A feline calicivirus (FCV) recombinant protein monoclonal antibody and a preparation method thereof

Abstract

The present application belongs to the technical field of bioengineering. The present application relates to a recombinant protein, which comprises two dominant antigen epitopes of feline calicivirus (FCV) protein. In order to improve the yield of the recombinant protein in a prokaryotic expression system, the amino acid sequence of the recombinant protein is converted into a corresponding nucleotide sequence by using E. coli preferred codons, the nucleotide sequence is chemically synthesized, and a recombinant expression vector is constructed. The present application also relates to the establishment of a phage library by immunizing mice with the recombinant protein, the screening of a single-chain antibody (scfv) sequence corresponding to the feline calicivirus recombinant protein by panning, the construction of a complete mouse IgG1 antibody sequence expression vector from the obtained scfv sequence, the expression of a monoclonal antibody by instantaneously transferring HEK293F cells, the purification of the monoclonal antibody, and the labeling of europium ions (Eu 3+ ) in the monoclonal antibody, respectively. The optimal monoclonal antibody pairing combination is determined by orthogonal experiments, and the monoclonal antibody can be used for the early diagnosis of upper respiratory tract infection in cats.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology. Specifically, this invention relates to a novel recombinant protein, to establishing a phage library by immunizing mice with the aforementioned recombinant protein, to screening for specific single-chain antibody scfv sequences, and to constructing the obtained scfv sequences into a eukaryotic expression vector to express a feline calicivirus recombinant protein monoclonal antibody, and applying it to the early diagnosis of upper respiratory tract infections in cats. Background Technology

[0002] Feline calicivirus (FCV) is a member of the Caliciviridae family and an important pathogen in cats. In recent years, highly pathogenic FCV strains (VSD-FCV) have broken out in several countries. This strain can cause systemic multi-systemic lesions in infected cats, with a high mortality rate. The main symptoms of feline calicivirus (FCV) infection are fever (around 40°C), oral ulcers (on the tongue or upper and lower palates), nasal inflammation, conjunctivitis, interstitial pneumonia, and polyarthritis. Sick cats exhibit lethargy, sneezing, increased oral and nasal secretions, drooling, and initially serous nasal and ocular discharge that becomes purulent after 4-5 days. Corneal inflammation can lead to blindness. Oral ulcers are the most prominent feature, especially on the tongue, hard palate, and around the middle cleft of the palate, with large areas of ulceration and granulation tissue proliferation. Sick cats have difficulty eating, easily leading to malnutrition, dehydration, and even death. The incubation period for feline calicivirus is generally two to three days. When the virus is highly virulent, pneumonia can occur, causing difficulty breathing and dry or wet rales in the lungs. Kittens under three months old can die from pneumonia. Calicivirus infection can usually resolve on its own after 7 to 10 days if it is not complicated by other viruses (infectious rhinotracheitis virus) or bacterial infections, but the infected cat often becomes a carrier.

[0003] Vaccination is the most effective way to prevent this disease, but the protective effect of existing vaccines is not ideal. Clinically, treatment mainly combines symptomatic and supportive care. In the early stages of feline viral infection, for respiratory infections, in addition to using antiviral drugs, interferon, and antibiotics to prevent secondary infections, oxygen therapy and nebulization are also necessary to help the cat clear phlegm and stop coughing. When breathing difficulties occur, oxygen therapy can be used to relieve them; it is recommended to use a combination of Synulox and Doxycycline. Therefore, the fundamental measures to control this disease lie in immunization and quarantine.

