Cat measles virus specific monoclonal antibody and application thereof

Abstract

The invention discloses a cat measles virus specific monoclonal antibody and application thereof, the monoclonal antibody is prepared by immunizing a cat measles virus N protein, and the monoclonal antibody specifically recognizes the cat measles virus N protein. The recognized feline measles virus N protein comprises a feline measles virus N protein-N fragment with a nucleotide sequence as shown in SEQ ID NO: 1 and a feline measles virus N protein-C fragment with a nucleotide sequence as shown in SEQ ID NO: 2. The monoclonal antibody provided by the invention can specifically recognize the feline measles virus N protein and is high in titer, good in affinity and high in sensitivity, a detection kit prepared based on the antibody can effectively detect feline measles viruses in body fluids such as cat blood and urine, and a technical support is provided for epidemiological monitoring and diagnosis of feline measles virus infection.

Description

Technical Field

[0001] The invention belongs to the technical field of biological detection, and in particular relates to a feline measles virus-specific monoclonal antibody and application thereof. Background Art

[0002] Feline morbillivirus (FeMV) is a single-stranded negative-sense RNA virus belonging to the genus Morbillivirus of the family Paramyxoviridae. Among them, the N protein is the nucleocapsid protein of feline morbillivirus. The nucleotide length is 1560bp, composed of 519 amino acids, and the molecular weight is 57KDa. The transmission host of feline morbillivirus is cats. The main clinical symptoms of infected cats are related to kidney disease. The mechanism by which the virus causes kidney disease is still unclear. Infected cats have a high infection rate and mortality rate.

[0003] Most members of the genus Morbillivirus are important pathogens for humans and animals. Measles virus is highly pathogenic and can cause death in animals and humans, so it is very necessary to carry out monitoring of feline measles virus. At present, most researchers mainly focus on the L gene of feline measles virus and carry out detection through RT-PCR detection method. This method has problems such as high requirements for the operating environment, long operation time, and easy false positives, and cannot be used in routine monitoring environments. Summary of the invention

[0004] In order to solve the defects of the prior art, the present invention provides a feline measles virus-specific monoclonal antibody and application thereof.

[0005] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0006] The first purpose of the present 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 a feline measles virus N protein-N fragment as shown in the nucleic acid sequence of SEQ ID NO:1 and a feline measles virus N protein-C fragment as shown in the nucleic acid sequence of SEQ ID NO:2.

[0007] Preferably, the monoclonal antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region are both composed of a complementary region determining region and a framework region; and the complementary region determining region of the heavy chain variable region and the light chain variable region are both composed of CDR1, CDR2 and CDR3;

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

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

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

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

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

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

[0014] Preferably, the amino acid sequence of the light chain variable region is shown as SEQ ID No.3, and the amino acid sequence of the heavy chain variable region is shown as SEQ ID No.4.

[0015] Preferably, the monoclonal antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region are both composed of a complementary region determining region and a framework region; and the complementary region determining region of the heavy chain variable region and the light chain variable region are both composed of CDR1, CDR2 and CDR3;

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

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

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

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

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

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

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

[0023] The second object of the present invention is to provide a kit containing monoclonal antibodies.

[0024] Preferably, the kit comprises immunomagnetic beads coupled with capture antibodies, feline measles virus N protein calibrator, alkaline phosphatase-labeled detection antibodies, and chemiluminescent substrates.

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

[0026] The monoclonal antibody provided by the present invention can specifically recognize the N protein of feline measles virus, has high antibody titer, good affinity and high sensitivity, and the detection kit prepared based on the antibody can effectively detect feline measles virus in cat blood, urine and other body fluids, providing technical support for the epidemiological monitoring and diagnosis of feline measles virus infection. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

[0029] Figure 3 It is a calibration curve diagram of the cat measles virus detection kit in Example 7 of the present invention. DETAILED DESCRIPTION

[0030] The preferred embodiments of the present invention are described below in conjunction with the accompanying drawings. It should be understood that the preferred embodiments described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention.

