Camel-derived nanometer antibody for feline calicivirus and application of nanometer antibody

Abstract

The invention discloses a feline calicivirus camel source nano antibody and application thereof, and belongs to the field of biological medicine. According to the invention, feline calicivirus is taken as an immunogen, through the steps of camel immunization, library construction, enrichment sequencing and the like, two nano antibodies VHH-V14 and VHH-V29 are obtained through screening, and the amino acid sequences of the two nano antibodies are respectively shown as SEQ ID NO.1-2. Experimental results show that the nanometer antibodies VHH-V14 and VHH-V29 are nanometer antibodies with outstanding neutralizing activity on the feline calicivirus, the IC50 of the nanometer antibodies is 0.6536 mg / mL, the IC50 of the nanometer antibodies is 0.6931 mg / mL, and the nanometer antibodies VHH-V14 and VHH-V29 have a good inhibiting effect on the feline calicivirus. The invention provides a new biological material for prevention and treatment of feline calicivirus infection, provides a new design theory and technical support for antibody treatment strategies of feline calicivirus, and has outstanding significance for clinical treatment of feline calicivirus infection.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a camel-derived nanoantibody derived from feline calicivirus and an application thereof. Background Art

[0002] Feline calicivirus (FCV) belongs to the Caliciviridae family and the Vesivirus genus. It is a spherical, non-enveloped virus with icosahedral symmetry and a diameter of 35-39 nm. The Caliciviridae family consists of five genera: Lagovirus, Newbovirus, Norovirus, Sapovirus, and Vesivirus, all of which can infect mammals.

[0003] Feline calicivirus mainly causes acute oral and upper respiratory diseases, chronic gingivostomatitis (CGS), lameness syndrome, claw and mouth disease, and virulent systemic feline calicivirus infection (VS-FCV) in felines. The pathological manifestations mainly include subcutaneous edema, oral ulcers, lameness, skin ulcers such as those on the claws. The prevalence of FCV is high, and the mortality rate of virulent systemic feline calicivirus infection caused by some strains is high, reaching 50%. The treatment of feline calicivirus is mainly based on antiviral treatment 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 antibodies.

[0004] Specific antibody therapy is currently recognized as a relatively effective treatment method, while conventional antibodies have a molecular weight of 150KDa, their stability is greatly affected by temperature, and they cannot cross the blood-brain barrier; Nanobodies (Nb) prepared based on the variable region of heavy chain antibodies of camelids have the advantages of small molecular weight, strong penetrability, and high temperature resistance. In recent years, they have been increasingly used in drug development and structural and functional research. Nanobodies benefit from their structural characteristics and have unique epitope binding properties that traditional whole-molecule antibodies do not have. They have high antigen binding activity and good tissue penetration. They also have advantages such as high water solubility, good immunogenicity, and easy genetic engineering, and have huge application prospects. However, the development and application of nanobodies against feline calicivirus have not been reported. Therefore, there is an urgent need to develop anti-feline calicivirus nanoantibodies in order to effectively prevent and control feline calicivirus infection and epidemics. Summary of the Invention

[0005] The purpose of the present invention is to provide camel-derived nanoantibodies against feline calicivirus and their applications to solve the problems existing in the above-mentioned prior art. The nanoantibodies VHH-V14 and VHH-V29 provided by the present invention are nanoantibodies with outstanding neutralizing activity against feline calicivirus and have a good inhibitory effect on feline calicivirus. The present invention provides new biomaterials for the prevention and treatment of feline calicivirus infection and provides new design theories and technical support for antibody treatment strategies for feline calicivirus.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides a feline calicivirus nanobody VHH-V14, the amino acid sequence of the feline calicivirus nanobody VHH-V14 is shown in SEQ ID NO.1.

[0008] The present invention also provides a feline calicivirus nanobody VHH-V29, the amino acid sequence of the feline calicivirus nanobody VHH-V29 is shown in SEQ ID NO.2.