[0004] Currently, monoclonal antibodies against feline calicivirus are mainly obtained through hybridoma technology. However, hybridoma technology has certain limitations in obtaining monoclonal antibodies: (1) It is not suitable to use hybridoma technology to prepare antibodies against animal toxic antigens, autoantigens, immune tolerance antigens, and weak immunogenic antigens because it cannot generate effective immunity. (2) The inherent affinity and limited biological activity of monoclonal antibodies limit their application range. Since monoclonal antibodies cannot undergo precipitation and agglutination reactions, many detection methods cannot be completed using monoclonal antibodies. (3) As heterologous diploid cells, candidate hybridoma clones need to be subcloned to become stable. Candidate hybridoma cells that are not subcloned in time are unstable and are prone to losing positive clones. Selecting positive clones, especially complex functional screening, requires a certain time window. The longer the window, the more mature and reliable the functional screening. Micro-crystallization of candidate clones can extend the functional screening time window, but functional detection requires a sufficient amount of antibody protein, so batch subcloning still requires a large workload. The solution after hybridoma screening failure is re-immunization or re-fusion (which is also limited by the feeding cycle of mice after immunization), which inevitably incurs time costs.

[0005] Therefore, the preparation of monoclonal antibodies against recombinant feline calicivirus (FCV) proteins for specific recognition and detection has become a primary method for early diagnosis. The conventional method for preparing FCV recombinant protein monoclonal antibodies involves preparing ascites fluid from Balb / c mice using FCV recombinant protein monoclonal cell lines, followed by purification of the monoclonal antibody using Protein A affinity chromatography. However, due to the uncertainty in ascites fluid yield per mouse and significant individual variability, the batch-to-batch variation of the obtained anti-FCV recombinant protein monoclonal antibodies is large, resulting in poor detection accuracy. Summary of the Invention

[0006] To address the shortcomings of the existing techniques for preparing monoclonal antibodies, a method was developed to design and synthesize recombinant feline calicivirus proteins and then prepare monoclonal antibodies by establishing phage libraries and expressing them in eukaryotic cells. This approach aims to save time, reduce batch-to-batch variability, and improve detection accuracy.

[0007] To achieve the above objectives, this application provides:

[0008] A feline calicivirus recombinant protein-specific monoclonal antibody 3C5 comprises a light chain and a heavy chain, wherein the amino acid sequence of the light chain is shown in SEQ ID NO.1 and the amino acid sequence of the heavy chain is shown in SEQ ID NO.2.

[0009] A feline calicivirus recombinant protein-specific monoclonal antibody 5E2 comprises a light chain and a heavy chain, wherein the amino acid sequence of the light chain is shown in SEQ ID NO.3; and the amino acid sequence of the heavy chain is shown in SEQ ID NO.4.

[0010] Beneficial effects: Using an immune antibody library to screen monoclonal antibodies against recombinant feline calicivirus protein, under the same immunization conditions, the more candidate clones there are, the easier it is to screen for highly active antibodies. Once the antibody library is built, it can be stored indefinitely. The original phage library is sufficient for repeated screening, and there is no need to rebuild the library. Only a small amount of phage particles need to be taken when screening monoclonal antibodies. In addition, the affinity panning method can specifically enrich antibodies that bind to the antigen and also minimize the removal of antibodies that bind to the control protein, effectively improving the screening efficiency. (1) Using recombinant feline calicivirus protein as the target antigen, two specific dominant antigenic epitopes of the antigen were analyzed and selected. The sequence alignment results showed that the two selected antigenic epitopes had no obvious homology with other protein sequences. (2) In order to promote the stimulation of the immune system of Balb / c mice by the selected dominant antigenic epitopes and enhance the immune effect, the sequences of the two selected dominant antigenic epitopes were tandemly linked by flexible fragments to form the amino acid sequence of the recombinant protein. (3) Using the E. coli preferred codon, the amino acid sequence of the recombinant protein was converted into the corresponding nucleotide sequence to facilitate the efficient expression of the recombinant protein in E. coli. (4) The nucleotide sequence obtained in the previous step was chemically synthesized and ligated by enzyme digestion. The synthesized nucleotide fragment was inserted into the prokaryotic expression vector PET-32a to construct a recombinant protein expression vector. (5) The recombinant protein expression vector was transformed into Escherichia coli ER2566 competent cells and screened with ampicillin resistance selection medium to obtain recombinant protein expression strains. (6) After large-scale culture of recombinant protein expression strains, the bacteria were sonicated and centrifuged at low temperature. The supernatant of the solution was taken and subjected to affinity chromatography on a nickel agarose column to elute and obtain purified recombinant protein. (7) After multiple immunizations of Balb / c mice with the purified recombinant protein, lymphocytes were isolated from their spleens to establish a single-chain antibody scfv phage display library. The single-chain antibody scfv sequence that can bind to recombinant feline calicivirus was finally obtained by multiple rounds of panning and screening using the anti-feline calicivirus recombinant protein. (8) The scfv sequence was used to construct a complete mouse IgG1 expression vector and expressed as a monoclonal antibody in HEK293 cells. The monoclonal antibody was purified by Protein A affinity chromatography and labeled with europium ions (Eu). 3+ (9) Orthogonal experimental screening showed that the combination of 3C5 monoclonal antibody coating and 5E2-Eu labeled monoclonal antibody was the best for detecting feline calicivirus recombinant protein. Detailed Implementation

