Monoclonal antibody specific for nipah virus n protein and preparation method and application thereof

Abstract

The application relates to a monoclonal antibody specific to a Nipah virus N protein and a preparation method and application thereof. The monoclonal antibody specific to the Nipah virus prepared by immunization of the Nipah virus N protein has an amino acid sequence as shown in SEQ ID NO. 2, and the Nipah virus N protein is transcribed from a Nipah virus N gene, and the nucleotide sequence of the Nipah virus N gene is as shown in SEQ ID NO. 1. The monoclonal antibody specific to the Nipah virus N protein can be specifically combined with the Nipah virus N protein, has high titer and good affinity, meanwhile, based on the antibody, a detection kit prepared can effectively carry out Nipah virus detection, and can provide technical support for epidemiological monitoring and diagnosis of Nipah virus infection.

Description

[0001] This invention belongs to the field of biomedical technology, specifically relating to a monoclonal antibody specific to the Nipah virus N protein, its preparation method, and its application. Background Technology

[0002] Nipah virus (NiV) is a novel zoonotic virus. It is an RNA virus belonging to the Paramyxoviridae family and causes widespread vasculitis. Infected individuals experience symptoms such as fever, severe headache, and meningitis, posing a serious threat to both humans and animals. As early as 2018, the World Health Organization listed Nipah virus as one of the 10 high-risk infectious diseases of concern. Globally, Nipah virus is mainly prevalent in Southeast Asia and Central Asia. It can be transmitted among mammals such as dogs, cats, pigs, horses, and humans via bats, fruit flies, feces, and saliva. Although the probability of short-term outbreaks of Nipah virus in animals is low due to geographical factors, routine monitoring in animals is essential. Currently, there are few diagnostic testing products for Nipah virus in the livestock and veterinary industry, and the application environment and detection efficiency of these products are also limited. Fruit flies and bats are mainly distributed in southern my country, bordering Southeast Asian epidemic areas, posing a potentially high risk. Therefore, developing monitoring and detection strategies for Nipah virus is urgently needed. Summary of the Invention

[0003] To address the aforementioned problems, this invention provides a monoclonal antibody specific to the Nipah virus N protein, its preparation method, and its application.

[0004] The technical solution of this invention is as follows:

[0005] A monoclonal antibody specific to the Nipah virus N protein, wherein the Nipah virus-specific monoclonal antibody is prepared by immunization with the Nipah virus N protein, the amino acid sequence of the Nipah virus N protein is shown in SEQ ID NO.2, the Nipah virus N protein is transcribed from the Nipah virus N gene, and the nucleotide sequence of the Nipah virus N gene is shown in SEQ ID NO.1.

[0006] The monoclonal antibody includes a VL domain and a VH domain;

[0007] The VL domain includes CDR-L1 of the amino acid sequence shown in SEQ ID No. 3, CDR-L2 of the amino acid sequence RMS, and CDR-L3 of the amino acid sequence shown in SEQ ID No. 4.

[0008] The VH domain includes CDR-H1 of the amino acid sequence shown in SEQ ID No. 5, CDR-H2 of the amino acid sequence shown in SEQ ID No. 6, and CDR-H3 of the amino acid sequence shown in SEQ ID No. 7.

[0009] The VL domain has the amino acid sequence shown in SEQ ID No. 8.

[0010] The VH domain has the amino acid sequence shown in SEQ ID No. 9.

[0011] The monoclonal antibody includes a VL domain and a VH domain;

[0012] The VL domain includes CDR-L1 of the amino acid sequence shown in SEQ ID No. 10, CDR-L2 of the LVS, and CDR-L3 of the amino acid sequence shown in SEQ ID No. 11.

[0013] The VH domain includes CDR-H1 of the amino acid sequence shown in SEQ ID No. 12, CDR-H2 of the amino acid sequence shown in SEQ ID No. 13, and CDR-H3 of the amino acid sequence shown in SEQ ID No. 14.

[0014] The VL domain has the amino acid sequence shown in SEQ ID No. 15.

[0015] The VH domain has the amino acid sequence shown in SEQ ID No. 16.

[0016] A kit containing the monoclonal antibody described above.

[0017] In the kit, the antibody is used as a capture antibody or as a detection antibody.

[0018] The kit contains immunomagnetic beads conjugated with the capture antibody, a Nipah virus N protein calibrator, an alkaline phosphatase-labeled detection antibody, and a chemiluminescent substrate.