[0009] The present invention also provides a use of the above-mentioned feline calicivirus nanobody VHH-V14 or the above-mentioned feline calicivirus nanobody VHH-V29 in the preparation of a product for detecting feline calicivirus.

[0010] Furthermore, the product is a kit.

[0011] The present invention also provides a kit for detecting feline calicivirus, comprising the above-mentioned feline calicivirus nanoantibody VHH-V14 or the above-mentioned feline calicivirus nanoantibody VHH-V29.

[0012] The present invention also provides a method for detecting feline calicivirus for purposes other than disease diagnosis or treatment, comprising the step of using the above-mentioned kit for detection.

[0013] The present invention also provides a use of the above-mentioned feline calicivirus nanobody VHH-V14 or the above-mentioned feline calicivirus nanobody VHH-V29 in the preparation of a medicament for preventing and / or treating feline calicivirus infection.

[0014] The present invention also provides a drug for preventing and / or treating feline calicivirus infection, which uses the above-mentioned feline calicivirus nanobody VHH-V14 and / or the above-mentioned feline calicivirus nanobody VHH-V29 as main active ingredients.

[0015] Furthermore, pharmaceutically acceptable excipients are also included.

[0016] Furthermore, the dosage form of the drug is an injection.

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

[0018] The present invention uses feline calicivirus as an immunogen, and through the steps of immunizing camels, constructing libraries, enriching and sequencing, two nanobodies VHH-V14 and VHH-V29 were screened and obtained, whose amino acid sequences are shown in SEQ ID NO.1-2 respectively. The experimental results show that nanobodies VHH-V14 and VHH-V29 are nanobodies with outstanding neutralizing activity against feline calicivirus, and their IC 50 The concentrations of the active ingredients in the active ingredients are 0.6536 mg / mL and 0.6931 mg / mL, respectively, showing a good inhibitory effect on feline calicivirus. The present invention provides a new biomaterial for the prevention and treatment of feline calicivirus infection, and provides new design theory and technical support for antibody treatment strategies for feline calicivirus, which has outstanding significance for the clinical treatment of feline calicivirus infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0020] Figure 1 Microscopic observation of F81 cells infected with FCV; A is a normal F81 cell; B is a pathological F81 cell after FCV inoculation;

[0021] Figure 2 Electrophoresis results for recombinant pCold-1-VHH-V14 and pCold-1-VHH-V29 plasmids. A is the electrophoresis analysis of PCR-amplified VHH-V14 and VHH-V29 fragments on a 1% agarose gel; M: DL 2000 Marker; 1: negative control; 2: amplified VHH-V14 fragment; 3: amplified VHH-V29 fragment; B is the electrophoresis analysis of the pCold-1 vector after double enzyme digestion on a 1% agarose gel; 4-5: pCold-1 vector after double enzyme digestion;

[0022] Figure 3 This figure shows the verification results of the recombinant pCold-1-VHH-V14 and pCold-1-VHH-V29 plasmids; M: DL2000 Marker; 1: PCR amplification of the VHH-V14 fragment inserted into the pCold-1 vector; 2: PCR amplification of the VHH-V29 fragment inserted into the pCold-1 vector;

[0023] Figure 4The results of SDS-PAGE identification of expressed VHH-V14 and VHH-V29 proteins are shown in the figure; 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 2 is the SDS-PAGE identification result after VHH-V14 and VHH-V29 protein purification; wherein, A is the VHH-V14 protein purification eluate (200 mM imidazole); M: protein marker; B is the VHH-V14 protein purification eluate (200 mM imidazole);

[0025] Figure 6 The Western-Blot results of VHH-V14 and VHH-V29 proteins after purification and concentration are shown in Figure 1. A shows VHH-V14 protein after purification and concentration; 1: negative control; M: protein marker; 2: VHH-V14 protein; B shows VHH-V29 protein after purification and concentration; 3: VHH-V14 protein.

