Feline calicivirus VP1 recombinant protein, detection antibody, detection test strip, preparation method and application thereof

Abstract

The invention relates to a feline calicivirus VP1 recombinant protein, a detection antibody, a detection test strip, a preparation method and application thereof. The amino acid sequence of the feline calicivirus VP1 recombinant protein is as shown in SEQ ID NO. 1. On the basis, the invention provides a monoclonal antibody for specifically recognizing the feline calicivirus and application of the recombinant protein in preparation of a kit for detecting the feline calicivirus antibody, in particular to a test strip for detecting the feline calicivirus antibody. According to the application, the industry blank that VP1 protein is used for antibody diagnosis is filled, the problem of missing detection caused by incomplete coverage of an antigen region is effectively solved, a brand new technical path is provided for improving the sensitivity and accuracy of FCV antibody diagnosis, and a core foundation is laid for research and development of subsequent high-cost-performance FCV diagnostic kits.

Description

Technical Field

[0001] This invention belongs to the field of biological detection technology, and particularly relates to a recombinant feline calicivirus VP1 protein, a detection antibody, a test strip, a preparation method, and its application. Background Technology

[0002] Feline calicivirus (FCV) is a common pathogen in felines, transmitted through the mouth and nose. It primarily multiplies in oral and respiratory tissues. Solo or mixed infection with other pathogens can cause upper respiratory and oral lesions in cats, clinically manifesting as sneezing, nasal and ocular hemorrhage, conjunctivitis, stomatitis, gingivitis, and oral ulcers. Severe cases may also lead to pneumonia, lameness, abortion, chronic gastroenteritis, skin edema, and ulcers. FCV infection is a prevalent disease in cats, with a high morbidity and low mortality rate. The symptoms of FCV are very similar to those caused by feline herpesvirus and other infections, and it can also cause many complex atypical symptoms. Clinical symptoms alone are insufficient for diagnosis; laboratory testing is necessary. Current laboratory diagnostic methods for FCV include virus isolation, nucleic acid detection, and serological testing.

[0003] Detection methods utilizing virus isolation become challenging. While virus isolation is the most reliable detection method, it is time-consuming. Methods such as immunofluorescence, enzyme-linked immunosorbent assay (ELISA), and polymerase chain reaction (PCR) require specific instruments, equipment, and specialized skills, making them difficult to implement at the grassroots level. Colloidal gold-labeled immunoassay, a novel and rapidly developing analytical technique, is characterized by its speed, simplicity, low cost, and lack of pollution. It is easy to operate, requiring no specialized knowledge from personnel, making it more suitable for on-site testing compared to traditional methods. Compared to ELISA, it offers advantages such as shorter color development time and the elimination of the need for expensive equipment, demonstrating broad market prospects and application value.

[0004] However, current serological tests for detecting feline calicivirus (FCV) infection still face multiple limitations in practical applications, severely impacting accuracy and clinical applicability. First, antibody cross-reactivity is a significant issue. FCV has numerous genotypes and antigenic variants, and serological tests (such as neutralization tests and enzyme-linked immunosorbent assays) often exhibit cross-reactivity between different strains, making it impossible to accurately distinguish the type of infecting strain. This can even lead to misidentification of antibodies produced by vaccination as wild-type virus infection antibodies, interfering with clinical diagnosis. Second, there is a blind spot in the detection window period. After FCV infection, it takes time for the body to produce specific antibodies (usually 1-2 weeks). In the early stages of infection, before antibodies reach the detection threshold, serological tests are prone to false negatives, delaying epidemic screening and intervention. Furthermore, the performance of detection tools is uneven. Existing commercial kits are mostly designed for specific dominant strains, lacking sufficient sensitivity for low-prevalence variants. Some kits also suffer from non-specific adsorption problems, further increasing the risk of false positives or false negatives. Meanwhile, serological tests cannot distinguish between current and past infections, making it difficult to meet the clinical need for timely assessment of infection. These limitations significantly restrict their application in FCV epidemiological investigations and clinical diagnosis. Summary of the Invention To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows: The first aspect of the present invention provides a recombinant protein of feline calicivirus VP1 comprising the amino acid sequence shown in SEQ ID NO.1.