[0011] Although the following embodiments provide a relatively detailed textual description of the design concept of the present invention, these textual descriptions are merely simple textual descriptions of the design concept of the present invention and are not intended to limit the design concept of the present invention. Any combination, addition, or modification that does not exceed the design concept of the present invention falls within the protection scope of the present invention.

[0012] Example 1: Selection of dominant antigenic epitopes in feline calicivirus recombinant proteins

[0013] Using recombinant feline calicivirus protein as the target antigen, the hydrophilicity and antigenicity of its epitope sequences were analyzed using the biological software DNAssist 2.0, and dominant epitopes A and B were selected. Sequence comparison results showed that the selected dominant epitope sequences A and B had high specificity and no significant homology with other protein sequences.

[0014] Example 2: Tandem of dominant antigenic epitopes in feline calicivirus recombinant protein

[0015] To enhance the stimulation of the mouse immune system by the selected antigenic epitopes and facilitate subsequent experiments, the A and B dominant antigenic epitope sequences of the feline calicivirus recombinant protein were repeated and then linked by a flexible fragment (four consecutive glycine residues) to obtain the amino acid sequence of the recombinant protein.

[0016] Example 3: Optimization of the nucleotide sequence encoding recombinant proteins

[0017] To improve the expression level of recombinant proteins in *E. coli*, the amino acid sequence encoding the recombinant protein was converted into the corresponding nucleotide sequence based on the codons preferred by *E. coli*, while maintaining the amino acid sequence unchanged. Nucleotide sequences corresponding to the BamHI and EcoRI restriction enzyme sites were then added upstream and downstream of the transverse codons, respectively. This process was performed by Hangzhou Xianzhi Biotechnology Co., Ltd. The synthesized target gene was cloned into the pMD19-T vector (Takara Bio Engineering Dalian Co., Ltd.).

[0018] Example 4: Construction of recombinant protein expression vector

[0019] The pMD19-T vector and PET-32a vector (Novagen, Germany) containing the target gene were double-digested at 37°C for 12 hours using restriction endonucleases BamHI and EcoRI (Takara Bio Engineering Dalian Co., Ltd.). The digestion products were subjected to 1% agarose gel electrophoresis, and the target gene and PET-32a vector were recovered by gel extraction (the gel extraction kits used in this invention were all purchased from Ningbo Zhongding Biotechnology Co., Ltd.). The recovered target gene and PET-32a vector were ligated at a certain ratio at 4°C for 12 hours using T4 ligase (Takara Bio Engineering Dalian Co., Ltd.). The ligation product was then transformed into DH5α competent cells (Hangzhou Xianzhi Biotechnology Co., Ltd.) and plated on LB agar plates containing ampicillin resistance (50 μg / mL). After incubation at 37°C for 12 hours, single clones were picked from the plates and transferred to LB liquid medium containing ampicillin resistance (50 μg / mL). After incubation at 37°C for 12 hours, plasmids were extracted using a plasmid purification kit (all plasmid purification kits used in this invention were purchased from Ningbo Zhongding Biotechnology Co., Ltd.). The correct recombinant expression vector was obtained after double digestion with BamHI and EcoRI.