[0026] Figure 7 IC for VHH-V14 and VHH-V29 proteins 50 Calculation result diagram. DETAILED DESCRIPTION

[0027] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0028] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0029] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0030] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

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

[0032] The feline calicivirus eye and nose swab of the present invention is stored in the Animal Disease Prevention and Control Laboratory of the Special Products Research Institute of the Chinese Academy of Agricultural Sciences.

[0033] Example 1 Preparation of Feline Calicivirus (FCV) Immunogen

[0034] 1. Sample processing and virus identification

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

[0036] Take F81 cells with a survival rate of more than 95% and a density of about 70%, discard the old culture medium in the cell bottle in the clean bench, add 10mL PBS solution, shake the bottle gently for washing, discard the PBS solution, and repeat once; add 8mL trypsin, shake the bottle gently at room temperature for digestion, and discard the trypsin; when the cells appear atomized and slightly detached, add 15mL DMEM cell culture medium containing 8% fetal bovine serum to stop digestion, and repeatedly blow the bottle wall until all cells fall off. Take 50μL of uniform cell suspension, mix it with trypan blue dye at a ratio of 1:9, and determine the cell concentration; take an appropriate amount of cell suspension as needed and dilute the cell suspension to 1×10 using DMEM cell culture medium containing 8% fetal bovine serum. 5 / mL, add it to a new cell bottle, culture it in a cell culture incubator at 37℃ and 5% CO2, and set up a normal cell control at the same time. After 1 hour of cell attachment, add 100μL of the above-prepared diseased material supernatant, continue to culture and observe the cytopathic effect; after about 70% of the cells show cytopathic effect, place the cell virus solution at -20℃ and freeze-thaw 3 times, mix well, and package and store at -80℃.

[0037] About 24 hours after virus inoculation, F81 cells showed typical cytopathic changes such as deformation, shedding, and grape-like appearance ( Figure 1 Middle B), while the control was established, the cell morphology was normal ( Figure 1 Middle A). Preliminary evidence shows that FCV proliferates in F81 cells.

[0038] 2. Purification Assay

[0039] Determination of TCID 50 (TCID 50 =10 -7.23 / 0.1mL) and PCR identification using specific primers that can amplify FCV conserved sequences successfully isolated an FCV strain; this virus was amplified in large quantities, and a total of approximately 20L of cell virus fluid 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 assay kit to obtain pure, high-concentration FCV virus particles.

[0040] Example 2 Construction of Nanobody Library

[0041] Bactrian camels were immunized with concentrated and purified FCV virus. Serum was collected one week after each immunization to monitor antibody levels. One week after the fifth immunization, serum affinity and neutralization titers reached high levels. At this time, approximately 200 mL of blood was collected, lymphocytes in the serum were separated, RNA was extracted, and reverse transcribed into cDNA. The VHH sequence was amplified using specific primers and inserted into the linearized pR2 phagemid through homologous recombination. After electroporation into E. coli TG1, an M13 phage display library displaying VHH was obtained, with a library capacity of 1.571×10 9 ; 30 clones were randomly selected from the library for DNA sequencing analysis. The sequence analysis results showed that the accuracy of the library was about 86.2%.

[0042] Example 3 Specific Nanobody Sequence Screening

[0043] The present invention uses rod-shaped expression of FCV-VP1 protein and purification for screening specific library phages, which are used as coating sources. Three rounds of screening are carried out through the specific binding of the coating source with the antibody to enrich specific phages. After each round of screening, the phages in the eluate are amplified, and the degree of enrichment is determined by measuring the titer and Phage ELISA. It is found that with the increase in the number of screening rounds, the P / N value continues to rise, proving that specific phages have been effectively enriched. By amplifying the VHH sequence by PCR and amplifying the phage in small batches and performing Phage ELISA detection, 51 phage strains with good affinity activity were screened from 96 monoclonal strains. The VHH sequences sequenced from the 51 strains were analyzed, and by deleting redundant sequences, analyzing their functional regions and performing homology analysis, two qualified FCV nanobodies were finally obtained, named VHH-V14 and VHH-V29, whose amino acid sequences are shown in SEQ ID NO.1-2, respectively.