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

[0032] Truncated feline measles virus N protein construction and expression: According to the amino acid sequence of feline measles virus N protein, the N-terminal and C-terminal sequences were designed, connected to the expression vector pET28a, and loaded into the expression strain BL21 (DE3). In LB medium, IPTG 1mmol / L was used to induce the soluble expression of feline measles virus N truncated protein overnight at 37 degrees. Prokaryotic recombinant expression was affinity purified by 6×His tag, and the purified protein was subjected to SDS-PAGE electrophoresis, with a purity of more than 90%.

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

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

[0035] BALB / c mice were immunized with feline measles virus N protein. For the first immunization, the antigen was mixed with Freund's complete adjuvant in equal proportions and injected subcutaneously. Each mouse was immunized with 50ug, and a total of 5 mice were immunized. After 14 days, the second and third immunizations were performed. The antigen was mixed with Freund's incomplete adjuvant in equal proportions and injected subcutaneously. Each mouse was immunized with 50ug. Ten days after the third immunization, blood was collected from the tail vein, and the serum was centrifuged and diluted stepwise. The pre-immunization serum was used as a negative control, and the serum titer was tested, as shown in Table 1.

[0036] Table 1: Mouse triple-immune serum titer detection.

[0037]

[0038] Mouse No. 2 with the highest serum titer was boosted with immunization, and spleen cells were removed under sterile conditions after 3 days. Cells with good growth status and higher serum titers were taken for cell fusion experiments. SP2 / 0 and spleen cells were mixed in a centrifuge tube in proportion. 1ml of preheated PEG was added to a 50ml centrifuge tube for 1 minute, and then preheated DMEM medium was added to 35mL. After 15 minutes in a 37℃ water bath, centrifuge at 800rpm / min for 10 minutes and discard the supernatant. Add 100mL of preheated HAT medium, gently blow and mix, transfer to a 96-well culture plate with feeder cells, 200μL per well, and culture in a carbon dioxide incubator. The cell supernatant was detected using feline measles virus N protein, and positive hybridomas were cloned and screened to obtain 4 hybridoma cell strains that secrete specific monoclonal antibodies, namely 1G1, 1B5, 3C8 and 4C6. The antibody subtype detection results showed that 1G1 and 1B5 were IgG 2b , 3C8 and 4C6 are IgG1.

[0039] Example 3: Epitope identification of monoclonal antibodies against feline measles virus N protein.

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

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

[0042] Liquid paraffin is injected intraperitoneally into mice. After 7-10 days, the obtained hybridoma cell line capable of stably secreting antibodies is inoculated into the mice. After the abdomen of the mice is swollen, ascites is collected.

[0043] The collected ascites was centrifuged at 8000rpm / min for 10min and the supernatant was collected. 10-20 volumes of ProteinA equilibrium buffer were added to the supernatant for ProteinA affinity chromatography purification. The antibody was eluted with 0.1M glycine-hydrochloric acid solution pH 2.7 and neutralized with 1M pH 9.0 Tris buffer. The purified antibody was dialyzed with 1×PBS solution and the antibody purity was detected by SDS-PAGE gel. The results showed that the heavy chain of the four antibodies had a band at 50KD and the light chain of the antibody had a band at 25KD. The purity of the grayscale analysis was greater than 90%, as shown in Figure 2. Figure 2 .

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

[0045] Example 5: Feline measles virus monoclonal antibody pairing verification.

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

[0047] Table 2: Validation of feline measles virus monoclonal antibody pairings.

[0048]

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

[0050] 2μg / ml of feline measles virus (FeMV) N protein, Nipah virus (NiV) N protein, bovine respiratory syncytial virus (BRSV) N protein, canine distemper virus (CDV) N protein 100μl / well were used to coat the ELISA plate at 4℃ overnight. After discarding the liquid in the wells of the ELISA plate, 10% skim milk powder was blocked in a 37℃ incubator for 2h and then the plate was washed. The antibody to be tested was diluted in a gradient, 100μl / well, and incubated in a 37℃ incubator for 1h. After washing the plate, 100μl of diluted goat anti-mouse IgG-HRP was added to each well, incubated in a 37℃ incubator for 1h, and the plate was washed. After adding 100ul TMB colorimetric solution to each well for 15min, the stop solution was added to stop. The OD450 absorbance value was read on the ELISA reader. The results showed that feline measles virus monoclonal antibodies 3C8 and 4C6 could specifically recognize the N protein of feline measles virus (FeMV), but had no immune response to the N protein of Nipah virus (NiV), bovine respiratory syncytial virus (BRSV), and canine distemper virus (CDV). 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: Feline measles virus chemiluminescent detection kit.