[0020] Example 5: Construction of a feline calicivirus recombinant protein expression strain

[0021] Transform the constructed recombinant expression vector E. coli ER2566 competent cells were plated on LB agar plates containing ampicillin-resistant (50 μg / mL) medium and cultured overnight at 37°C. The next day, single clones were picked from the plates and cultured in LB liquid medium containing ampicillin-resistant (50 μg / mL) medium at 37°C for 8 hours. One mL of the culture was stored, and the remaining medium was induced with IPTG (isopropylthio-β-D-galactopyranoside) (final concentration 1.0 mmol / L) for 4 hours to prepare protein electrophoresis samples. 11% polyacrylamide gel electrophoresis results showed successful expression of the recombinant protein, yielding a recombinant protein expression strain against feline calicivirus.

[0022] Example 6: Purification of recombinant feline calicivirus protein

[0023] The recombinant protein expression strain was inoculated into LB liquid medium, and ampicillin was added to a final concentration of 50 μg / mL. After incubation at 37°C in a shaker for 8 hours, the strain was diluted 1:100 with LB liquid medium containing 50 μg / mL ampicillin and aliquoted into bacterial culture flasks. The flasks were then incubated at 37°C in a shaker until OD600 = 0.8. IPTG (isopropylthio-β-D-galactopyranoside) was added to a final concentration of 1.0 mmol / L, and induction was continued for 4 hours. After centrifugation to collect the bacterial cells, the cells were lysed by sonication at 4°C. The supernatant was then passed through a nickel-agarose affinity chromatography column, and after washing and elution, the purified anti-cat calicivirus recombinant protein was obtained.

[0024] Example 7: Construction of a single-chain antibody scfv phage library

[0025] Female Balb / c mice aged 4-6 weeks were used for the primary immunization. Each mouse received a subcutaneous injection of 100 μg of recombinant feline calicivirus protein emulsified with Freund's complete adjuvant, totaling 400 μl per mouse. A second booster immunization was administered 20 days later, using 80 μg of recombinant feline calicivirus protein emulsified with Freund's incomplete adjuvant, totaling 400 μl per mouse, injected subcutaneously at multiple sites. A third booster immunization was administered 15 days later, using the same method as the second booster. Twenty days later, a booster injection of 120 μg of recombinant feline calicivirus antigen was administered intraperitoneally. 72 hours later, blood was collected from the orbital sinus, and the mice were euthanized. Spleen lymphocytes were isolated using a mouse spleen lymphocyte isolation kit (Tianjin Haoyang Biological Products Technology Co., Ltd.). Total RNA was extracted from isolated lymphocytes using an RNA extraction kit (Tiangen Biotech Co., Ltd.). cDNA was synthesized by reverse transcription using a reverse transcription kit (Takara). The heavy chain variable region and light chain variable region genes were amplified using mouse single-chain antibody SCFV universal degenerate primers. The PCR products were subjected to 1% agarose gel electrophoresis, and the target genes were recovered by gel excision. The recovered target genes were then analyzed by overlap... PCR was used to ligate scfv. The PCR product was subjected to 1% agarose gel electrophoresis, and the target gene was recovered by gel excision. After digestion with NotI and SfiI enzymes, the ligation was carried out using T4 ligase and pCANTAB5e (Beijing Baokewei Food Safety Biotechnology Co., Ltd.) vector at a certain ratio at 4℃ for 12 hours. The ligation product was recovered using a gel recovery kit to remove enzymes and buffer substances. The recovered product was electroporated into E. coli TG1 electroporation competent cells multiple times using a bacterial electroporator (Biorad). The cells were then plated on 2×YT-AG plates containing ampicillin resistance (50 μg / mL) and 2% glucose. After incubation at 30℃ for 12 hours, an appropriate amount of 2×YT medium was taken and all colonies on the plate were scraped off with a sterile glass rod and the bacterial suspension was collected. This is the constructed phage antibody library.