[0019] The beneficial effects of this invention are as follows:

[0020] The Nipah virus N protein-specific monoclonal antibody described in this invention can specifically bind to the Nipah virus N protein, exhibiting high titer and good affinity. Furthermore, the detection kit prepared based on this antibody can effectively detect Nipah virus and provide technical support for the epidemiological monitoring and diagnosis of Nipah virus infection. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 The diagram shows an SDS-PAGE analysis of antibodies 1D6, 3F9, 4B3, and 4E9 in Example 2 of this invention.

[0023] Figure 2 The figure shown is a calibration curve of the Nipah virus detection kit in Example 6 of the present invention. Detailed Implementation

[0024] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be described in detail below. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. Based on the embodiments of this invention, all other implementation methods obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0025] The above technical solution will be described in detail below with reference to specific embodiments.

[0026] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0027] Example 1: Nipah virus N protein expression and purification

[0028] The N gene sequence of the Ind-Nipah-07-FG strain, reported in GenBank, was used as a reference. The N gene was cloned into a pcDNA3.1 vector containing a 6×His tag, and the vector was named pcDNA3.1-NiV-N. pcDNA3.1-NiV-N was transformed into TOP10 cells, and plasmid extraction was performed according to the instructions of the endotoxin-free plasmid extraction kit. The plasmid was transfected into 293 suspension cells to express the Nipah virus N protein.

[0029] The Nipah virus N gene sequence is shown in SEQ ID NO.1, specifically:

[0030]

[0031] The cell supernatant after expression was centrifuged at 12,000 rpm for 10 min at 4 °C and filtered through a 0.45 μm filter membrane. The filtrate was then purified by affinity chromatography.

[0032] The Nipah virus N protein sequence is shown in SEQ ID NO.2, specifically:

[0033] .

[0034] Example 2: Preparation of hybridoma cell lines for Nipah virus N protein monoclonal antibodies

[0035] BALB / c mice were immunized with Nipah virus N protein. For the initial immunization, the antigen and Freund's complete adjuvant were mixed in an equal ratio and administered subcutaneously, with each mouse receiving 50 μg, for a total of 3 mice. Every 14 days, the antigen and Freund's incomplete adjuvant were mixed in an equal ratio and administered subcutaneously, with each mouse receiving 50 μg. Ten days after the third immunization, blood was collected from the tail vein, centrifuged, and the serum was serially diluted. The pre-immunization serum served as a negative control. Serum titers were measured (see Table 1).

[0036] Table 1 - Mouse serum titer detection

[0037] Dilution ratio Mouse number 1 Mouse number 2 Mouse number 3 1：1000 2.901 3.119 2.932 1：2000 2.708 2.829 2.838 1：4000 2.653 2.776 2.713 1：8000 2.219 2.498 2.539 1：16000 1.988 1.809 1.913 1：32000 1.604 1.471 1.544 1：64000 1.401 1.228 1.39 1：128000 1.001 0.976 0.851 negative control 0.101 0.119 0.113

[0038] Mice with the highest serum titer 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. The cell supernatant was analyzed using Nipah virus N protein detection. Positive hybridomas were screened for clones, yielding four hybridoma cell lines secreting specific monoclonal antibodies: 1D6, 3F9, 4B3, and 4E9. Antibody subtype results showed that 1D6, 3F9, and 4E9 were IgG. 2b 4B3 is IgG1.

[0039] Example 3: Preparation, purification, and titer detection of Nipah virus monoclonal antibodies

[0040] 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.

[0041] 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 1 .

[0042] Nipah virus N protein was diluted to 2 μg / ml with 1×CBS, coated overnight at 4°C, and then blocked with 10% skim milk powder 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 plates were incubated at 37°C for 1 h. The liquid in the wells was discarded, and after washing, TMB was added for color development. The OD450 value was read after stopping the reaction with stop solution. The titers of monoclonal antibodies 1D6, 3F9, 4B3, and 4E9 were all 6.4 × 10⁻⁶. 7 1.6×10 7 6.4×107 3.2×10 7 .

