Cathepsin s and uses thereof

Abstract

The application discloses camel-derived nanobodies of feline calicivirus and application thereof, and belongs to the field of biological medicine. The application takes feline calicivirus as an immunogen, and through steps of immunizing a camel, constructing a library, enriching sequencing and the like, two nanobodies VHH-V14 and VHH-V29 are screened, and the amino acid sequences of the two nanobodies are shown as SEQ ID NO. 1-2. Experimental results show that the nanobodies VHH-V14 and VHH-V29 are nanobodies with outstanding neutralization activity to feline calicivirus, and the IC 50 of the two nanobodies is 0.6536 mg / mL and 0.6931 mg / mL respectively, and the two nanobodies have good inhibiting effect on feline calicivirus. The application provides new biological materials for prevention and treatment of feline calicivirus infection, and provides new design theory and technical support for antibody treatment strategy of feline calicivirus, and has outstanding significance for clinical treatment of feline calicivirus infection.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, in particular to a camel-derived nanobody against feline calicivirus and use thereof. BACKGROUND

[0002] Feline calicivirus (FCV) belongs to Caliciviridae and Vesivirus. The virus structure is a 20-sided symmetrical spherical non-enveloped virus with a diameter of 35-39 nm. Caliciviridae is composed of five genera, namely Leporid virus, Neuburg virus, Norovirus, Sapporo virus and Vesivirus, which can all infect mammals.

[0003] Feline calicivirus mainly causes acute oral and upper respiratory tract disease, chronic gingivostomatitis (CGS), lameness syndrome, claw mouth disease and virulent systemic feline calicivirus infection (VS-FCV) in cats. The pathological manifestations mainly include subcutaneous edema, oral ulcer, lameness, and skin ulceration in the claw part. FCV has a high prevalence rate, and some strains cause high mortality of virulent systemic feline calicivirus infection, which can reach 50%. The treatment of feline calicivirus is mainly antiviral therapy and symptomatic treatment, and there is currently a lack of specific and effective drugs. Existing vaccines have weak cross-protection against the disease and cannot fully cover the currently prevalent strains. Therefore, there is an urgent need to develop a new generation of highly effective and broad-spectrum feline calicivirus neutralizing antibody.

[0004] Specific antibody therapy is currently recognized as a relatively effective treatment method, while conventional antibody molecules have a molecular weight of 150 KDa, and their stability is greatly affected by temperature, and they cannot pass through the blood-brain barrier. Nanobodies (Nb) prepared based on the variable region of heavy chain antibodies of Camelidae have the advantages of small molecular weight, strong penetration, and high temperature resistance, and have been increasingly applied in drug research and development as well as structure and function research in recent years. Nanobodies, due to their structural characteristics, have unique epitope binding properties that traditional whole molecule antibodies do not have, and have high binding activity with antigens, good tissue penetration, high water solubility, good immunogenicity, easy genetic engineering modification, and other advantages, and have great application prospects. However, the development and application of nanobodies against feline calicivirus have not been reported. Therefore, it is urgent to develop anti-feline calicivirus nanobodies to effectively prevent and control feline calicivirus infection and prevalence. SUMMARY

[0005] The application aims to provide cat calicivirus camel-derived nanobodies and applications thereof to solve the problems in the prior art.

[0006] To achieve the above-mentioned purposes, the application provides the following solutions.

[0007] The application provides a cat calicivirus nanobody VHH-V14, and the amino acid sequence of the cat calicivirus nanobody VHH-V14 is shown as SEQ ID NO. 1.

[0008] The application also provides a cat calicivirus nanobody VHH-V29, and the amino acid sequence of the cat calicivirus nanobody VHH-V29 is shown as SEQ ID NO. 2.

[0009] The application also provides application of the cat calicivirus nanobody VHH-V14 or the cat calicivirus nanobody VHH-V29 in preparation of a product for detecting cat calicivirus.

[0010] Further, the product is a kit.

[0011] The application also provides a kit for detecting cat calicivirus, which comprises the cat calicivirus nanobody VHH-V14 or the cat calicivirus nanobody VHH-V29.

[0012] The application also provides a method for detecting cat calicivirus for non-disease diagnosis or treatment purposes, which comprises the step of detecting by using the kit.