[0026] Example 8: Panning and Screening of Single-Chain Antibody SCFV

[0027] A certain amount of bacterial culture was taken from the phage antibody library and inoculated into 2×YT-AG medium to achieve an OD600 of 0.3. The culture was incubated at 37°C with shaking at 250 rpm for approximately 1 hour until the OD600 reached 0.5. Then, helper phage M13K07 was added for superinfection at a ratio of M13K07 / TG1 = 20:1. After shaking at 37°C with shaking at 250 rpm for 1 hour, the bacteria were centrifuged at 3300 g for 10 minutes to precipitate the bacteria, and the supernatant was carefully discarded. The bacteria were resuspended in 2×YT-AK medium containing ampicillin-resistant (50 μg / mL) and ampicillin-resistant (50 μg / mL) medium and incubated overnight at 30°C with shaking at 250 rpm. The next day, the bacteria were centrifuged at 10800 g for 20 minutes to precipitate the bacteria. The supernatant was transferred to a clean centrifuge tube, and 1 / 5 volume of PEG / NaCl was added. After mixing, the mixture was incubated on ice for 2 hours. Centrifuge at 10800 g, 4℃ for 20 min to precipitate cells. Carefully discard the supernatant, remove excess water, and resuspend the precipitate in PBS. Filter through a 0.45 μm membrane to remove bacterial debris for panning. Dilute the purified recombinant feline calicivirus antigen to 8 μg / ml with coating buffer and coat 4 ml of each immunotube (Thermo). Incubate overnight at 4℃. The next day, discard the coating buffer and unadsorbed antigen, wash three times with sterile PBST, add 5 ml of blocking buffer to each immunotube, and incubate at 37℃ for 2 h. Discard the blocking buffer, wash three times with sterile PBST, and add 4 ml of PEG-precipitated phage to each immunotube. Incubate at 37℃ for 1 h. The liquid in the immunoassay tubes was discarded. The tubes were washed 10 times with sterile PBST, then 10 times with sterile PBS. 1 ml of 100 mM triethylamine was added to elute the bound phages. Immediately afterward, 500 μl of 1 M Tris-HCl was added to neutralize the phages at pH 7.4. The neutralized phages were then added to a certain amount of TG1 E. coli in the logarithmic growth phase for superinfection; this was the first round of panning and enrichment. After three rounds of panning, feline calicivirus-specific SCFV was enriched. After the final round of elution and neutralization, the phages were infecting TG1 *E. coli* and plated onto 2×YT-AG plates. After incubation at 30°C for 12 hours, 400-600 single colonies were randomly selected and placed in 96-well deep-well plates. The plates were then incubated with 2×YT-AG medium at 37°C with shaking at 250 rpm for 2 hours. A certain amount of M13K07 helper phage was added for superinfection, followed by incubation at 37°C with shaking at 250 rpm for 1 hour. The supernatant was discarded by centrifugation, and 2×YT-AK medium containing ampicillin resistance (50 μg / mL) and ampicillin resistance (50 μg / mL) was added. The plates were incubated overnight at 30°C with shaking at 250 rpm. Single-clone ELISA screening was performed the following day:

[0028] Coating: Dilute the feline calicivirus recombinant protein with coating buffer to a final concentration of 1 μg / mL, add 100 μL / well to the microplate (Shenzhen Jincanhua Industrial Co., Ltd.), incubate overnight at 4°C, and then wash once with washing buffer using a DEM-3 plate washer (Sun Yat-sen University Da An Gene Co., Ltd.).

[0029] Blocking: Add 200 μL of blocking solution to each well, block at 37°C for 2 hours, and wash once with washing solution using a plate washer;

[0030] Sample loading: Add overnight bacterial culture supernatant and control serum, 100 μL / well, incubate at 37°C for 1 h, and wash 3 times with washing buffer using a plate washer;

[0031] Add enzyme-labeled antibody: Add 100 μL / well of freshly diluted rabbit anti-M13 phage HRP enzyme-labeled secondary antibody (purchased from Beijing Yiqiao Shenzhou Biotechnology Co., Ltd.), incubate at 37°C for 30 minutes, and then wash 4 times with washing buffer using a plate washer.