[0043] Example 4: Verification of Nipah virus monoclonal antibody pairing

[0044] Purified monoclonal antibodies 1D6, 3F9, 4B3, and 4E9 were used as capture and detection antibodies, respectively, to verify antibody pairing. Microplates were coated with 2 μg / ml antibody and incubated overnight at 4°C. After blocking with 10% skim milk powder at 37°C for 2 h, the plates were washed, and 100 μl of different concentrations (0-100 ng / ml) of Nipah virus N protein calibrator were added. The plates were incubated at 37°C for 60 min, washed again, and then 100 μl of 0.1 μg / ml HRP-labeled 1D6, 3F9, 4B3, and 4E9 were added and incubated at 37°C for 60 min. After washing, substrate was added, and the reaction was allowed to proceed for 15 min. The reaction was terminated with stop solution, and the absorbance was measured using a microplate reader. The results are shown in Table 2. Ultimately, 4B3 was selected as the capture antibody for the kit, and 4E9 was selected as the detection antibody for Nipah virus detection.

[0045] Table 2 - Nipah virus monoclonal antibody pairing verification

[0046]

[0047] The full-length amino acid sequence of the light chain (L chain) variable region of the monoclonal antibody 4B3 is shown in SEQ ID No. 8.

[0048] SEQ ID No. 8 is as follows:

[0049] DIVMTQATPSVSVTPGESVSISCRSSKSLLHSDGNTFLFWFLQRPGQSPQ LLIHRMSNLASGVPDRFSGSGSGTTFTLRISRVEAEDVGVYYCLRHLFYPLTF GAGTKLELK.

[0050] The three CDR sequences of the light chain (L chain) are as follows:

[0051] CDR-L1 amino acid sequence: KSLLHSDGNT (SEQ ID No. 3);

[0052] CDR-L2 amino acid sequence: RMS;

[0053] CDR-L3 amino acid sequence: LRHLFYPLT (SEQ ID No. 4).

[0054] The full-length amino acid sequence of the heavy chain (H chain) variable region of the monoclonal antibody 4B3 is shown in SEQ ID No. 9. SEQ ID No. 9 is as follows:

[0055] EVQLQQSGAELVKPGASIKLSCTASGFNIKDTYMHWVKQRPEQGLEWIGWIDPATGNTKYDPKFQGKATLTADTSSNTAYLQLNSLTSEDTAVYYCYGKYDKWGQGTTLTVSS

[0056] The three CDR sequences of the heavy chain (H chain) are as follows:

[0057] CDR-H1 amino acid sequence: GFNIKDTY (SEQ ID No. 5);

[0058] CDR-H2 amino acid sequence: IDPATGNT (SEQ ID No. 6);

[0059] CDR-H3 amino acid sequence: YGKYDK (SEQ ID No. 7).

[0060] The full-length amino acid sequence of the light chain (L chain) variable region of the monoclonal antibody 4E9 is shown in SEQ ID No. 15. SEQ ID No. 15 is as follows:

[0061] DVVMTQTPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPR RLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWTGTHFPQTF GGGTKLEIK.

[0062] The three CDR sequences of the light chain (L chain) are as follows:

[0063] CDR-L1 amino acid sequence: KSLLHSDGNT (SEQ ID No. 10);

[0064] CDR-L2 amino acid sequence: LVS;

[0065] CDR-L3 amino acid sequence: WTGTHFPQT (SEQ ID No. 11).

[0066] The full-length amino acid sequence of the heavy chain (H chain) variable region of the monoclonal antibody 4E9 is shown in SEQ ID No. 16. SEQ ID No. 16 is as follows:

[0067] EVQLQQSGPELVKPGASVKKSCKTSGYTFTEYTMHWVKQSHGKSLEWI GGISPNNGDTTYNQKFKGKATLTVDKSSSTAYMELRSLTFEDSAVYYCARG YFGYWGQGTLVTVSA.

[0068] The three CDR sequences of the heavy chain (H chain) are as follows:

[0069] CDR-H1 amino acid sequence: GYTFTEYT (SEQ ID No. 12);

[0070] CDR-H2 amino acid sequence: ISPNNGDT (SEQ ID No. 13);

[0071] CDR-H3 amino acid sequence: ARGYFGY (SEQ ID No. 14).