[0013] The application also provides application of the cat calicivirus nanobody VHH-V14 or the cat calicivirus nanobody VHH-V29 in preparation of a medicine for preventing and / or treating cat calicivirus infection.

[0014] The application also provides a medicine for preventing and / or treating cat calicivirus infection, and the cat calicivirus nanobody VHH-V14 and / or the cat calicivirus nanobody VHH-V29 are main effective components.

[0015] Further, the medicine further comprises a pharmaceutically acceptable excipient.

[0016] Further, the dosage form of the medicine is an injection.

[0017] The present application discloses the following technical effects:

[0018] The present application takes feline calicivirus as an immunogen, and through the steps of immunizing a camel, constructing a library, enriching sequencing and the like, two nanobodies VHH-V14 and VHH-V29 are screened, and the amino acid sequences thereof are shown as SEQ ID NO. 1-2. The experimental results show that the nanobodies VHH-V14 and VHH-V29 are nanobodies with outstanding neutralization activity on feline calicivirus, and the IC 50 50 of the nanobodies VHH-V14 and VHH-V29 are 0.6536 mg / mL and 0.6931 mg / mL respectively, and the nanobodies have good inhibitory effect on feline calicivirus. The present application provides a new biological material for the prevention and treatment of feline calicivirus infection, and provides a new design theory and technical support for the antibody treatment strategy of feline calicivirus, and has outstanding significance for the clinical treatment of feline calicivirus infection. BRIEF DESCRIPTION OF DRAWINGS

[0019] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed in the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor.

[0020] Figure 1 is a micrograph of FCV infected F81 cells; wherein, A is a normal F81 cell; B is a lesioned F81 cell after inoculation of FCV;

[0021] Figure 2 is an electrophoresis result map of recombinant pCold-1-VHH-V14 and pCold-1-VHH-V29 plasmids; wherein, A is 1% agarose gel electrophoresis identification of PCR amplified VHH-V14 and VHH-V29 fragments; M: DL 2000 Marker; 1: negative control; 2: amplified VHH-V14 fragment; 3: amplified VHH-V29 fragment; B is 1% agarose gel electrophoresis identification of double enzyme digested pCold-1 vector; 4-5: double enzyme digested pCold-1 vector;

[0022] Figure 3 is an electrophoresis result map of recombinant pCold-1-VHH-V14 and pCold-1-VHH-V29 plasmids; wherein, M: DL 2000 Marker; 1: PCR amplified VHH-V14 fragment inserted into pCold-1 vector; 2: PCR amplified VHH-V29 fragment inserted into pCold-1 vector;

[0023] Figure 4Figure for SDS-PAGE identification result of VHH-V14 and VHH-V29 protein expression; wherein, M: protein Marker; 1: negative control of VHH-V14 protein; 2: expressed VHH-V14 protein; 3: negative control of VHH-V29 protein; 4: expressed VHH-V29 protein;

[0024] Figure 5 Figure for SDS-PAGE identification result of VHH-V14 and VHH-V29 protein purification; wherein, A is VHH-V14 protein purification eluent (200 mM imidazole); M: protein Marker; B is VHH-V29 protein purification eluent (200 mM imidazole);

[0025] Figure 6 Figure for Western-Blot identification result of VHH-V14 and VHH-V29 protein purification concentration; wherein, A is VHH-V14 protein purification concentration; 1: negative control; M: protein Marker; 2: VHH-V14 protein; B is VHH-V29 protein purification concentration; 3: VHH-V29 protein;

[0026] Figure 7 Figure for IC50 of VHH-V14 and VHH-V29 protein; 50 Figure for calculation result. DETAILED DESCRIPTION

[0027] The following detailed description is provided to understand certain aspects, features and embodiments of the present application. It should be understood that the detailed description and specific examples, while indicating certain embodiments of the application, are given by way of illustration only, and should not be construed in any manner limiting the broad aspects of the application.

[0028] It should be understood that the terms used in the specification are for the purpose of describing particular embodiments only and are not intended to be limiting of the present application. In addition, where particular ranges of values are given, understand that each intervening value, to the upper or lower limit of the ranges is also specifically included. Each smaller range that falls within the broader ranges is also specifically included in the present application. The upper and lower limits of these smaller ranges can independently be included or excluded in the range.