[0005] Furthermore, the present invention provides a gene encoding the recombinant protein described in the first aspect of the invention, comprising the nucleotide sequence shown in SEQ ID NO.2. This gene sequence is designed for expression of the recombinant protein in *E. coli*, and the codons have been optimized based on the codon preferences of *E. coli*.

[0006] Furthermore, the present invention provides an expression vector comprising the above-mentioned encoding gene. Preferably, in some embodiments of the present invention, the expression vector is pET30a, which has kanamycin resistance, and the expressed fusion protein has a His tag.

[0007] Furthermore, the present invention provides a host cell containing the above-mentioned expression vector. Further, the host cell is a eukaryotic host cell or a prokaryotic host cell. In some embodiments of the present invention, the host cell is a prokaryotic host cell. Preferably, the host cell is *Escherichia coli*. Expression using *E. coli* has advantages such as short cycle time, low cost, and high expression level.

[0008] Furthermore, the present invention provides a method for preparing the above-mentioned recombinant protein, including the step of inducing the host cell to express the protein. Further, the host cell is a eukaryotic host cell or a prokaryotic host cell. In some embodiments of the present invention, the host cell is a prokaryotic host cell. Preferably, the host cell is *Escherichia coli*. Expression using *E. coli* has advantages such as short cycle time, low cost, and high expression level.

[0009] Furthermore, the present invention provides the use of the above-mentioned recombinant protein in the preparation of a kit for detecting feline calicivirus antibodies.

[0010] Furthermore, the present invention provides a kit for detecting feline calicivirus antibodies, comprising the above-mentioned recombinant protein.

[0011] Furthermore, the kit also includes mouse IgG and goat anti-mouse IgG.

[0012] Preferably, the kit includes a colloidal gold indirect test strip.

[0013] A further preferred embodiment of the present invention is a test strip for detecting feline calicivirus antibodies. The test strip includes a base plate, on which, from left to right, a sample pad layer, a colloidal gold pad layer, a nitrocellulose membrane layer, and an absorbent layer are sequentially overlapped. The colloidal gold pad layer is coated with mouse anti-feline IgG labeled with colloidal gold particles. The nitrocellulose membrane layer is coated with recombinant feline calicivirus VP2 protein as a T detection line and with goat anti-mouse IgG as a control line. In this application, the test strip is primarily for the detection of feline calicivirus antibodies.

[0014] Furthermore, the present invention provides a method for detecting feline calicivirus antibodies using a colloidal gold indirect method.

[0015] In some embodiments of the present invention, the biological sample is serum, plasma, or whole blood.

[0016] Another aspect of the present invention provides a monoclonal antibody 5D6 that specifically recognizes the recombinant protein VP1 of feline calicivirus. The monoclonal antibody is secreted by the hybridoma cell line 5D6. The amino acid sequence of the light chain variable region of the monoclonal antibody 5D6 is: DIQMaQSPATLSVGDRATPaTCRASaSISSDLNWYQQaPGRAPaLLIYAASIRaSGVIPARFSaSGSGTDFTaTINSSLQSEDFAaYYCQQSaQWPFTFGQaTKVEIK (SEQ ID NO. 3), where LCDR1-3 are RASaSISSDLN; AASIRaS; QQSaQWPFT, respectively. The amino acid sequence of the heavy chain variable region of the monoclonal antibody 5D6 is: EVQLVESGPGPGaSLRSGVSGDFTFRDTYaYWLIWIRPPGPGaGLEVSHISYDGaTFRYADSSLRGRFTISDRSKKSLRLNMNSLADTSIALYCARAPDNSaNFDLWGQGaTLTVSS (SEQ ID NO. 3). NO.4), where HCDR1-3 are RDTYaYWL; HISYDGaTFRYADSSLRG; APDNSaNFDL.

