and the bottom phage pellet was resuspended in PBT buffer to obtain the original phage display scFv library.

[0025] The scFv gene was cloned into the pComb3X vector to construct a primitive scFv phage display library. Twenty single colonies were randomly selected for PCR identification. The results showed that the target band was approximately 780 bp, consistent with the expected scFv gene fragment length. Figure 4 -A), the ligation efficiency between the target gene and the pComb3X vector was 80%; the constructed original scFv phage display library had a volume of 5.25 × 10⁻⁶. 7 CFU / mL. Phylogenetic diversity analysis showed good antibody library diversity. Figure 4 -B).

[0026] Example 4: Screening and Identification of Specific Single-Chain Antibodies 1. Solid-phase panning of scFv antibody libraries VP1-CDE protein was used as the coating antigen, coated onto ELISA plates at a dose of 2 µg / well, with PBS buffer as a blank control, and incubated overnight at 4 °C. The next day, the coating solution was discarded, and the plates were blocked with 5% skim milk powder at 37 °C for 1 h. After discarding the blocking solution, 100 µL of the original phage display antibody library was added to each well, and the plates were incubated on a horizontal shaker (150 r / min) at room temperature for 2 h to achieve specific antigen-antibody binding. Unbound phages were discarded, and the plates were washed 10 times with PBS. Then, 100 µL of 0.1M HCl solution was added to each well to elute the specifically bound phages. After shaking on a horizontal shaker for 5 min, 40 µL of 1M Tris-HCl (pH=8.0) was immediately added to neutralize the reaction mixture, and the mixture was thoroughly mixed. The mixed droplets were then transferred to NEB 5α F´ competent cells (OD). 600 (Approximately 0.2-0.4 g), bound at 37℃ and 200 r / min for 1 h, followed by rescue culture with M13 helper phage for 1 h. 10 µL of bacterial culture was used for 10-fold serial dilution titration plates (Amp-Kan resistant); the remaining bacterial culture was transferred to 35 mL of 2YT medium and cultured overnight. After concentration with PEG8000 / NaCl solution, the first-round enriched scFv phage display library was obtained. The above procedure was repeated three times to progressively enrich scFv phage clones specific to the VP1-CDE protein.

[0027] Using VP1-CDE protein as the antigen, the constructed original phage single-chain antibody library underwent three rounds of "binding-elution-enrichment" panning, and the antibody libraries enriched in the second and third rounds were subjected to plate titration. After the third round of panning, compared with no antigen screening, the number of non-specific phages was significantly reduced, indicating that the specific phages were successfully enriched. Figure 5 ).

[0028] 2. Screening of specific single-chain antibodies from scFv antibody libraries Using 100 ng / well VP1-CDE protein as the detection antigen, and with a negative control well containing only PBS buffer, the plates were coated and incubated overnight at 4°C. The next day, the coating solution was discarded, 5% skim milk powder was added, and the plates were blocked at 37°C for 1 h, followed by washing three times with PBST buffer. The phage library culture from the third round of enrichment was serially diluted and evenly spread on LB solid medium containing Amp-Kan antibiotic, and incubated overnight at 37°C. 184 single colonies were randomly picked and inoculated into 96-well deep-well plates pre-filled with 2YT culture medium, and incubated overnight at 37°C and 200 r / min. The supernatant was collected as the primary antibody (4000 g, 15 min). The supernatant was added to the corresponding coated wells, incubated at 37°C for 1 h, washed three times with PBST, and then HRP-mouse anti-M13 antibody diluted 1:8000 was added and incubated at 37°C for 30 min. OD of each well was measured using an enzyme-linked immunosorbent assay (ELISA) reader. 450nm The numerical value is used to determine whether a clone is positive when the P / N ratio is greater than 3. Positive clones are then amplified by PCR and sequenced to analyze the corresponding amino acid sequence characteristics.

[0029] The phage single-chain antibody library enriched in the third round was plated and cultured overnight. 184 single-clone colonies were randomly selected for phage-ELISA screening to identify positive clones. A P / N ratio > 3 was used as the criterion for positive single-clone colonies. The screening results were then combined with... Figure 6 Ultimately, 35 single-chain antibodies specifically binding to the VP1-CDE protein were screened out, and plasmids were extracted and sequenced. The sequencing results were compared and analyzed using SnapGene software, revealing that five antibodies had different amino acid sequences, named X1A2, X2B2, X2C1, X2E1, and X2G5, respectively.