[0029] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although preferred methods and materials are described, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application. All documents mentioned herein are incorporated by reference to disclose and describe the methods and / or materials in connection with which the documents are cited. In the event of conflict between the present specification and any document incorporated by reference, the present specification controls.

[0030] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0031] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0032] The feline calicivirus ocular and nasal swabs of the present invention were preserved by the Animal Disease Prevention and Control Laboratory of the Institute of Special Agricultural Products, Chinese Academy of Agricultural Sciences.

[0033] Example 1: Preparation of feline calicivirus (FCV) immunogen

[0034] 1. Pathogen processing and virus identification

[0035] Eye and nasal swabs were collected from cats suspected of having FCV at an animal hospital in Changchun City, Jilin Province. The swabs were stored in sterile PBS at -80°C. Before use, the swabs were slowly thawed at 4°C, centrifuged at 2000 rpm for 5 min, and the supernatant was collected, filtered through a 0.22 μm filter membrane, and stored at -80°C.

[0036] F81 cells with a viability of over 95% and a density of approximately 70% were collected. In a clean bench, the old culture medium in the cell culture flask was discarded, and 10 mL of PBS solution was added. The flask was gently agitated and washed, and the PBS solution was discarded. This process was repeated once. 8 mL of trypsin was added, and the flask was gently agitated at room temperature to digest the cells. The trypsin was discarded. Once the cells were atomized and slightly detached, 15 mL of DMEM cell culture medium containing 8% fetal bovine serum was added to stop the digestion. The flask walls were repeatedly rinsed until all cells detached. 50 μL of the homogeneous cell suspension was mixed with trypan blue staining solution at a 1:9 ratio to determine the cell concentration. As needed, an appropriate amount of cell suspension was taken and diluted to 1×10⁻⁶ using DMEM cell culture medium containing 8% fetal bovine serum. 5 Add cells / mL to a new cell culture flask and culture in a cell culture incubator at 37℃ and 5% CO2. Set up a normal cell control at the same time. After the cells adhere to the wall for 1 hour, add 100 μL of the above-prepared pathogen supernatant and continue to culture and observe cytopathic effects. When about 70% of the cells show cytopathic effects, freeze and thaw the cell virus solution three times at -20℃, mix well, aliquot and store at -80℃.

[0037] Approximately 24 hours after viral inoculation, F81 cells exhibited typical cytopathic effects such as deformity, shedding, and grape-like clustering. Figure 1 (Medium B), while the control group was established, and the cell morphology was normal ( Figure 1FCV was preliminarily proved to proliferate in F81 cells.

[0038] 2. Purification assay

[0039] TCID assay 50 (TCID 50 = 10 -7.23 / 0.1 mL) and PCR identification using specific primers capable of amplifying the conserved sequence of FCV, a strain of FCV was successfully isolated; the virus was amplified in large quantities, and about 20 L of cell virus liquid was collected, which was concentrated using PEG6000, and then purified by sucrose density gradient centrifugation; the purified product was identified by SDS-PAGE and transmission electron microscopy, and the concentration of the purified virus was determined using a BCA protein concentration determination kit, thereby obtaining pure and high-concentration FCV virus particles.

[0040] Example 2 Construction of nanobody library

[0041] The FCV virus after concentration and purification was used to immunize a bactrian camel, and serum was taken 1 week after each immunization to monitor the antibody level; 1 week after the fifth immunization, the serum affinity and neutralization titer reached a high level; at this time, about 200 mL of blood was collected, the lymphocytes in the serum were separated, RNA was extracted and reverse transcribed into cDNA, VHH sequences were amplified using specific primers, and were inserted into linearized pR2 phagemid through homologous recombination; after electrotransformation into E. coli TG1, an M13 phage display library displaying VHH was obtained, and the library capacity was 1.571 x 10 9 ; 30 clones were randomly picked from the library for DNA sequencing analysis, and the sequence analysis results showed that the accuracy of the library was about 86.2%.

