A feline measles virus-specific monoclonal antibody and its application

Abstract

This invention discloses a feline measles virus-specific monoclonal antibody and its applications. The monoclonal antibody is prepared by immunization with the feline measles virus N protein. The monoclonal antibody specifically recognizes the feline measles virus N protein, which includes the feline measles virus N protein-N fragment with the nucleic acid sequence shown in SEQ ID NO:1 and the feline measles virus N protein-C fragment with the nucleic acid sequence shown in SEQ ID NO:2. The monoclonal antibody provided by this invention can specifically recognize the feline measles virus N protein, exhibiting high titer, good affinity, and high sensitivity. Detection kits prepared based on this antibody can effectively detect feline measles virus in feline blood, urine, and other bodily fluids, providing technical support for the epidemiological monitoring and diagnosis of feline measles virus infection.

Description

Technical Field

[0001] This invention belongs to the field of biological detection technology, specifically relating to a feline measles virus-specific monoclonal antibody and its application. Background Technology

[0002] Feline morbillivirus (FeMV) is a single-stranded, negative-sense RNA virus belonging to the genus Measlesvirus in the family Paramyxoviridae. The N protein is the nucleocapsid protein of feline morbillivirus. It has a nucleotide length of 1560 bp, consists of 519 amino acids, and has a molecular weight of 57 kDa. The primary host for feline morbillivirus transmission is the cat. The main clinical symptoms in infected cats are related to kidney disease. The mechanism by which this virus causes kidney disease is not yet fully understood. Infected cats have a high infection and mortality rate.

[0003] Most members of the measlesvirus genus are important pathogens in humans and animals. Measles viruses are highly pathogenic and can cause death in both animals and humans, making surveillance of feline measles virus (FMV) essential. Currently, most researchers focus on the L gene of FMV and use RT-PCR for detection. However, this method has drawbacks such as demanding operating conditions, long processing times, and a high risk of false positives, making it unsuitable for routine surveillance environments. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides a feline measles virus-specific monoclonal antibody and its applications.

[0005] To solve the above-mentioned technical problems, the present invention provides the following technical solution:

[0006] The first objective of this invention is to provide a feline measles virus-specific monoclonal antibody, which is prepared by immunization with feline measles virus N protein. The monoclonal antibody specifically recognizes feline measles virus N protein, and the recognized feline measles virus N protein includes the feline measles virus N protein-N fragment with a nucleic acid sequence as shown in SEQ ID NO:1 and the feline measles virus N protein-C fragment with a nucleic acid sequence as shown in SEQ ID NO:2.

[0007] Preferably, the monoclonal antibody contains a heavy chain variable region and a light chain variable region, both of which are composed of a determinant complementary region and a framework region; the determinant complementary regions of both the heavy chain variable region and the light chain variable region are composed of CDR1, CDR2 and CDR3.

[0008] The amino acid sequence of CDR1 in the light chain variable region is shown as positions 27-32 of SEQ ID No. 3;

[0009] The amino acid sequence of CDR2 in the light chain variable region is shown as positions 50-52 of SEQ ID No. 3;

[0010] The amino acid sequence of CDR3 in the light chain variable region is shown as positions 89-97 of SEQ ID No. 3;

[0011] The amino acid sequence of CDR1 in the heavy chain variable region is shown as positions 26-33 of SEQ ID No. 4;

[0012] The amino acid sequence of CDR2 in the heavy chain variable region is shown as positions 51-60 of SEQ ID No. 4;

[0013] The amino acid sequence of CDR3 in the heavy chain variable region is shown as positions 99-105 of SEQ ID No. 4.

[0014] Preferably, the amino acid sequence of the light chain variable region 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.

[0015] Preferably, the monoclonal antibody contains a heavy chain variable region and a light chain variable region, both of which are composed of a determinant complementary region and a framework region; the determinant complementary regions of both the heavy chain variable region and the light chain variable region are composed of CDR1, CDR2 and CDR3.

[0016] The amino acid sequence of CDR1 in the light chain variable region is shown as positions 27-37 of SEQ ID No. 5;

[0017] The amino acid sequence of CDR2 in the light chain variable region is shown as positions 55-57 of SEQ ID No. 5;

[0018] The amino acid sequence of CDR3 in the light chain variable region is shown as positions 94-102 of SEQ ID No. 5;

[0019] The amino acid sequence of CDR1 in the heavy chain variable region is shown as positions 26-33 of SEQ ID No. 6;

[0020] The amino acid sequence of CDR2 in the heavy chain variable region is shown as positions 51-58 of SEQ ID No. 6;

[0021] The amino acid sequence of CDR3 in the heavy chain variable region is shown as positions 97-109 of SEQ ID No. 6.