[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 N YADFVKGRFTISRDNVKNTVYLQMNSLKPEDTARYYC HAYRQTGRGPLHS WGQGTQVTV SS.

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

[0049] Example 4 Expression and identification of nanobodies

[0050] 1. Experimental Methods

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

[0052] Using the pCold-1 plasmid as the vector, two restriction enzyme sites, upstream (EcoRI) and downstream (XbalI), were selected. Start and stop sequences were added to the upstream and downstream primers, respectively. A His-tag was placed after the start sequence of the upstream primer. VHH-V14 and VHH-V29 fragments were inserted, and specific primers were designed (Table 1). The orders were sent to a biological company for primer synthesis.

[0053] Table 1 Primers for specific amplification of VHH-V14 and VHH-V29 sequences

[0054]

[0055] PrimeSTAR Max Premix (2×) was used to prepare the PCR system (Table 2). The stored V14 and V29 bacterial cultures were used as templates to amplify the VHH-V14 and VHH-V29 fragments. The target fragment sizes were 372 bp and 405 bp, respectively. The PCR reaction procedure was as follows: pre-denaturation at 98°C for 2 min; denaturation at 98°C for 10 s, annealing at 60°C for 10 s, and extension at 72°C for 1 min, repeated for 35 cycles; extension at 72°C for another 5 min, and storage at 4°C.

[0056] Table 2 PCR reaction system for VHH-V14 and VHH-V29 fragment amplification

[0057]

[0058] The pCold-1 vector with known concentration stored in the laboratory was double-digested with EcoRI and XbalI restriction enzymes at 37°C for 1 h (Table 3).

[0059] Table 3 Reaction system for enzyme digestion of pCold-1 vector

[0060]

[0061] Mix the PCR-amplified FCV-VP1 fragment and the double-digested pCold-1 linear vector in a 1:10 ratio with Loading Buffer. Add a 1% agarose gel and perform electrophoresis in 1× TAE buffer at 180V for 15 minutes. Observe the results using a gel imager and record the results. Recover the fragments according to the gel recovery and purification kit instructions, determine the concentration, and store at -40°C. Prepare the ligation system (Table 4) according to the Seamless Cloning Kit instructions. Ligate the recovered VHH-V14 and VHH-V29 fragments to the disconnected double-digested pCold-1 linear vector, respectively, at 50°C for 1 hour.

[0062] Table 4 Seamless cloning connection system

[0063]

[0064] Take out the BL21 competent cells from the -80℃ freezer and place them on ice prepared in advance to melt; take 20μL of the ligation product and add it to the BL21 competent cells in the clean bench, shake it gently, do not shake it violently, and place it on ice for 25 minutes; then place it in a pre-prepared 42℃ water bath, heat shock it for 45 seconds, quickly take it out of the water bath and put it back on ice; after 2 minutes, add 900μL of non-resistant liquid SOC medium in the clean bench, pipet and mix it, and place it in a shaker at 225rpm and 37℃ for overnight; dilute it 10 times in the clean bench -1 , 10 -2 , take about 100 μL of the culture product and apply it to the prepared LB plate with ampicillin resistance; transfer it to a 37°C incubator, place it upright for 0.5 hours, then invert it for culture, and observe the results after 12-16 hours; pick up the original bacterial solution with a concentration of 10 in the clean bench and dilute it to -2 A single transformed colony was inoculated into 5 mL of ampicillin-resistant LB liquid medium and incubated at 37°C, 180 rpm, for 10-16 hours. Gently shake the tube to resuspend the pellet. Within the clean bench, 2 μL of the bacterial solution from each tube was used as a template for PCR analysis, using the same reaction system and procedures as above (Tables 1 and 2). The PCR product was mixed with Loading Buffer at a ratio of 1:10, added to a 1% agarose gel, and electrophoresed in 1× TAE buffer at 180V for 15 minutes. The results were visualized using a gel imager and recorded. Gel extraction and purification were performed according to the gel extraction and purification kit instructions. The concentration was determined, the fragments were stored at -40°C, and the fragments were sent to the company for sequencing to verify the sequence accuracy of the amplified VHH-V14 and VHH-V29 fragments.