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

[0056] Table 4: Feline measles virus chemiluminescent detection kit.

[0057]

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

[0059] 8.1 Sensitivity of the kit: The positive control sample was diluted in a gradient manner and tested by chemiluminescence assay (CLIA) and enzyme-linked immunosorbent assay (ELISA). The results are shown in Table 5: The analysis showed that the linear range of chemiluminescence assay was 50-8000 pg / ml, while the linear range of ELISA assay was 250-2000 pg / ml. The chemiluminescence assay had a wider linear range and higher detection sensitivity than ELISA.

[0060] Table 5: Sensitivity test results.

[0061]

[0062]

[0063] 8.2 Detection rate of positive samples: The test kit was used to detect 10 positive samples and 10 negative samples, and the detection results were consistent with the rate of 100%; the results are shown in Table 6.

[0064] Table 6: Positive sample test results.

[0065]

[0066]

[0067] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in 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, and the monoclonal antibody specifically recognizes feline measles virus N protein. The recognized feline measles virus N protein includes a feline measles virus N protein-N fragment as shown in the nucleic acid sequence of SEQ ID NO:1 and a feline measles virus N protein-C fragment as shown in the nucleic acid sequence of SEQ ID NO:

2.

2. The feline measles virus-specific monoclonal antibody according to claim 1, characterized in that The monoclonal antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region are both composed of a complementary region determining a cluster and a framework region; the complementary region determining a cluster 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 of the light chain variable region is shown in positions 27-32 of SEQ ID No. 3; The amino acid sequence of CDR2 of the light chain variable region is shown in positions 50-52 of SEQ ID No. 3; The amino acid sequence of CDR3 of the light chain variable region is shown in positions 89-97 of SEQ ID No. 3; The amino acid sequence of CDR1 of the heavy chain variable region is shown in positions 26-33 of SEQ ID No. 4; The amino acid sequence of CDR2 of the heavy chain variable region is shown in positions 51-60 of SEQ ID No. 4; The amino acid sequence of CDR3 of the heavy chain variable region is shown in positions 99-105 of SEQ ID No.

4.

3. The feline measles virus-specific monoclonal antibody according to claim 2, 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.

4. The feline measles virus-specific monoclonal antibody according to claim 1, characterized in that The monoclonal antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region are both composed of a complementary region determining a cluster and a framework region; the complementary region determining a cluster 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 of the light chain variable region is shown in positions 27-37 of SEQ ID No. 5; The amino acid sequence of CDR2 of the light chain variable region is shown in positions 55-57 of SEQ ID No. 5; The amino acid sequence of CDR3 of the light chain variable region is shown in positions 94-102 of SEQ ID No.5; The amino acid sequence of CDR1 of the heavy chain variable region is shown in positions 26-33 of SEQ ID No. 6; The amino acid sequence of CDR2 of the heavy chain variable region is shown in positions 51-58 of SEQ ID No.6; The amino acid sequence of CDR3 of the heavy chain variable region is shown in positions 97-109 of SEQ ID No.

6.

5. The feline measles virus-specific monoclonal antibody according to claim 4, 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.

6. A kit comprising the monoclonal antibody according to any one of claims 1 to 5.

7. The kit according to claim 6, characterized in that: The kit comprises immunomagnetic beads coupled with the capture antibody according to claim 2 or 3, a feline measles virus N protein calibrator, an alkaline phosphatase-labeled detection antibody according to claim 4 or 5, and a chemiluminescent substrate.

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