[0022] Preferably, the amino acid sequence of the light chain variable region is shown in SEQ ID No. 5, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID No. 6.

[0023] A second objective of this invention is to provide a kit containing monoclonal antibodies.

[0024] Preferably, the kit contains immunomagnetic beads conjugated with capture antibodies, feline measles virus N protein calibrator, alkaline phosphatase-labeled detection antibody, and chemiluminescent substrate.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] The monoclonal antibody provided by this invention can specifically recognize the N protein of feline measles virus. It has high antibody titer, good affinity, and high sensitivity. The detection kit prepared based on this antibody can effectively detect feline measles virus in feline blood, urine and other bodily fluids, providing technical support for the epidemiological monitoring and diagnosis of feline measles virus infection. Attached Figure Description

[0027] Figure 1 This is a schematic diagram of the recognition of feline measles virus N protein by antibodies 3C8 and 4C6 in Embodiment 3 of the present invention.

[0028] Figure 2 This is a schematic diagram of SDS-PAGE analysis of antibodies 1G1, 1B5, 3C8 and 4C6 in Example 4 of the present invention.

[0029] Figure 3 This is the calibration curve of the feline measles virus detection kit in Embodiment 7 of the present invention. Detailed Implementation

[0030] The preferred embodiments of the present invention will be described below with reference to the accompanying drawings. It should be understood that the preferred embodiments described herein are for illustration and explanation only and are not intended to limit the present invention.

[0031] Example 1: Expression and purification of feline measles virus N protein.

[0032] Construction and expression of the truncated feline measles virus (FMR) N protein: Based on the amino acid sequence of the FMR N protein, N-terminal and C-terminal sequences were designed and ligated into the expression vector pET28a, which was then inserted into the expression strain BL21(DE3). Soluble expression of the truncated FMR N protein was induced overnight in LB medium at 37°C with 1 mmol / L IPTG. Prokaryotic recombinant expression was purified by 6×His tag affinity chromatography, and the purified proteins were subjected to SDS-PAGE electrophoresis, with purity exceeding 90%.

[0033] The amino acid sequence of the feline measles virus N protein-N fragment is shown in SEQ ID NO: 1. The amino acid sequence of the feline measles virus N protein-C fragment is shown in SEQ ID NO: 2.

[0034] Example 2: Preparation of hybridoma cell line for monoclonal antibody against feline measles virus N protein.

[0035] BALB / c mice were immunized with feline measles virus N protein. For the primary immunization, the antigen and Freund's complete adjuvant were mixed in an equal proportion and injected subcutaneously. Each mouse received 50 μg of the mixture, and a total of 5 mice were immunized. Fourteen days later, a second and third immunization were performed. The antigen and Freund's incomplete adjuvant were mixed in an equal proportion and injected subcutaneously. Each mouse received 50 μg of the mixture. Ten days after the third immunization, blood was collected from the tail vein, centrifuged, and the serum was serially diluted. The pre-immunization serum was used as a negative control. Serum titers were measured (see Table 1).

[0036] Table 1: Serum titer of mice receiving triple immunity.

[0037]

[0038] Mice with the highest serum titer (sample number 2) were boosted with immunization, and spleen cells were harvested under aseptic conditions 3 days later. Cell fusion experiments were performed on cells with good growth status and high serum titers. SP2 / 0 and spleen cells were mixed in centrifuge tubes according to the specified ratio. 1 ml of pre-warmed PEG was added to a 50 ml centrifuge tube and incubated for 1 min. Then, pre-warmed DMEM medium was added to a final volume of 35 mL. The mixture was incubated at 37°C for 15 min, followed by centrifugation at 800 rpm for 10 min, and the supernatant was discarded. 100 mL of pre-warmed HAT medium was added, and the mixture was gently pipetted to mix. The mixture was then transferred to 200 μL of feeder cells in 96-well plates and cultured in a CO2 incubator. Feline measles virus N protein was used to detect the cell supernatant. Positive hybridomas were screened for clones, yielding four hybridoma cell lines secreting specific monoclonal antibodies: 1G1, 1B5, 3C8, and 4C6. Antibody subtype detection results showed that 1G1 and 1B5 were IgG. 2b 3C8 and 4C6 are IgG1.

[0039] Example 3: Identification of monoclonal antibody epitopes for feline measles virus N protein.