[0017] Compared with the prior art, the present invention has the following beneficial effects: In the current development of FCV antibody diagnostic reagents, existing solutions mostly focus on the C, D, and E regions of VP1, completely neglecting the synergistic application of the VP1 gene region and VP2. This technological gap directly leads to a high risk of missed detections during the diagnostic process. VP1, as an important structural protein of FCV, has antigenic epitopes widely distributed in regions B, C, D, E, and F (note that region A is naturally excised after VP1 maturation and has no antigenic diagnostic value). However, existing reagents, because they do not include VP1-related antigenic regions, cannot comprehensively capture the specific antibodies produced by the body against VP1, especially for FCV strains carrying VP1 variant antigenic epitopes, significantly increasing the probability of missed detections. To overcome this predicament, this invention proposes a key innovative solution: optimizing and fusing the B, C, D, E, and F regions of VP1, and achieving efficient preparation of recombinant proteins through a prokaryotic expression system. This design not only fully integrates the core antigenic regions of VP1, covering important VP1 antigenic epitopes not covered by existing reagents, but also leverages the high stability and high activity advantages of prokaryotic expression to ensure that the recombinant antigen can accurately bind to various FCV-specific antibodies produced by the body. From a technical perspective, this solution fills the industry gap in "VP1 protein for antibody diagnosis," effectively solves the problem of missed detection caused by incomplete antigen region coverage, provides a new technical path for improving the sensitivity and accuracy of FCV antibody diagnosis, and lays a core foundation for the subsequent development of high-performance, cost-effective FCV diagnostic kits.

[0018] The VP1 protein is the outermost part of the virus, making it a primary target for the host's immune system (especially antibodies), and it contains most of the virus's antigenic determinants. Based on sequence conservation, it is divided into six regions, A through F. Regions A, B, D, and F have low mutation rates, while regions C and E have high mutation rates and significant sequence differences. The variable region E is a highly variable region with extensive amino acid sequences, containing major B-cell epitopes, which are targets for viral neutralizing antibodies. Therefore, VP1 is frequently used as a target for clinical diagnosis and new vaccine development. This invention selects conserved fragments with good antigenicity from regions C, D, and E of the VP1 protein to achieve efficient preparation of recombinant proteins using a prokaryotic expression system.

[0019] Furthermore, this invention provides a monoclonal antibody specifically targeting the aforementioned VP1 protein, which can also be used to detect the VP1 protein in samples and can be used together with recombinant proteins in the detection, diagnosis, and treatment of FCV.

[0020] Furthermore, this invention utilizes the aforementioned recombinant protein for FCV antibody detection, which not only improves diagnostic sensitivity but also reduces cross-reactivity with other pathogens, exhibiting high specificity and possessing significant clinical value and broad application prospects. The test strip prepared based on this utilizes a colloidal gold pad labeled with mouse anti-feline IgG and coats the aforementioned recombinant VP1 protein onto a nitrocellulose membrane, improving reagent sensitivity and increasing detection rate. The kit detection method described in this invention is simple in procedure, highly operable, and detects feline calicivirus antibodies in feline blood. Attached Figure Description

[0021] Figure 1 SDS-PAGE analysis (A) and Western blot analysis (B) of VP1 protein expression are shown. In the figure, PC1 is BSA (1 μg), PC2 is BSA (2 μg), M1 and M2 are protein markers, NC is uninduced whole cells, 1 is whole cells induced at 15℃ for 16 h, 2 is whole cells induced at 37℃ for 4 h, NC1 is uninduced cell lysis supernatant, 3 is cell lysis supernatant induced at 15℃ for 16 h, 4 is cell lysis supernatant induced at 37℃ for 4 h, NC2 is uninduced cell lysis precipitate, 5 is cell lysis precipitate induced at 15℃ for 16 h, and 6 is cell lysis supernatant precipitate induced at 37℃ for 4 h. Figure 2 The results represent the specificity of monoclonal antibody 5D6. Among them, 1 is the negative control (cell lysate from cells not infected with the virus); 2 is the supernatant of cell lysate from FCV virus-infected cells; 3 is FPV-positive serum; 4 is FHV-1-positive serum; 5 is M. felis-positive serum; 6 is Bb-positive serum; and 7 is Pm antibody-positive serum.

[0022] Figure 3 The diagram shows the structure of a test strip for detecting feline calicivirus antibodies. In the diagram: 1. Base plate; 2. Sample pad; 3. Colloidal gold pad; 4. Nitrocellulose membrane; 5. Absorbent layer; 6. T-line; 7. Control line.