[0042] Example 3 Screening of specific nanobody sequences

[0043] The present application adopts the rod-shaped FCV-VP1 protein expressed and purified, is used for screening specific phage library, takes it as a coating agent, and carries out three rounds of screening through specific binding of the coating agent and the antibody, so as to enrich specific phages. After each round of screening, the phages in the eluate are amplified, and the enrichment degree is determined by determining the titer and Phage ELISA. It is found that with the increase of the screening rounds, the P / N value rises continuously, proving that the specific phages are effectively enriched. Through PCR amplification of VHH sequences and small-batch amplification of phages and Phage ELISA detection, 51 phage strains with good affinity activity are screened from 96 monoclonal strains. The VHH sequences of the 51 strains are analyzed, and through deletion of redundant sequences, analysis of the functional region and homology analysis, finally 2 FCV nanobodies meeting the conditions are obtained, and are named as VHH-V14 and VHH-V29 respectively, and the amino acid sequences are shown as SEQ ID NO. 1-2.

[0044] VHH-V14 (SEQ ID NO. 1):

[0045] QLQLVESGGGTVQPGGSLKLSCAAS AVTFRWSS MNWYRQAPGKERELVAT ISSFGDTT YADSVKGRFTVSRDNFKHTMYLEMNGLEPEDTAVYYC ATPHY WGRGTQVTVSS.

[0046] VHH-V29 (SEQ ID NO. 2):

[0047] QLQLVESGGGLVQPGESLRLSCVVS GLTFSSSD MIWDRQAPGKERERVAS ISGADGST NYADFVKGRFTISRDNVKNTVYLQMNSLKPEDTARYYC HAYRQTGRGPLHS WGQGTQVTVSS.

[0048] In SEQ ID NO. 1-2, the underlined sequence is the CDR fragment of the antibody, and the CDR fragment includes CDR1, CDR2 and CDR3.

[0049] Example 4 Expression and identification of nanobody

[0050] 1. Experimental method

[0051] 1.1 Construction of recombinant plasmids pCold-1-VHH-V14 and pCold-1-VHH-V29

[0052] The pCold-1 plasmid was used as a carrier, and the upstream (EcoRI) and downstream (XbalI) restriction enzyme cutting sites were selected. The start and end sequences were added in the upstream and downstream primers, respectively. His-tag was present after the start sequence in the upstream primer. The VHH-V14 and VHH-V29 fragments were inserted. Specific primers were designed (Table 1), and the primers were ordered from a biological company.

[0053] Table 1 Specific primers for amplifying VHH-V14 and VHH-V29 sequences

[0054] Primer name Primer sequence (5'→3') VHH-V14-F ACCCTCGAGGGATCCGAATTCCAGGTGCAGCTCGTGGAG (SEQ ID NO.3) VHH-V14-R AGCAGAGATTACCTATCTAGATCAGGAGACGGTGACCTGG (SEQ ID NO.4) VHH-V29-F ACCCTCGAGGGATCCGAATTCGATGTGCAGCTGCAGGAGTC (SEQ ID NO.5) VHH-V29-R AGCAGAGATTACCTATCTAGATCAGGAGACGGTGACCTGG (SEQ ID NO.4)

[0055] A PCR system was prepared using PrimeSTAR Max Premix (2x) (Table 2). The V14 and V29 bacterial liquid were used as templates to amplify the VHH-V14 and VHH-V29 fragments. The size of the target fragments was 372 bp and 405 bp. The PCR reaction program was as follows: 98℃ pre-denaturation for 2 min; 98℃ denaturation for 10 s, 60℃ annealing for 10 s, 72℃ extension for 1 min, repeated for 35 cycles; 72℃ extension for 5 min, and 4℃ storage.

[0056] Table 2 PCR reaction system for amplifying VHH-V14 and VHH-V29 fragments

[0057] Reaction system component Volume VHH-V14-F / VHH-V29-F 1 μL VHH-V14-R / VHH-V29-R 1 μL Bacterial solution 2 μL PrimeSTAR Max Premix (2×) 8 μL ddH2O Added to 20 μL

[0058] EcorI and XbalI restriction enzymes were used to double-enzyme cut the pCold-1 vector with known concentration preserved in the laboratory at 37℃ for 1 h (Table 3).