[0032] Add colorimetric reagents: Add 50 μL of colorimetric reagent A and 50 μL of colorimetric reagent B to each well, and develop the color at 37°C in the dark for 10 minutes.

[0033] To terminate the reaction, add 2M H2SO4 at a rate of 50 μL / well;

[0034] Result Interpretation: OD values ​​were read at 450 nm using a microplate reader after zeroing the blank wells. Immune mouse serum was used as a positive control. Results showed 14 positive clones with high OD values; sequencing yielded 5 SCFV sequences: 3C5, 6B1, 2D8, 5E2, and 7H4. The relevant solution formulations are as follows:

[0035] Coating solution: 1.5g Na2CO3, 2.9g NaHCO3, add ddH2O to make up to 1000mL (pH 9.6).

[0036] Blocking solution: Na2HPO4·12H2O 2.68g, NaH2PO4·2H2O 0.39g, NaCl 8.5g, 20g bovine serum albumin, add ddH2O to bring the volume to 1000mL (pH 7.4).

[0037] Washing solution: Na2HPO4·12H2O 2.68g, NaH2PO4·2H2O 0.39g, NaCl 8.5g, Tween-20 0.5mL, add ddH2O to make up to 1000mL (pH 7.4).

[0038] Colorimetric solution A: Dissolve 200 mg TMB in 100 mL of anhydrous ethanol, and add ddH2O to bring the volume to 1000 mL.

[0039] Colorimetric solution B: 2.1g citric acid, 71g Na2HPO4·12H2O, add ddH2O to make up to 1000mL.

[0040] When using: 1 mL of colorimetric solution A + 1 mL of colorimetric solution B + 0.4 μL of 30% H2O2

[0041] Stop solution: 2M H2SO4, 21.7mL concentrated H2SO4, add ddH2O to make up to 1000mL.

[0042] Example 9: Construction of eukaryotic expression vector and transient transduction expression and purification in HEK293F cells

[0043] Five feline calicivirus single-chain antibody (scfv) sequences were used to construct complete mouse IgG1 antibody sequences. Specifically, the heavy and light chain variable regions of the scfv were bridged to the heavy and light chain constant regions of mouse IgG1, respectively, via PCR, and then inserted into the pcDNA3.1 plasmid (Novagen, Germany). HEK293F cells were co-transfected with the constructed heavy and light chain plasmids via PEI. After 7 days of expression on a shaker at 37°C, 5% CO2, and 120 rpm, the cells were centrifuged, and the supernatant was collected and filtered through a 0.45 μm filter. An agarose affinity medium Protein A chromatography column (Nanjing Genscript Biotech Co., Ltd.) was equilibrated with 50 mL of PBS (pH 7.4) and sputtered onto a computer-controlled nucleic acid and protein analyzer (Shanghai Huxi Analytical Instrument Factory Co., Ltd.), where the absorbance was 0. After loading the supernatant, wash with PBS until the absorbance reaches 0, then elute with 0.1M glycine (pH 3.0). Collect the eluent and neutralize to approximately pH 7.0 with 500mM Tris-HCl (pH 8.5) buffer to obtain purified monoclonal antibodies 3C5, 6B1, 2D8, 5E2, and 7H4.

[0044] Example 10: Eu 3+ Preparation of labeled monoclonal antibodies

[0045] Take 1 mg of purified monoclonal antibody and dialyze three times in 0.05 mol / L carbonate buffer (pH 9.6) at 4°C. Add 1 mg of DTPA (diethyltriaminepentaacetic acid), mix immediately, and react at room temperature for 1 h. Add 200 μL of EuCl3 (33 mmol / L), react at room temperature for 1 h, and then dialyze overnight at 4°C with 10 mmol / L PBS (pH 7.4). Use the above method to react monoclonal antibodies 3C5, 6B1, 2D8, 5E2, and 7H4 with EuCl3. 3+ Marking. The relevant solution formulation is as follows:

[0046] Carbonate buffer (pH 9.6): 1.5g Na2CO3, 2.9g NaHCO3, add double-distilled water to a final volume of 1000mL.