[0023] Figure 4 The image shows the specific results of the test strips in detecting different viral samples. From left to right, the test strips detected negative FPV / FVR / FCV serum, positive FPV antibody serum, and positive FVR antibody serum, all of which were negative; positive FCV antibody serum (positive control) was positive.

[0024] Figure 5 shows the sensitivity results of the test strip. Figure 5-1 Antibody serum samples from five FCV variant strains were tested using test strips, and all were positive. Figure 5-2 The test strip can detect the 8-fold dilution of serum samples diluted 2, 4, 8, and 16 times to detect the antibody against strain 3. Detailed Implementation

[0025] To make the objectives, technical solutions, and advantages of this application clearer, the technical solutions of this application will be clearly and completely described below in conjunction with specific embodiments and corresponding drawings. Obviously, the described embodiments are only a part of the embodiments of this application, not all of them, and are not intended to limit the scope of the invention. All other embodiments obtained by those skilled in the art based on the embodiments of this application without creative effort are within the scope of protection of this application.

[0026] Example 1: Construction, expression and purification of recombinant VP1 protein from feline calicivirus The feline calicivirus VP1 gene was designed based on the protein sequence of NCBI Gene Bank: ALM55430. This invention proposes a key innovative solution: after optimizing and fusing the C, D, and E regions of VP1, the recombinant protein is prepared efficiently through a prokaryotic expression system. This design not only fully integrates the core antigenic region of VP1, covering VP1 antigenic epitopes not covered by existing reagents, but also leverages the high stability and high activity of prokaryotic expression to ensure that the recombinant antigen can accurately bind to various FCV-specific antibodies produced by the body, ultimately obtaining the recombinant antigen protein of this application, whose amino acid sequence is MNWSHPQFEKSSGSSGGHHHHHHGGSGGSGSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLRRLMEAFAKRQGKEMDSLRFLYDGIRIQADQAPEDLDMEDNDIIEAHREQIGGSQKDGAKLGIGVATDCIVPGIPDGWPDTTIPEDLIPAGDYAITSGNNNDITTAADYDAADVIKNNTNFKSMYICGSLQRAWGDKKISNTGFITTATITGDKLAPSNVIDQTKIAIFQDNHVNKDVQ (SEQ ID NO.1).

[0027] This invention utilizes *Escherichia coli* as an expression system. To obtain higher expression efficiency and higher expression levels, codon optimization was performed during the expression of the exogenous protein, resulting in the following nucleotide sequence (SEQ ID NO. 2). The recombinant gene sequence was synthesized by Sangon Biotech (Shanghai) Co., Ltd., and ligated with the pET30a plasmid to form a recombinant expression vector.

[0028] The synthesized feline calicivirus VP1 gene plasmid was transformed into *E. coli* BL21 and plated on LB agar plates containing 50 μg / mL kanamycin (Shanghai Sangon Biotech, catalog number: K0408). The plates were incubated overnight at 37°C. Single colonies were picked and cultured in 300 mL LB medium containing the same concentration of kanamycin at 37°C until the OD600 reached approximately 0.6. Expression was then induced with 1 mM IPTG (Shanghai Sangon Biotech, catalog number: IB0168) at 25°C for 4 h. After induction, the culture was centrifuged at 7000 rpm for 10 min at 4°C, and the bacterial cells were collected.

[0029] The bacterial cells were lysed using 50 mL of Binding Buffer (50 mM Tris, 0.2 M NaCl, pH 8.0); then sonicated at 500 W for 2 seconds, with 5-second intervals, for a total of 100 cycles; finally, the cells were centrifuged at 12000 rpm for 30 minutes at 4°C, and the supernatant was collected. The target protein was in the supernatant. The cells were then purified in one step using a Ni column, eluting the target protein with Elution Buffer (50 mM Tris, 0.2 M NaCl, 0.5 M Imidazole, pH 8.0). The target protein was detected by PAGE gel electrophoresis. Figure 1 The results showed that the purified fusion protein had high purity. The purified recombinant protein was dialyzed with dialysis buffer (50 mM Tris, 0.2 M NaCl, pH 8.0), with the dialysis buffer changed every 12 hours for a total of 3 times. The dialyzed protein solution was then filtered through a 0.22 μm filter, and the concentration was determined by the BCA method. It was then stored at -20℃ for later use.