[0059] Table 3 Reaction system for cutting pCold-1 vector

[0060] Reaction system component Volume Restriction Enzyme: Ecor Ⅰ HF 1 μL Restriction Enzyme: Xbal I HF 1 μL pCold-1 2 μL 10× NEBuffer: rCutSmart 5 μL ddH2O Added to 50 μL

[0061] The PCR-amplified FCV-VP1 fragment and the double-enzyme cut pCold-1 linear vector were mixed with Loading Buffer at a ratio of 1:10, and 1% agarose gel was added. Electrophoresis was performed in 1xTAE buffer at 180 V for 15 min. The results were observed using a gel imaging instrument and were recorded. The gel was recovered and purified according to the instructions of the Gel Recovery and Purification Kit. The concentration was determined, and the sample was stored at -40℃. According to the instructions of the Seamless Cloning Kit, a ligation system was prepared (Table 4). The recovered VHH-V14 and VHH-V29 fragments were ligated with the disconnected double-enzyme cut pCold-1 linear vector, respectively, at 50℃ for 1 h.

[0062] Table 4 Ligation system for seamless cloning

[0063] Reaction system component Volume Double enzyme-digested pCold-1 linear vector 5 μL VHH-V14 / VHH-V29 fragment 5 μL 2×Seamless Cloning Mix Added to 20 μL

[0064] Remove BL21 competent cells from the -80℃ freezer and thaw them on ice. In a clean bench, add 20 μL of the ligation product to each BL21 competent cell, gently shake to mix (avoiding vigorous shaking), and incubate on ice for 25 min. Then, place the cells in a pre-prepared 42℃ water bath for heat shock for 45 s, and quickly remove them from the water bath and return them to ice. Two minutes later, add 900 μL of antibiotic-free liquid SOC medium to the clean bench, mix well by pipetting, and incubate overnight at 37℃ with a shaker at 225 rpm. Serially dilute 10⁻⁶ cells in a clean bench. -1 10 -2 Approximately 100 μL of the culture product was spread onto a pre-prepared LB agar plate containing ampicillin; it was then transferred to a 37°C incubator and incubated upright for 0.5 h, followed by inverted incubation. The results were observed after 12-16 h; a portion of the original bacterial culture was picked and diluted to 10 in a clean bench. -2 A single transformed colony was inoculated into 5 mL of LB liquid medium containing ampicillin and incubated at 37°C and 180 rpm for 10–16 h. The precipitate was gently agitated and resuspended. 2 μL of the bacterial culture was taken from each tube in a clean bench as a template for PCR detection, following the same reaction system and procedure as above (Tables 1 and 2). The PCR product was mixed with Loading Buffer at a 1:10 ratio, added to a 1% agar gel, and electrophoresed in 1×TAE buffer at 180 V for 15 min. The results were observed and recorded using a gel imaging system. The gel was purified according to the instructions of the gel extraction kit, the concentration was determined, and the gel was stored at -40°C and sent to the company for sequencing to verify the sequence correctness of the amplified VHH-V14 and VHH-V29 fragments.

[0065] The bacterial suspensions that tested positive were preserved by mixing 500 μL of bacterial suspension and 500 μL of 50% glycerol in a clean bench, aliquoting the mixture into 1.5 mL sterile centrifuge tubes, and storing at -80°C.

[0066] 1.2 Prokaryotic expression of VHH-V14 and VHH-V29 proteins

[0067] The strains containing pCold-1-VHH-V14 and pCold-1-VHH-V29 plasmids were respectively induced for small batch prokaryotic expression: first, the stored bacterial liquid identified as positive by PCR was taken in the clean bench, and was plated on LB plates with ampicillin resistance, and a negative control was set, which was transferred to a 37°C incubator, and was cultured vertically after 0.5 h of vertical placement, and the results were observed after 12-16 h; single colonies were picked in the clean bench, and were inoculated in 5 mL of LB liquid medium with ampicillin resistance, and were cultured at 37°C and 180 rpm to the logarithmic growth phase, and were induced for expression by adding IPTG (isopropyl-beta-D-thiogalactoside) at a final concentration of 1 mM, and the expression conditions were 16°C, 180 rpm, and culture for 24 h; the bacteria were collected by centrifugation at 8000 rpm for 20 min, the bacteria were resuspended with about 10 times the volume of PBS solution, and were mixed by blowing and sucking, and were placed on ice for ultrasonic crushing, and the solution was clarified, and the supernatant and precipitate were collected by centrifugation at 8000 rpm for 20 min, and the precipitate was resuspended with about 10 times the volume of PBS solution, and was mixed by blowing and sucking. The supernatant and precipitate were used as samples for SDS-PAGE identification.