[0072] Example 5: Specificity analysis of monoclonal antibodies against Nipah virus N protein

[0073] Coat each well with 100 μl of 2 μg / ml Nipah virus (NiV) N protein, respiratory syncytial virus (RSV) N protein, human parainfluenza virus type 3 (HPIV-3) N protein, and human parainfluenza virus type 4 (HPIV-4) 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 h, then wash the plates. Serially dilute the test antibodies to 100 μl / well and incubate at 37°C for 1 h. After washing, add 100 μl / well of diluted goat anti-mouse IgG-HRP and incubate at 37°C for 1 h, then wash the plates. Add 100 μl of TMB chromogenic buffer to each well and develop for 15 min, then add stop solution to terminate the reaction. Read the OD450 values ​​on a microplate reader. The results showed that Nipah virus monoclonal antibodies 4B3 and 4E9 could specifically recognize the Nipah virus antigen (NiV) but did not elicit an immune response against respiratory syncytial virus (RSV), human parainfluenza virus type 3 (HPIV-3), and human parainfluenza virus type 4 (HPIV-4). The results are shown in Table 3.

[0074] Table 3: Specificity of Nipah virus N protein monoclonal antibodies

[0075]

[0076] Example 6: Nipah virus chemiluminescence detection kit

[0077] The Nipah virus chemiluminescence detection kit includes: immunomagnetic beads labeled with monoclonal antibodies against the Nipah virus N protein, monoclonal antibodies against the Nipah virus N protein labeled with alkaline phosphatase, and Nipah virus N protein calibrators. The kit components are shown in Table 4, and the standard curve is shown in [Table 4]. Figure 2 As shown.

[0078] Table 4: Nipah virus chemiluminescence detection kit

[0079]

[0080] Example 7: Sensitivity and Specificity of the Nipah Virus Chemiluminescence Detection Kit

[0081] The sensitivity of the kit: Positive control samples were serially diluted and detected by chemiluminescence immunoassay (CLIA) and ELISA, respectively. The results are shown in Table 5. The analysis showed that the linear range of the chemiluminescence immunoassay was 12.5-4000 pg / ml, while the linear range of the ELISA method was 250-4000 pg / ml. The chemiluminescence immunoassay method has a wider linear range and higher detection sensitivity than the ELISA method.

[0082] Table 5 - Sensitivity Detection Results

[0083]

[0084]

[0085] Positive sample detection rate: 10 positive samples and 10 negative samples were detected for the N protein of the sennipa virus using the kit, and the detection results and the concordance rate were 100%; the results are shown in Table 6.

[0086] Table 6 - Detection Results of Positive Samples

[0087] Sample number Concentration value (pg / ml) determination 1 5.13 - 2 6.47 - 3 10.21 - 4 16.09 - 5 7.14 - 6 21.27 - 7 2.86 - 8 20.54 - 9 2.04 - 10 9.18 - 11 223.91 + 12 145.47 + 13 466.67 + 14 315.45 + 15 108.59 + 16 358.72 + 17 204.32 + 18 218.29 + 19 307.34 + 20 251.38 +

[0088] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A monoclonal antibody specific for the N protein of a Nipah virus, characterized in that, The monoclonal antibody comprises a VL domain and a VH domain; The VL domain comprises CDR-L1 of the amino acid sequence shown in SEQ ID No. 3, CDR-L2 of the amino acid sequence RMS, and CDR-L3 of the amino acid sequence shown in SEQ ID No. 4; The VH domain comprises CDR-H1 of the amino acid sequence shown in SEQ ID No. 5, CDR-H2 of the amino acid sequence shown in SEQ ID No. 6, and CDR-H3 of the amino acid sequence shown in SEQ ID No.

7.

2. The monoclonal antibody specific for Nipah virus N protein according to claim 1, characterized in that, The VL domain has the amino acid sequence shown in SEQ ID No. 8, and the VH domain has the amino acid sequence shown in SEQ ID No.

9.

3. A monoclonal antibody specific for the N protein of a Nipah virus, characterized in that, The monoclonal antibody comprises a VL domain and a VH domain; The VL domain has the amino acid sequence shown in SEQ ID No. 15, and the VH domain has the amino acid sequence shown in SEQ ID No.

16.

4. A kit comprising the monoclonal antibody according to any one of claims 1-3.

5. The kit of claim 4, wherein In the kit, the antibody according to any one of claims 1-2 is used as a capture antibody, and the antibody according to claim 3 is used as a detection antibody.

6. The kit of claim 5, wherein The kit comprises immunomagnetic beads coupled with the capture antibody, a Nipah virus N protein calibrator, the detection antibody labeled with alkaline phosphatase, and a chemiluminescent substrate.

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