[0065] The bacterial solution identified as positive was preserved by mixing 500 μL of bacterial solution with 500 μL of 50% glycerol in a clean bench, dispensing 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 the pCold-1-VHH-V14 and pCold-1-VHH-V29 plasmids were induced to express prokaryotes in small batches: first, the bacterial suspension identified as positive by PCR was taken out from the clean bench and plated on LB plates with ampicillin resistance. At the same time, a negative control was set up and transferred to a 37°C incubator. After being placed upright for 0.5 hours, the plate was inverted and cultured. The results were observed after 12-16 hours. Monoclonal colonies were picked up from the clean bench and inoculated into 5 mL of LB liquid culture medium with ampicillin resistance at 37°C for 18 hours. The cells were cultured at 100 rpm until the logarithmic growth phase. IPTG (isopropyl-β-D-thiogalactopyranoside) was added to induce expression at a final concentration of 1 mM. The expression conditions were 16°C, 180 rpm, and cultured for 24 hours. The cells were collected by centrifugation at 8000 rpm for 20 minutes, resuspended in PBS solution approximately 10 times the cell volume, and mixed by pipetting. The cells were then ultrasonically disrupted on ice. After the solution clarified, the supernatant and precipitate were collected by centrifugation at 8000 rpm for 20 minutes. The precipitate was resuspended in PBS solution approximately 10 times the cell volume, and mixed by pipetting. The supernatant and precipitate were used as samples for SDS-PAGE analysis.

[0068] Using the above conditions and methods, bacterial cultures that were positive by SDS-PAGE were selected for large-scale induction of prokaryotic expression. 100 mL of LB liquid medium containing ampicillin resistance was used for culture, and 10 mL of supernatant was finally harvested from each culture.

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

[0070] The supernatant collected in 1.2 was used as a sample to purify the VHH-V14 and VHH-V29 proteins. Finally, ultrafiltration and concentration were performed according to the instructions of the ultrafiltration tube (3 kDa). The replacement solution was PBS. Finally, highly pure VHH-V14 and VHH-V29 proteins were obtained, and their concentrations were determined using the BCA protein concentration assay kit.

[0071] Western-Blot identification was performed using the purified and concentrated VHH-V14 and VHH-V29 proteins as primary antibodies.

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

[0073] The neutralization potency of purified and concentrated VHH-V14 and VHH-V29 proteins was determined using the following conditions and steps:

[0074] Take a 96-well cell culture plate and add serum-free MEM medium from the second column, 50 μL / well, take the filtered VHH-V14 and VHH-V29 proteins as samples, add 100 μL / well to the first column of wells, set up 8 replicate wells as controls, and make two-fold dilutions from the second column of wells until the last dilution; dilute the FCV virus solution to 200 TCID 50 After adding 50 μL / well to a 96-well cell culture plate, the plate was placed in a 37°C, 5% CO2 cell culture incubator for neutralization for 1 hour. Virus dilution titration verification: the diluted virus solution was diluted 10 times, 100 times, and 1000 times, and added to a 96-well cell culture plate together with the original virus dilution solution at 100 μL / well. The prepared concentration was 1×10 5 A suspension of F81 cells (100 μL / well) at a concentration of 1 cell / mL was added to a 96-well cell culture plate at 100 μL / well. The cells were cultured in a cell culture incubator at 37°C and 5% CO2. The cytopathic effect was observed daily. Observation was stopped on the 5th day and the results were recorded. The neutralization potency of the VHH-V14 and VHH-V29 proteins was calculated according to the Reed-Muench method based on the number of wells with cytopathic effect.