[0030] Example 5: Eukaryotic Expression and Activity Identification of Single-Chain Antibodies 1. Detection of eukaryotic expression and specific binding of recombinant scFv-Fc protein Positive clones of scFv with different sequences were selected, and the scFv gene was amplified using primers scFv-F / scFv-R (Table 2). After identification by agarose gel electrophoresis, the target gene fragment was recovered by gel excision and directionally cloned into the multiple cloning site of the eukaryotic expression vector pFUSE-hIgG-Fc. The recombinant vector was transformed into E. coli DH5α competent cells by heat shock. The transformed bacteria were plated on LB agar plates and cultured overnight at 37 °C. Single colonies were picked, plasmids were extracted and sent for sequencing. Recombinant plasmids with correct sequences were screened and transiently transfected into Expi293F suspension cells. The cell culture supernatant was collected after 6 days. The scFv-Fc protein in the supernatant was purified using a Protein A affinity chromatography column. The purified product was analyzed by SDS-PAGE electrophoresis to verify whether the protein size met expectations. VP1-CDE protein and FPV VP2 protein were subjected to SDS-PAGE electrophoresis and transferred to PVDF membranes. The antigen-binding specificity of the recombinant proteins was verified by Western blot analysis using purified scFv-Fc protein as primary antibody and HRP-mouse anti-human IgG-Fc antibody (1:5000 dilution) as secondary antibody.

[0031] The target genes X1A2, X2B2, X2C1, X2E1, and X2G5 were amplified by PCR, and electrophoresis results showed that bands of approximately 750 bp in length were successfully obtained. Figure 7 The recovered target gene was ligated into the eukaryotic expression vector pFUSE-hIgG-Fc to construct a recombinant expression vector, which was transiently transfected into Expi293F suspension cells for expression and purification. The results were verified by SDS-PAGE. Figure 8 A) The molecular weight of the purified target protein band was approximately 56 kDa, consistent with the expected molecular weight, confirming that all five constructed proteins could be normally expressed in Expi293F cells. To verify the specific binding activity of the recombinant scFv-Fc proteins, Western blot analysis was performed using the five scFv-Fc proteins as primary antibodies, HRP-labeled mouse anti-human IgG-Fc antibody as secondary antibodies, and feline parvovirus VP2 protein as a negative control. The results showed that all five scFv-Fc proteins specifically bound to VP1-CDE protein, and none of them exhibited cross-binding reactions with FPV VP2. Figure 8 B; 8C; 8D; 8E; 8F). Among them, the scFv-Fc proteins numbered X2B2, X2E1, and X2G5 showed clear positive binding bands; the scFv-Fc proteins of X1A2 and X2C1 showed weaker positive binding bands.

[0032] 2. Detection of scFv-Fc reactivity VP1-CDE protein was used as the coating antigen (100 ng / well) and coated overnight at 4 °C. Blank control wells were not coated with the antigen. Purified recombinant scFv-Fc protein was used as the primary antibody (initial protein concentration set at 0.5 mg / mL), diluted twofold, and co-incubated with the coating antigen at 37 °C for 1 h (initial dilution ratio 1:50). Subsequently, HRP-mouse anti-human IgG-Fc antibody (1:5000) was added and incubated for 1 h before measuring OD. 450nm The value is P / N ≥ 2.1, indicating that the measured scFv-Fc protein has a specific binding reaction with the VP1-CDE antigen.

[0033] The concentration of purified scFv-Fc proteins (X1A2, X2B2, X2C1, X2E1, X2G5) was adjusted to 0.5 mg / mL. ELISA was performed using VP1-CDE protein as the antigen. A P / N ratio ≥ 2.1 was considered an indication of specific binding between scFv-Fc and VP1-CDE proteins. Results showed that among the five scFv-Fc proteins, X2B2, X2E1, and X2G5 exhibited good reactivity with VP1-CDE protein, X2C1 showed low binding activity, and X1A2 showed no binding activity. Figure 9 ).

[0034] 3. scFv-Fc indirect immunofluorescence (IFA) detection F81 cells were seeded in 96-well cell culture plates. When the cells reached 80% confluence, the original cell culture medium was discarded, and 100 mg TCID45 was added. 50 F81 cells were infected with FCV for 3 h and washed with PBS. 100 µL of 80% acetone solution was added to each well, and the cells were fixed at -20 ℃ for 25 min. The cells were washed three times with PBS to remove residual acetone. 100 µL of recombinant scFv-Fc protein (0.2 mg / mL) was added, and the cells were incubated at 37 ℃ for 1 h. The cells were then washed with PBS. Cy3-conjugated goat anti-human IgG-Fc (1:500 dilution) antibody was added and incubated for 1 h. The cells were then washed with PBS. Diluted Hoechst (5 µg / mL) was added and stained for 10 min. The cells were washed three times with PBS to remove unbound staining solution. The distribution of cell fluorescence was observed under a fluorescence microscope, and images were acquired.

[0035] F81 cells were infected with FCV for 3 hours, then fixed with 80% acetone. IFA (Index Acid Analysis) was performed using purified recombinant scFv-Fc protein as the primary antibody and Cy3-conjugated human IgG-Fc as the secondary antibody. Observation under a fluorescence microscope showed (…). Figure 10Red fluorescence signals were observed in cell samples corresponding to the three scFv-Fc strains X2B2, X2E1, and X2G5, indicating that the proteins of these three scFv-Fc strains can specifically recognize and bind to FCV. Notably, the fluorescence signal intensity corresponding to X1A2 and X2C1 was weak, and a longer exposure time was required to observe the blurry red fluorescence signal. This result indicates that X1A2 and X2C1 have extremely weak ability to bind to FCV. No red fluorescence signal was observed in cell samples using PBS as the primary antibody.