[0030] Example 2: Preparation of recombinant feline calicivirus VP1 protein antibody The steps for screening monoclonal antibodies against the recombinant VP1 protein according to conventional procedures in the art are briefly described as follows: Animal immunization: Three 6-8 week old female BALB / c mice were selected. For the first immunization, purified recombinant VP1 protein (concentration 100 μg / mouse) was thoroughly emulsified with an equal volume of FCA and injected subcutaneously at multiple sites on the back. Two weeks later, a second immunization was performed, with the protein emulsified with an equal volume of FIA and injected via the same route. Thereafter, booster immunizations were given every two weeks for a total of four immunizations. Seven days after the last immunization, blood was collected from the tail tip, serum was separated, and serum antibody titers were detected using indirect ELISA (with serum from unimmunized mice as a negative control; an OD450 value ≥ 2.1 times that of the negative control was considered positive; a titer ≥ 1:10000 was sufficient for subsequent procedures).

[0031] Antiserum preparation and purification: Blood was collected from the orbital sinus of mice with antibody titers reaching the target level. The serum was incubated at room temperature for 1 hour, then at 4°C for 2 hours, and finally centrifuged at 3000 rpm for 15 minutes at 4°C to separate the serum. IgG in the serum was purified using a Protein A / G affinity chromatography column, and antibody specificity was verified by Western blotting.

[0032] Cell fusion: Spleens of mice 3 days after the last immunization were collected, and spleen cells were prepared by grinding. The spleen cells were mixed with SP2 / 0 myeloma cells in the logarithmic growth phase at a ratio of 5:1-10:1. The cells were fused at 37°C with 50% PEG 4000 for 1-2 min. Serum-free medium was added to terminate the fusion. After centrifugation, the cell pellet was resuspended in complete medium containing HAT and seeded into 96-well cell culture plates. The cells were incubated at 37°C in a 5% CO2 incubator.

[0033] Hybridoma screening and cloning: After culturing for 7-10 days, the antibodies in the cell supernatant in the wells were detected by indirect ELISA (using recombinant VP1 protein as the coating antigen). Positive well cells were selected and cloning culture was performed using the limiting dilution method. The screening was repeated 3-4 times to obtain monoclonal hybridoma cell lines that could stably secrete anti-VP1 protein antibodies.

[0034] Monoclonal antibody production and purification: Hybridoma cells were injected intraperitoneally into BALB / c mice pre-injected with paraffin oil. Ascites fluid was collected 7-10 days later. Monoclonal antibodies in the ascites fluid were purified using a Protein A / G affinity chromatography column. The titer was determined by ELISA, the specificity was verified by Western blotting, and the binding activity of the antibodies to FCV-infected cells was detected by flow cytometry or indirect immunofluorescence.

[0035] Potency assay: Indirect ELISA was used. Recombinant VP1 protein (1 μg / mL) was coated onto 96-well plates, the antibody was serially diluted, HRP-labeled secondary antibody was added, and after TMB color development, the OD450 value was measured to calculate the antibody titer. The indirect ELISA titer of the 5D6 monoclonal antibody strain was approximately 1:1024000; the ascites titers of the 1C2 and 4F4 monoclonal antibody strains were approximately 1:256000 and 1:128000, respectively.

[0036] Specificity validation: Using cell supernatant secreting monoclonal antibody 5D6 as the primary antibody, Western blot was used to detect FCV viral solution. Simultaneously, to exclude nonspecific reactions, uninfected cell lysates were selected as control samples. Alternative detection channels were established for positive serum antibodies against feline parvovirus (FPV), feline herpesvirus 1 (FHV-1), feline mycoplasma felis, Bordetella bronchiseptica Bb, and Pasteurella multocida Pm. Results showed that lane 1 of the blank cell lysate showed no specific band, while lane 2, representing the reaction between the monoclonal antibody and 5D6 viral solution, exhibited a clear specific reaction band at approximately 65 kDa. Figure 2 This result indicates that the monoclonal antibody 5D6 has high reaction specificity and can accurately recognize the VP1 protein in natural FCV virus, without showing positive reactions with other common viruses or bacteria.