[0075] 2. Experimental Results

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

[0077] Using SnapGene software, pCold-1 plasmid was used as the vector. Two restriction enzyme sites, upstream (EcoR I) and downstream (Xbal I), were selected, His-tag was added, and the selected VHH-V14 and VHH-V29 fragments were inserted, respectively. Specific primers were designed, and a PCR system was prepared using high-fidelity enzymes to amplify the VHH-V14 and VHH-V29 fragments. The pCold-1 vector stored in this laboratory was double-digested with the selected EcoR I and Xbal I restriction enzymes. The above results were identified by 1% agarose gel electrophoresis, and the target fragments of approximately 372 bp, 405 bp, and 4383 bp were obtained, respectively, which were in line with the expected sizes ( Figure 2 The concentrations of the purified and recovered amplified VHH-V14 and VHH-V29 fragments and the double-enzyme-digested pCold-1 vector were determined to be 93.52 ng / μL, 98.37 ng / μL, and 33.22 ng / μL, respectively, and stored at -40°C.

[0078] The purified and recovered fragments and vectors were appropriately diluted using Elution Bffuer, and a ligation system was prepared. The ligation products were transformed into BL21 competent cells, and a single colony was picked for culture. The inserted VHH-V14 and VHH-V29 fragment sequences were amplified using the bacterial solution as a template. The results were identified by 1% agarose gel electrophoresis, and the target fragments of approximately 372 bp and 405 bp were obtained, respectively ( Figure 3 The PCR products were purified and recovered, and sent to the company for sequencing. Sequencing results showed that the VHH-V14 and VHH-V29 fragments had no mutations or deletions, resulting in the correct recombinant pCold-1-VHH-V14 and pCold-1-VHH-V29 plasmids.

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

[0080] First, the VHH-V14 and VHH-V29 proteins were induced to express in prokaryotes in small batches: positive bacterial liquid was plated and cultured at 37°C for 12-16 hours; single clones were picked and inoculated into 5 mL of LB liquid medium with ampicillin resistance, cultured at 37°C and 180 rpm until the logarithmic growth phase, IPTG was added at a final concentration of 1 mM, and the expression conditions were 16°C and 180 rpm for 24 hours; the bacteria were collected by centrifugation, resuspended in 1 mL of PBS solution, disrupted by ultrasonication, and centrifuged to collect the supernatant. The VHH-V14 and VHH-V29 proteins in the supernatant were identified by SDS-PAGE ( Figure 4 ), the results showed that VHH-V14 and VHH-V29 proteins were well expressed. Subsequently, large-scale prokaryotic expression of VHH-V14 and VHH-V29 proteins was induced.

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

[0082] Purify the supernatant of the previous large-scale prokaryotic expression of VHH-V14 and VHH-V29 proteins according to the instructions of the His tag purification medium, and perform SDS-PAGE identification on the flow-through, equilibrium solution and eluate collected during the purification process ( Figure 5 ), the results showed that the purification effect of VHH-V14 and VHH-V29 proteins was good; the eluate identified as positive was centrifuged using an ultrafiltration tube (3kDa), and the replacement solution was PBS. The concentrations of VHH-V14 and VHH-V29 proteins were determined to be 1.03 mg / mL and 1.23 mg / mL, respectively. Western-Blot identification was performed on them ( Figure 6 ), the results showed that both VHH-V14 and VHH-V29 could specifically bind to FCV-VP1, and relatively pure prokaryotic expression VHH-V14 and VHH-V29 proteins were obtained.

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

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

[0085] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

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

2.

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

4. The use according to claim 3, characterized in that The product is a test kit.

5. A kit for detecting feline calicivirus, characterized in that It comprises the feline calicivirus nanobody VHH-V14 according to claim 1 or the feline calicivirus nanobody VHH-V29 according to claim 2.

6. A method for detecting feline calicivirus for non-disease diagnosis or treatment purposes, characterized in that The method comprises the step of using the kit according to claim 5 to perform detection.

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

8. A drug for preventing and / or treating feline calicivirus infection, characterized in that The feline calicivirus nanobody VHH-V14 according to claim 1 and / or the feline calicivirus nanobody VHH-V29 according to claim 2 are used as main active ingredients.

9. The drug according to claim 8, wherein Pharmaceutically acceptable excipients are also included.

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

Images