[0068] The bacterial liquid identified as positive by SDS-PAGE was selected for large batch prokaryotic expression induction under the above conditions and methods, and 100 mL of LB liquid medium with ampicillin resistance was used for culture, and finally 10 mL of supernatant was collected.

[0069] 1.3 Purification and identification of VHH-V14 and VHH-V29 proteins

[0070] The VHH-V14 and VHH-V29 proteins were purified using the collected supernatant as a sample, and finally ultrafiltration and concentration were performed according to the instructions of the ultrafiltration tube (3 kDa), and the replacement solution was PBS, and finally high-purity VHH-V14 and VHH-V29 proteins were obtained, and the concentration was determined using a BCA protein concentration determination kit.

[0071] The purified and concentrated VHH-V14 and VHH-V29 proteins were used as primary antibodies for Western-Blot identification.

[0072] 1.4 Neutralization titer determination of VHH-V14 and VHH-V29 proteins

[0073] The purified and concentrated VHH-V14 and VHH-V29 proteins were used as samples for neutralization titer determination, and the conditions and steps were as follows:

[0074] Take a 96-well cell culture plate and add serum-free MEM medium, 50 μL / well, starting from the second column. Add filtered VHH-V14 and VHH-V29 proteins, 100 μL / well, to the first column of wells. Set up 8 replicate wells as controls. Perform serial dilutions starting from the second column of wells until the final dilution. Dilute the FCV virus solution to 200 TCID. 50 After adding 100 μL, the solution was added to 96-well cell culture plates at 50 μL / well, and neutralized in a 37℃, 5% CO2 cell culture incubator for 1 h. Virus dilution back-titration verification: The diluted virus solution was diluted 10-fold, 100-fold, and 1000-fold, and added to 96-well cell culture plates at 100 μL / well along with the original virus dilution. The prepared concentration was 1×10⁻⁶. 5 F81 cell suspension of 100 μL / well was added to a 96-well cell culture plate and cultured in a cell culture incubator at 37°C and 5% CO2. Cytopathic effects were observed daily. Observation was stopped and results were recorded on day 5. The neutralizing titers of VHH-V14 and VHH-V29 proteins were calculated according to the number of wells with cytopathic effects according to the Reed-Muench method.

[0075] 2. Experimental Results

[0076] 2.1 Construction of recombinant plasmids pCold-1-VHH-V14 and pCold-1-VHH-V29

[0077] Using SnapGene software and the pCold-1 plasmid as a vector, two restriction enzyme sites (EcoRI upstream and XbalI downstream) were selected, and His-tags were added. The selected VHH-V14 and VHH-V29 fragments were inserted, respectively. Specific primers were designed, and a high-fidelity enzyme PCR system was prepared to amplify the VHH-V14 and VHH-V29 fragments. The pCold-1 vector stored in our laboratory was double-digested with the selected EcoRI and XbalI restriction endonucleases. The results were confirmed by 1% agarose gel electrophoresis, yielding target fragments of approximately 372 bp, 405 bp, and 4383 bp, respectively, which were in line with expectations. Figure 2 The concentrations of the purified and recovered amplified VHH-V14 and VHH-V29 fragments and the double-digested pCold-1 vector were determined to be 93.52 ng / μL, 98.37 ng / μL, and 33.22 ng / μL, respectively, and were stored at -40℃.

[0078] The purified fragments and vectors were diluted with Elution Buffer B according to the instructions, a ligation system was prepared, the ligation product was transformed into BL21 competent cells, and a single colony was picked and cultured; the inserted VHH-V14 and VHH-V29 fragment sequences were amplified using the bacterial solution as a template, and the results were identified using 1% agarose gel electrophoresis, obtaining the target fragments of about 372 bp and 405 bp respectively (the sizes were consistent with the expectations). Figure 3 The PCR products were purified and recovered, and sent to a company for sequencing. The sequencing results showed that the VHH-V14 and VHH-V29 fragments had no mutations and deletions, and the correct recombinant pCold-1-VHH-V14 and pCold-1-VHH-V29 plasmids were obtained.