[0047] PBS buffer (pH 7.4): KH2PO4 0.29g, Na2HPO4·12H2O 2.9g, NaCl 8.2g, add double-distilled water to a final volume of 1000mL.

[0048] Example 11: Screening of paired monoclonal antibodies

[0049] Five monoclonal antibodies (3C5, 6B1, 2D8, 5E2, 7H4) were diluted with coating buffer (final concentration 1 μg / mL) and added to ELISA plates (Wuxi Guosheng Biotechnology Co., Ltd.) at 100 μL / well. After coating at 4°C for 12 hours, the plates were washed twice with washing buffer using a DEM-3 plate washer (Sun Yat-sen University Da An Gene Co., Ltd.). Blocking buffer was added at 150 μL / well, and the plates were blocked at 37°C for 1 hour, followed by one wash. Ocular, nasal, and oral secretions from feline calicivirus-infected cats and normal cats were dissolved in diluent at 100 μL / well, incubated with shaking at room temperature for 30 minutes, and then washed five times with washing buffer. Eu, prepared in Example 10, was added. 3+ Labeled monoclonal antibodies, 100 μL / well, incubated at room temperature with shaking for 30 minutes, then washed 5 times with washing buffer; enhancement buffer, 100 μL / well, incubated at room temperature with shaking for 5 minutes, and read using a time-resolved imager (Shanghai Xinbo Biotechnology Co., Ltd.). The relevant solution formulations are as follows:

[0050] Coating solution: 1.5 g Na2CO3, 2.9 g NaHCO3, diluted to 1000 mL with double-distilled water (pH 9.6).

[0051] Blocking solution: Na2HPO4·12H2O 2.68g, NaH2PO4·2H2O 0.39g, NaCl 8.5g, 20g bovine serum albumin, add double-distilled water to a final volume of 1000mL (pH 7.4).

[0052] Washing solution: Na2HPO4·12H2O 2.68g, NaH2PO4·2H2O 0.39g, NaCl 8.5g, Tween-20 0.5mL, add double-distilled water to a final volume of 1000mL (pH 7.4).

[0053] Enhancement solution: 6 mL glacial acetic acid, 1 mL Triton X-100, 50 μmol TOPO (Tri-Octyl Phosphine Oxide), 15 μmol β-NTA (N(CH2COOH)3 aminotriacetic acid), adjust pH to 3.2 with 0.1 mol / L potassium hydrogen phthalate, and add double-distilled water to a final volume of 1000 mL.

[0054] The above method was used to orthogonally test the pairing of each coated monoclonal antibody with europium-labeled monoclonal antibody, and the P / N value (the ratio of the mean of positive sample test to the mean of negative sample test) was calculated, as shown in Table 1.

[0055]

[0056] SEQ ID NO1: 3C5 light chain amino acid sequence of the recombinant protein-specific monoclonal antibody against feline calicivirus;

[0057] SEQ ID NO2: 3C5 heavy chain amino acid sequence of a recombinant monoclonal antibody against feline calicivirus protein;

[0058] SEQ ID NO3: The 5E2 light chain amino acid sequence of the recombinant protein-specific monoclonal antibody against feline calicivirus;

[0059] SEQ ID NO4: 5E2 heavy chain amino acid sequence of the anti-feline calicivirus recombinant protein-specific monoclonal antibody;

Claims

1. Feline calicivirus recombinant protein-specific monoclonal antibody 3C5, comprising a light chain and a heavy chain, characterized in that: The light chain amino acid sequence is shown in SEQ ID NO.1; The heavy chain amino acid sequence is shown in SEQ ID NO.

2.

2. Feline calicivirus recombinant protein-specific monoclonal antibody 5E2, comprising a light chain and a heavy chain, characterized in that: The light chain amino acid sequence is shown in SEQ ID NO.3; The heavy chain amino acid sequence is shown in SEQ ID NO.4.

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