[0040] Western blotting (WB) was used to confirm antibody recognition epitopes: 5 μg of each of the three proteins (N, C, and full-length) of feline measles virus (FMD) N protein were loaded and subjected to 15% SDS-PAGE electrophoresis. After electrophoresis, the proteins were transferred to 0.45 μm PVDF under constant voltage of 150 V. After transfer, the membrane was blocked at 37°C for two hours with PBST solution containing 5% BSA. The membrane was then incubated at 37°C for one hour with 1 μg / mL of 3C8 and 4C6 monoclonal antibody solutions, respectively. After incubation, 10 ng / mL of goat anti-mouse IgG-HRP was added and incubated for one hour. After washing, ECL chemiluminescence was applied for color development. The full-length FMD N protein served as a positive control. The appearance of a band at the N / C fragment of the FMD N protein indicated that the antibody recognized the target fragment. The results showed that the 3C8 antibody recognized the N fragment of the FMD N protein, and the 4C6 antibody recognized the C fragment of the FMD N protein. Figure 1 As shown.

[0041] Example 4: Preparation, purification, and titer detection of feline measles virus monoclonal antibody.

[0042] Liquid paraffin was injected into mice via intraperitoneal injection. After 7-10 days, the hybridoma cell line that could stably secrete antibodies was inoculated into the mice. Ascites fluid was collected after the mice's abdomens swelled.

[0043] The collected ascites fluid was centrifuged at 8000 rpm for 10 min, and the supernatant was collected. 10-20 volumes of Protein A equilibration buffer were added to the supernatant for Protein A affinity chromatography purification. The antibody was eluted with 0.1 M glycine-hydrochloric acid solution (pH 2.7) and neutralized with 1 M pH 9.0 Tris buffer. The purified antibody was dialyzed against 1×PBS, and the purity was determined by SDS-PAGE gel chromatography. The results showed that the heavy chains of all four antibody strains exhibited bands at 50 KD, and the light chains at 25 KD. Gray-scale analysis showed a purity greater than 90% for all strains. Figure 2 .

[0044] Feline measles virus N protein was diluted to 2 μg / ml with 1×CBS, coated overnight at 4°C, and the coating solution was discarded. The plate was then blocked with 10% skim milk powder at 37°C for 2 h. The purified antibodies were serially diluted, 100 μL per well, and incubated at 37°C for 1 h. After washing, goat anti-mouse IgG-HRP was added, and the plate was incubated at 37°C for 1 h. The liquid in the wells was discarded, and after washing, TMB was added for color development. After adding stop solution, the OD450 value was read. The titers of monoclonal antibodies 1G1, 1B5, 3C8, and 4C6 were all 5.12 × 10⁻⁶. 5 1.02×10 6 6.4×10 7 3.2×10 7 .

[0045] Example 5: Pairing verification of feline measles virus monoclonal antibody.

[0046] The purified monoclonal antibodies 1G1, 1B5, 3C8, and 4C6 were used as capture and detection antibodies, respectively, to verify antibody pairing. Microplates were coated overnight with 2 μg / ml of antibody. After washing and blocking, 100 μl of feline measles virus N protein calibrator at different concentrations (0-100 ng / ml) was added, and the plates were incubated at 37°C for 60 min. After washing, 100 μl of HRP-labeled 1G1, 1B5, 3C8, and 4C6 at a concentration of 0.1 μg / ml was added, and the plates were incubated at 37°C for 60 min. After washing, substrate was added, and the reaction was carried out for 15 min. The reaction was terminated with stop solution, and the OD450 absorbance was measured using a microplate reader. The results are shown in Table 2. Ultimately, 3C8 was selected as the capture antibody for the kit, and 4C6 was selected as the detection antibody for the detection of feline measles virus.

[0047] Table 2: Pairing verification of feline measles virus monoclonal antibodies.

[0048]

[0049] Example 6: Specificity analysis of monoclonal antibody against feline measles virus N protein.

[0050] Coat each well with 100 μl of 2 μg / ml feline measles virus (FeMV) N protein, Nipah virus (NiV) N protein, bovine respiratory syncytial virus (BRSV) N protein, and canine distemper virus (CDV) N protein, respectively, and incubate at 4°C overnight. After discarding the liquid in the wells, block with 10% skim milk powder at 37°C for 2 hours, then wash the plates. Serially dilute the test antibodies to 100 μl / well and incubate at 37°C for 1 hour. After washing, add 100 μl of diluted goat anti-mouse IgG-HRP to each well and incubate at 37°C for 1 hour, then wash again. Add 100 μl of TMB chromogenic buffer to each well and develop for 15 minutes, then add stop solution to terminate the reaction. Read the OD450 absorbance on a microplate reader. The results showed that feline measles virus monoclonal antibodies 3C8 and 4C6 could specifically recognize the feline measles virus (FeMV) N protein, but had no immune response to the Nipah virus (NiV), bovine respiratory syncytial virus (BRSV), or canine distemper virus (CDV) N proteins. The results are shown in Table 3.

[0051] Table 3: Specificity of monoclonal antibodies against feline measles virus N protein.

[0052]

[0053]

[0054] Example 7: Chemiluminescent detection kit for feline measles virus.