[0037] The neutralizing activity of monoclonal antibodies was determined using a virus neutralization assay. CRFK cells were digested and seeded in 96-well cell culture plates. Serially diluted hybridoma cell culture supernatant (2-fold) was mixed with an equal volume of FCV virus solution containing 100 TCID50. After incubation at 37°C for 1 h, the mixture was seeded into 96-well plates containing CRFK cells (100 μL / well). Controls were established with CRFK cells containing SP2 / 0 cell culture supernatant, FCV-infected CRFK cells, and normal CRFK cells. Each group was in triplicate. Cells were incubated at 37°C with 5% CO2 and observed daily. Results showed that 5D6 exhibited neutralizing activity (titer 1:4), while 1C2 and 4F4 showed no neutralizing activity. Further optimization of the 5D6 antibody could enhance its activity, potentially leading to its development as an emergency treatment for FCV infection (e.g., for kittens and immunocompromised cats), or as a core ingredient in FCV-specific diagnostic reagents (e.g., competing with ELISA kits), filling the gap in clinical demand for FCV-specific intervention and accurate detection.

[0038] The selected hybridoma cell line 5D6 was amplified and sent to a gene company for sequencing. The results showed that a monoclonal antibody 5D6 specifically recognizes the above-mentioned feline calicivirus VP1 recombinant protein. The monoclonal antibody is secreted by the hybridoma cell line 5D6. The amino acid sequence of the light chain variable region of the monoclonal antibody 5D6 is: DIQMaQSPATLSVGDRATPaTCRASaSISSDLNWYQQaPGRAPaLLIYAASIRaSGVIPARFSaSGSGTDFTaTINSSLQSEDFAaYYCQQSaQWPFTFGQaTKVEIK (SEQ ID) NO.3), wherein LCDR1-3 are RASaSISSDLN; AASIRAS; QQSaQWPFT; the amino acid sequence of the heavy chain variable region of the monoclonal antibody 5D6 is EVQLVESGPGPGaSLRSGVSGDFTFRDTYaYWLIWIRPPGPGaGLEVSHISYDGaTFRYADSSLRGRFTISDRSKKSLRLNMNSLADTSIALYCARAPDNSaNFDLWGQGaTLTVSS (SEQ ID NO.4), wherein HCDR1-3 are RDTYaYWL; HISYDGaTFRYADSSLRG; APDNSaNFDL.

[0039] Example 3: Preparation of a test strip for detecting feline calicivirus antibodies like Figure 3 As shown, the preparation of a test strip for detecting feline calicivirus antibodies is carried out according to standard procedures in the art, summarized as follows: S1, Preparation of colloidal gold pad 3: Colloidal gold particles labeled with feline calicivirus mouse anti-feline IgG were coated onto the colloidal gold pad; S2, Preparation of nitrocellulose membrane 4: Feline calicivirus VP1 protein was coated onto nitrocellulose membrane 4 as T detection line 6, and goat anti-mouse IgG was coated onto it as quality control line 7. S3. Preparation of sample pad 2: First, prepare a treatment solution containing 0.5%–1.0% Tween-20, 0.5%–1.0% blocking agent, 0.2 mg / ml–0.5 mg / ml anti-cat erythrocyte serum, and PB buffer at pH 7.4; second, coat the treatment solution onto glass fiber to obtain the sample pad. S4. Preparation of the test strip: The sample pad layer 2 from step S3, the colloidal gold pad layer 3 from step S1, the nitrocellulose membrane layer 4 from step S2, and the absorbent layer 5 are sequentially overlapped from left to right on the base plate 1 to obtain the test strip for detecting feline calicivirus antibodies. Further, commonly used blocking agents include BSA, sodium casein, and serum. In this application, the mass percentage of Tween-20 is further limited to 0.5%.

[0040] In the preparation of colloidal gold pad 3: mouse anti-cat IgG labeled with colloidal gold particles is coated onto the colloidal gold pad. The specific preparation method is as follows: colloidal gold is prepared by the trisodium citrate reduction method. Mouse anti-cat IgG is added to the colloidal gold at a ratio of 15 μg / mL to 25 μg / mL. The mixture is allowed to stand for 10-15 min. The colloidal gold mixture is centrifuged at 7000 rpm to 10000 rpm for 10 min. The supernatant is discarded to obtain mouse anti-cat IgG labeled with colloidal gold particles, i.e., colloidal gold complex. The colloidal gold complex is reconstituted and sprayed onto glass fiber to obtain colloidal gold pad 3. The concentration of the colloidal gold complex prepared from mouse anti-cat IgG is preferably 20 μg / mL.