[0079] 2.2 Prokaryotic expression and identification of VHH-V14 and VHH-V29 proteins

[0080] First, small-batch prokaryotic induction expression of VHH-V14 and VHH-V29 proteins: plate positive bacterial solution, 37°C, culture for 12-16 h; pick single colonies and inoculate into 5 mL LB liquid medium with ampicillin resistance, 37°C, 180 rpm culture to logarithmic growth phase, add IPTG with a final concentration of 1 mM, expression conditions are 16°C, 180 rpm, culture for 24 h; centrifugal collection of bacterial bodies, resuspend the bacterial bodies with 1 mL PBS solution respectively, break by ultrasonic disrupter and centrifugal collection of supernatant, identify VHH-V14 and VHH-V29 proteins in supernatant using SDS-PAGE (see Figure 4 ), the results show that VHH-V14 and VHH-V29 proteins are well expressed. Then, large-batch prokaryotic induction expression of VHH-V14 and VHH-V29 proteins.

[0081] 2.3 Purification and identification of VHH-V14 and VHH-V29 proteins

[0082] Purify the supernatant of the large-batch prokaryotic induction expression of VHH-V14 and VHH-V29 proteins according to the His tag purification filler instructions, and identify the flow-through, equilibration liquid and eluate collected during the purification process using SDS-PAGE (see Figure 5 ), the results show that VHH-V14 and VHH-V29 proteins are well purified; centrifugal the eluate identified as positive using ultrafiltration tube (3 kDa), replace the solution with PBS, and determine the concentrations of VHH-V14 and VHH-V29 proteins to be 1.03 mg / mL and 1.23 mg / mL respectively, and perform Western-Blot identification (see Figure 6), the results showed that VHH-V14 and VHH-V29 could specifically bind to FCV-VP1, and the prokaryotic expression VHH-V14 and VHH-V29 proteins were relatively pure.

[0083] 2.4 Neutralization titer detection of VHH-V14 and VHH-V29 proteins

[0084] The purified and concentrated VHH-V14 and VHH-V29 proteins were diluted to 1 mg / mL with PBS solution, filtered with a 0.22 μm filter membrane in a clean bench, and used as samples. After the antigen-antibody neutralization, the F81 cells were inoculated, the cytopathic effect was observed, the neutralization titer was calculated according to the Reed-Muench method, and the data were recorded and counted. Figure 7 The results showed that the IC 50 of VHH-V14 was 0.6536 mg / mL, and the IC 50 of VHH-V29 was 0.6931 mg / mL, both of which had good neutralization activity.

[0085] The above-described embodiments are only used to describe the preferred modes of the present application, and do not limit the scope of the present application. Without departing from the design spirit of the present application, various modifications and improvements of the technical solutions of the present application made by those skilled in the art shall fall within the protection scope determined by the claims of the present application.

Claims

1. A feline calicivirus nanobody VHH-V14, characterized in that, The amino acid sequence of the feline calicivirus nanobody VHH-V14 is shown as SEQ ID NO.

1.

2. A feline calicivirus nanobody VHH-V29, characterized in that, The amino acid sequence of the feline calicivirus nanobody VHH-V29 is shown as SEQ ID NO.

2.

3. Use of the feline calicivirus nanobody VHH-V14 of claim 1 or the feline calicivirus nanobody VHH-V29 of claim 2 in the preparation of a product for detecting feline calicivirus.

4. The use according to claim 3, wherein the compound is ###0002### The product is a kit.

5. A kit for detecting feline calicivirus, characterized by comprising: The product comprises the feline calicivirus nanobody VHH-V14 of claim 1 or the feline calicivirus nanobody VHH-V29 of claim 2.

6. A method for detecting feline calicivirus for non-diagnostic or therapeutic purposes, characterized in that, The product comprises the step of detection using the kit of claim 5.

7. Use of the feline calicivirus nanobody VHH-V14 of claim 1 or the feline calicivirus nanobody VHH-V29 of claim 2 in the preparation of a medicine for preventing and / or treating feline calicivirus infection.

8. A medicine for preventing and / or treating feline calicivirus infection, characterized by, The feline calicivirus nanobody VHH-V14 of claim 1 and / or the feline calicivirus nanobody VHH-V29 of claim 2 is the main effective component.

9. The medicament according to claim 8, wherein The medicine further comprises a pharmaceutically acceptable excipient.

10. The medicament according to claim 8, wherein The dosage form of the medicine is injection.

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