[0055] The feline measles virus chemiluminescence detection kit includes: immunomagnetic beads labeled with monoclonal antibody against feline measles virus N protein, monoclonal antibody against feline measles virus N protein labeled with alkaline phosphatase, and feline measles virus N protein calibrator. The kit components are shown in Table 4, and the standard curve is shown in Table 5. Figure 3 As shown.

[0056] Table 4: Chemiluminescent detection kit for feline measles virus.

[0057]

[0058] Example 8: Sensitivity and specificity of the feline measles virus chemiluminescent detection kit.

[0059] 8.1 Sensitivity of the kit: Positive control samples were serially diluted and detected by chemiluminescence immunoassay (CLIA) and enzyme-linked immunosorbent assay (ELISA), respectively. The results are shown in Table 5. The analysis showed that the linear range of the chemiluminescence method was 50-8000 pg / ml, while the linear range of the ELISA method was 250-2000 pg / ml. The chemiluminescence method has a wider linear range and higher detection sensitivity than the ELISA method.

[0060] Table 5: Sensitivity test results.

[0061]

[0062]

[0063] 8.2 Positive sample detection rate: 10 positive samples and 10 negative samples were tested using the kit, and the detection results and the concordance rate were 100%; the results are shown in Table 6.

[0064] Table 6: Detection results of positive samples.

[0065]

[0066]

[0067] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A feline measles virus-specific monoclonal antibody, characterized in that, The monoclonal antibody is prepared by immunization with feline measles virus N protein. The monoclonal antibody specifically recognizes feline measles virus N protein. The recognized feline measles virus N protein includes the feline measles virus N protein-N fragment with an amino acid sequence as shown in SEQ ID NO:1 and the feline measles virus N protein-C fragment with an amino acid sequence as shown in SEQ ID NO:

2. The monoclonal antibody contains a heavy chain variable region and a light chain variable region, both of which are composed of a determinant complementary region and a framework region; the determinant complementary regions of the heavy chain variable region and the light chain variable region are both composed of CDR1, CDR2 and CDR3. The amino acid sequence of CDR1 in the light chain variable region is shown as positions 27-32 of SEQ ID No. 3; The amino acid sequence of CDR2 in the light chain variable region is shown as positions 50-52 of SEQ ID No. 3; The amino acid sequence of CDR3 in the light chain variable region is shown as positions 89-97 of SEQ ID No. 3; The amino acid sequence of CDR1 in the heavy chain variable region is shown as positions 26-33 of SEQ ID No. 4; The amino acid sequence of CDR2 in the heavy chain variable region is shown as positions 51-60 of SEQ ID No. 4; The amino acid sequence of CDR3 in the heavy chain variable region is shown as positions 99-105 of SEQ ID No.

4.

2. The feline measles virus-specific monoclonal antibody according to claim 1, characterized in that, The amino acid sequence of the light chain variable region 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.

3. A feline measles virus-specific monoclonal antibody, characterized in that, The monoclonal antibody is prepared by immunization with feline measles virus N protein. The monoclonal antibody specifically recognizes feline measles virus N protein. The recognized feline measles virus N protein includes the feline measles virus N protein-N fragment with an amino acid sequence as shown in SEQ ID NO:1 and the feline measles virus N protein-C fragment with an amino acid sequence as shown in SEQ ID NO:

2. The monoclonal antibody contains a heavy chain variable region and a light chain variable region, both of which are composed of a determinant complementary region and a framework region; the determinant complementary regions of the heavy chain variable region and the light chain variable region are both composed of CDR1, CDR2 and CDR3. The amino acid sequence of CDR1 in the light chain variable region is shown as positions 27-37 of SEQ ID No. 5; The amino acid sequence of CDR2 in the light chain variable region is shown as positions 55-57 of SEQ ID No. 5; The amino acid sequence of CDR3 in the light chain variable region is shown as positions 94-102 of SEQ ID No. 5; The amino acid sequence of CDR1 in the heavy chain variable region is shown as positions 26-33 of SEQ ID No. 6; The amino acid sequence of CDR2 in the heavy chain variable region is shown as positions 51-58 of SEQ ID No. 6; The amino acid sequence of CDR3 in the heavy chain variable region is shown as positions 97-109 of SEQ ID No.

6.

4. The feline measles virus-specific monoclonal antibody according to claim 3, characterized in that, The amino acid sequence of the light chain variable region is shown in SEQ ID No. 5, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID No.

6.

5. A kit containing a monoclonal antibody as described in any one of claims 1-4.

6. The reagent kit according to claim 5, characterized in that, The kit contains immunomagnetic beads conjugated with any of the monoclonal antibodies as described in claims 1-2, feline measles virus N protein calibrator, alkaline phosphatase-labeled monoclonal antibodies as described in any of claims 3-4, and chemiluminescent substrate.

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