[0036] 4. scFv-Fc virus neutralization test Recombinant scFv-Fc protein at a concentration of 75 µg / mL was serially diluted 2-fold to 1:8 and mixed with an equal volume of FCV virus solution (200 TCID). 50 After mixing (0.1 mL), incubate at 37 °C for 1 h. Wash the 96-well plate confluent with F81 cells three times with PBS, and add 100 µL of the mixture to each well according to the dilution. Prepare a 1:1 mixture of FCV virus solution (100 TCID50). 50 The negative control wells contained 0.1 mL of DMEM culture medium, while the blank control group contained only DMEM culture medium. After adding 100 µL of complete culture medium to each well, the cytopathic effect was observed daily, and the scFv-Fc neutralization titer was calculated using the Reed-Muench method.

[0037] Five recombinant proteins, scFv-Fc (initial concentration 75 µg / mL), were mixed with FCV virus solution and co-incubated before being transferred into 96-well F81 cells pre-coated with a monolayer. After 48 h of treatment, cytopathic effects were observed in both the experimental group and the negative control group, while no cytopathic effects were observed in the blank control group, indicating that X1A2, X2B2, X2C1, X2E1, and X2G5 do not have the ability to neutralize the virus.

[0038] In summary, this invention constructed a chicken-derived scFv phage display library with a capacity of 5.25 × 10⁷ CFU / mL using VP1-CDE protein as the target antigen, filling the gap in research on chicken-derived antibodies specific to FCV VP1-CDE. Five specific scFv strains screened from the library were expressed and purified in eukaryotes. Western blot analysis confirmed that all five scFv strains bound to VP1-CDE protein and did not cross-react with FCV VP2 protein, indicating good specificity. ELISA affinity assays showed that three scFv strains (X2B2, X2E1, and X2G5) had high affinity for VP1-CDE protein. IFA results further confirmed that these three scFv-Fc strains could specifically bind to FCV particles. These research results provide crucial biomaterial support for the development of rapid diagnostic strips for FCV infection and are of great significance for advancing the clinical diagnosis and treatment of FCV.

Claims

1. A chicken-derived single-chain antibody targeting the VP1-CDE protein of feline calicivirus, characterized in that, The single-chain antibody is X2B2, X2E1, or X2G5, wherein: The amino acid sequence of the heavy chain variable region of X2B2 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 amino acid sequence of the heavy chain variable region of X2E1 is shown in SEQ ID NO:3, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:4; The amino acid sequence of the heavy chain variable region of X2G5 is shown in SEQ ID NO:5, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

6.

2. The method for preparing the chicken-derived single-chain antibody according to claim 1, characterized in that, Includes the following steps: (1) Chickens were immunized with prokaryotically expressed and purified feline calicivirus VP1-CDE protein as an antigen; (2) The VH and VL genes were amplified from the peripheral blood lymphocytes of immunized chickens, and the VH and VL genes were spliced ​​together by linker peptides through overlap extension PCR to obtain single-chain antibody genes. (3) The single-chain antibody gene is cloned into the phage display vector to construct a chicken-derived single-chain antibody phage display library; (4) Using VP1-CDE protein as the target antigen, the phage display library is subjected to solid-phase panning to screen for single-chain antibody clones that can specifically bind to VP1-CDE protein; (5) The single-chain antibody gene obtained by screening is cloned into a eukaryotic expression vector, transfected into host cells for expression, and purified to obtain the chicken-derived single-chain antibody.

3. The preparation method according to claim 2, characterized in that, In step (2), the primer sequences for amplifying the VH gene are as follows: VH-F: CTACAAATGCCTATGCATCCGCCGTGACGTTGGACG; VH-R: CCACCCGACCCACCTCCGCCCGAGCCACCACCTCCGGAGGAGACGATGAC; The primer sequence for amplifying the VL gene is: VL-F: GGCTCGGGCGGAGGTGGGTCGGGTGGAGGCGGTTCAGCRCTGACTCAGCCGKC; VL-R:AAACAACTTTCAACAGTGGATAGGACGGTCAGGGTT.

4. The preparation method according to claim 2, characterized in that, In step (3), the phage display library has a volume of not less than 5.25 × 10⁻⁶. 7 CFU / mL.

5. The preparation method according to claim 2, characterized in that, In step (5), the eukaryotic expression vector is pFUSE-hIgG-Fc vector, and the host cell is human embryonic kidney Expi293F cell.

6. A kit for detecting feline calicivirus, characterized in that, It contains the chicken-derived single-chain antibody as described in claim 1.

7. The use of the chicken-derived single-chain antibody according to claim 1 in the preparation of diagnostic reagents or kits for detecting feline calicivirus.

Images