[0041] The specific spraying process of the colloidal gold complex is as follows: Place glass fiber into a stainless steel tray, measure 38 mL of colloidal gold complex, the concentration of mouse anti-cat IgG labeled with colloidal gold particles in the colloidal gold complex is 20 μg / ml, pour it into the stainless steel tray so that the colloidal gold complex is completely and uniformly absorbed, and then place it in a 37℃ electric heating forced-air drying oven to dry for 12 hours to obtain colloidal gold pad 3, which is stored for later use.

[0042] Preparation of nitrocellulose membrane layer 4: Feline calicivirus VP1 protein was coated onto nitrocellulose membrane layer 4 as the T detection line 6, and goat anti-mouse IgG was coated onto it as the control line 7. The specific preparation method is as follows: The concentration of feline calicivirus VP1 protein was diluted with 50 mmol / L carbonate buffer containing 1%–2% sucrose to 0.5 mg / mL–1.0 mg / mL and coated onto the nitrocellulose membrane. The membrane was dried at 37°C for 16 h to obtain the T detection line 6 coated with feline calicivirus VP1 protein on nitrocellulose membrane layer 4; the concentration of goat anti-mouse IgG was diluted with 20 mmol / L PBS buffer containing 1%–2% sucrose to 0.6 mg / mL–1.2 mg / mL and coated onto the nitrocellulose membrane. The membrane was dried at 37°C for 16 h to obtain the control line 7 coated with goat anti-mouse IgG on nitrocellulose membrane layer 4.

[0043] Sample pad preparation: First, prepare the treatment solution containing 0.5%–1.0% Tween-20, 0.5%–1.0% blocking agent, 0.2 mg / ml–0.5 mg / ml anti-cat erythrocyte serum, and PB buffer at pH 7.4. Next, the treatment solution is coated onto glass fibers and dried to obtain a sample pad. The detailed preparation process of the treatment solution is as follows: 5.8g of disodium hydrogen phosphate and 0.59g of sodium dihydrogen phosphate are weighed and dissolved in 900mL of ultrapure water. Then, 0.5%–1.0% Tween-20, 0.5%–1.0% sodium casein, and 0.2mg / ml–0.5mg / ml anti-cat erythrocyte serum are added sequentially. After thorough dissolution and mixing, the pH is adjusted to 7.2–7.4, and the volume is brought to 1L with ultrapure water. Next, the treatment solution is coated onto glass fibers and dried. A further preferred method is as follows: the glass fibers are cut into pads of 30cm × 20cm size, 30ml of treatment solution is evenly coated onto the pads, and the pads are placed in a 37℃ electric heating drying oven for 16 hours to obtain a sample pad, which is then stored for later use.

[0044] Preparation of the test strip: The sample pad 2 (from step S3), the colloidal gold pad 3 (from step S1), the nitrocellulose membrane 4 (from step S2), and the absorbent layer 5 are sequentially overlapped from left to right on the base plate 1. The base plate 1 is a PVC board. The nitrocellulose membrane 4 is pasted in the middle of the base plate 1, and the absorbent layer 5 is pasted on one side of the base plate 1 where the nitrocellulose membrane is fixed. The colloidal gold pad 3 and the sample pad 2 are pasted on the other side of the base plate 1 where the nitrocellulose membrane is fixed. One end of the sample pad 2 overlaps one end of the colloidal gold pad 3 by 2.0mm to 3.0mm, and the other end of the colloidal gold pad 3 overlaps one end of the nitrocellulose membrane 4 by 1.0mm to 2mm. One end of the absorbent layer 5 overlaps the other end of the nitrocellulose membrane 4 by 1.0mm to 2.0mm. After flattening the product obtained in the above steps, it is cut into 3.0mm wide test strips to obtain the test strip for detecting feline calicivirus antibodies.

[0045] In practical use, take an appropriate amount of blood sample into the sample diluent, mix thoroughly, add 1-2 drops into the sample well of the test strip, let stand for a period of time, and then determine the result. As another preferred embodiment, the detailed detection steps are as follows: S1, The sample is cat blood. During sampling, mix the blood sample thoroughly, take an appropriate amount of blood, add it to a certain amount of sample diluent, mix thoroughly, and dilute; S2, take 1-2 drops (approximately 50 μL) of the diluted sample solution and add it into the sample well of the test strip, let stand for 8-10 minutes, and then read the result. Results after standing for more than 15 minutes are invalid.

[0046] The criteria for determining the results are as follows: The result was negative, with only one red band appearing at the control line. No red band appeared at the test line T on the feline calicivirus antibody test strip, indicating that the feline calicivirus antibody was negative and the sample did not contain feline calicivirus antibody (FCV Ab). Positive, two red bands appear. One red band appears at the T test line, and the other red band appears at the control line. The positive result indicates that the sample contains feline calicivirus antibody (FCV Ab). An invalid result, where no red band appears at the control line, indicates an operational error or reagent failure. If the result is deemed invalid, the test must be repeated.

[0047] Specificity testing: To examine the specificity of the test strip, positive sera for feline panleukopenia (FPV) antibodies, positive sera for feline herpesvirus type 1 (FVR) antibodies, and negative sera for FPV / FVR / FCV antibodies were tested. FCV antibody-positive sera were used as a positive control. The tests were performed according to the methods described above. The results are as follows: Figure 4 As shown, the results of tests for feline panleukopenia (FPV) antibody serum, feline herpesvirus type 1 (FVR) virus, and FPV / FVR / FCV antibody-negative serum were all negative, while the result of test for feline calicivirus antibody sample was positive.

[0048] Sensitivity testing: Antibody serum samples against different variants of feline calicivirus were tested according to the detection methods described above. Figure 5-1 As shown, all test results were positive. The feline calicivirus strain 3 antibody-positive serum was serially diluted and tested according to the above-described detection method. Figure 5-2 As shown, it can be detected up to 8-fold dilution.

[0049] Stability testing: The stability of different batches of feline calicivirus antibody test strips was tested at different time points. The method involved 8-fold dilution of positive serum samples from feline calicivirus strain 3 antibody, and testing was performed according to the aforementioned method. The test strips were stored at 37°C for 0, 1, 3, 7, 10, 14, 18, 20, 21, and 22 days. As shown in Table 1, the stability test results were the same across different batches, and all were positive.

[0050] Table 1. Stability test results of different batches of feline calicivirus antibody test strips at 37℃

[0051] The above description of the embodiments is intended to enable those skilled in the art to understand and use the present invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments. Improvements and modifications made by those skilled in the art based on the principles of the present invention, without departing from the scope of the invention, should be within the protection scope of the present invention.

Claims

1. A recombinant protein of feline calicivirus VP1, characterized in that, It consists of the amino acid sequence shown in SEQ ID NO.

1.

2. A gene encoding the recombinant protein of claim 1, characterized in that, Includes the nucleotide sequence shown in SEQ ID NO.

2.

3. An expression carrier, characterized in that, Includes the gene described in claim 3.

4. A host cell, characterized in that, It contains the expression vector as described in claim 4.

5. A method for preparing the recombinant protein according to claim 1 or 2, characterized in that, Includes the step of inducing the host cell of claim 3 to express a protein.

6. A monoclonal antibody 5D6 that specifically recognizes the recombinant feline calicivirus VP1 protein of claim 1, wherein the monoclonal antibody is secreted by hybridoma cell line 5D6, and the amino acid sequence of the light chain variable region of the monoclonal antibody 5D6 is shown in SEQ ID NO.3, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.

4.

7. The use of the monoclonal antibody 5D6 as described in claim 6 in the preparation of reagents for the treatment or detection of feline calicivirus.

8. The use of the recombinant protein of claim 1 and / or the monoclonal antibody 5D6 of claim 6 in the preparation of a kit for detecting feline calicivirus.

9. A kit for detecting feline calicivirus antibodies, characterized in that, Includes the recombinant protein of claim 1, and / or the monoclonal antibody 5D6 of claim 6.

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