Nipah virus N protein specific monoclonal antibody as well as preparation method and application thereof

Abstract

The invention relates to a Nipah virus N protein specific monoclonal antibody as well as a preparation method and application thereof. The nipah virus specific monoclonal antibody is prepared by immunizing nipah virus N protein, the amino acid sequence of the nipah virus N protein is as shown in SEQ ID NO.2, the nipah virus N protein is obtained by transcription of a nipah virus N gene, and the nucleotide sequence of the nipah virus N gene is as shown in SEQ ID NO.1. The nipah virus specific monoclonal antibody has the advantages that the nipah virus specific monoclonal antibody is prepared by immunizing the nipah virus N protein; the monoclonal antibody with the nipah virus N protein specificity can be specifically combined with the nipah virus N protein and is high in titer and good in affinity, meanwhile, a detection kit prepared based on the antibody can effectively carry out nipah virus detection, and technical support can be provided for epidemiological monitoring and diagnosis of nipah virus infection.

Description

[0001] The present invention belongs to the field of biomedical technology, and in particular relates to a monoclonal antibody specific to the Nipah virus N protein and a preparation method and application thereof. Background Art

[0002] Nipah virus (NiV) is a new type of zoonotic virus. It is an RNA virus belonging to the Paramyxoviridae family. It can cause widespread vasculitis. Infected people have symptoms such as fever, severe headache, meningitis, etc., which bring serious harm to humans and animals. As early as 2018, the World Health Organization listed Nipah virus as one of the 10 high-risk infectious diseases that need attention. In the world, Nipah virus is mainly prevalent in Central and South Asia. It can be transmitted between mammals such as dogs, cats, pigs, horses, and humans through bats, fruit flies, feces, saliva and other transmission routes. Although the probability of a Nipah virus outbreak among animals in the short term is not high due to the influence of geographical environment, it is very necessary to carry out regular monitoring in animals. There are not many Nipah virus diagnostic and detection products for the animal husbandry and veterinary industry, and the use environment and detection efficiency of related products are not high. Fruit flies and bats are mainly distributed in the southern region of my country, and are adjacent to the epidemic areas in Southeast Asia, which has potential high-risk situations. Therefore, it is urgent to develop monitoring and detection strategies for Nipah virus. Summary of the invention

[0003] In order to solve the above problems, the present invention provides a monoclonal antibody specific to Nipah virus N protein and a preparation method and application thereof.

[0004] The technical solution of the present invention is:

[0005] A monoclonal antibody specific for Nipah virus N protein, wherein the Nipah virus specific monoclonal antibody is prepared by immunization with 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 comprises a VL domain and a VH domain;

[0007] The VL domain includes CDR-L1 with an amino acid sequence as shown in SEQ ID No.3, CDR-L2 with an amino acid sequence as RMS, and CDR-L3 with an amino acid sequence as 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 comprises a VL domain and a VH domain;

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

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

[0018] The kit comprises immunomagnetic beads coupled to the capture antibody, Nipah virus N protein calibrator, alkaline phosphatase-labeled detection antibody and chemiluminescent substrate.

[0019] The beneficial effects of the present invention are:

[0020] The monoclonal antibody specific for the Nipah virus N protein of the present invention can specifically bind to the Nipah virus N protein, has high titer and good affinity, and the detection kit prepared based on the antibody pair can effectively carry out Nipah virus detection, and can provide technical support for the epidemiological monitoring and diagnosis of Nipah virus infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0022] Figure 1 Shown is a schematic diagram of SDS-PAGE analysis of 1D6, 3F9, 4B3 and 4E9 antibodies in Example 2 of the present invention;

[0023] Figure 2 Shown is a calibration curve diagram of the Nipah virus detection kit in Example 6 of the present invention. DETAILED DESCRIPTION

[0024] To make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described in detail below. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other implementation methods obtained by ordinary technicians in this field without creative work belong to the scope of protection of the present invention.

[0025] The above technical solution is described in detail below in conjunction with specific embodiments.

[0026] The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples are all commercially available unless otherwise specified.

[0027] Example 1 Expression and purification of Nipah virus N protein

[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 the pcDNA3.1 vector containing a 6×His tag and the vector was named pcDNA3.1-NiV-N. The pcDNA3.1-NiV-N was transformed into TOP10 and the plasmid was extracted 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 expressed cell supernatant was centrifuged at 4°C, 12000 rpm / min for 10 min, filtered with a 0.45 μm filter membrane, and the filtrate was 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 of monoclonal antibodies against Nipah virus N protein

[0035] BALB / c mice were immunized with Nipah 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 50 μg. A total of 3 mice were immunized. Every 14 days, the antigen was mixed with Freund's incomplete adjuvant in equal proportions for the second and third subcutaneous injections, and each mouse was immunized with 50 μg. Ten days after the third immunization, blood was collected from the tail vein, and serum was centrifuged to obtain serum. The serum was gradiently diluted, and the pre-immunization serum was used as a negative control to detect the serum titer, as shown in Table 1.

[0036] Table 1 - Mouse serum titer detection

[0037] Dilution ratio Mouse No. 1 Mouse No. 2 Mouse No. 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] Mouse No. 1 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 Nipah virus N protein was used to detect the cell supernatant, and the positive hybridomas were cloned and screened to obtain 4 hybridoma cell strains that secreted specific monoclonal antibodies, namely 1D6, 3F9, 4B3 and 4E9. The 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 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.

[0041] The collected ascites was centrifuged at 8000rpm / min for 10min and the supernatant was collected. 10-20 times the volume of ProteinA equilibrium buffer was added to the supernatant for ProteinA affinity chromatography purification. The antibody was eluted with 0.1M glycine-hydrochloric acid solution pH2.7 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 1 .

[0042] The Nipah virus N protein was diluted to 2 μg / ml with 1×CBS, coated overnight at 4°C, and blocked with 10% skim milk powder for 2 hours. The purified antibodies were diluted in series, 100ul per well, and incubated at 37°C for 1 hour. After washing the plate, goat anti-mouse IgG-HRP was added, incubated at 37°C for 1 hour, the liquid in the well was discarded, TMB was added for color development, and the OD450 value was read after the stop solution was stopped. The titers of monoclonal antibodies 1D6, 3F9, 4B3, and 4E9 were 6.4×10 7 , 1.6×10 7 , 6.4×107 , 3.2×10 7 .

[0043] Example 4: Nipah virus monoclonal antibody pairing verification

[0044] Purified 1D6, 3F9, 4B3 and 4E9 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 at 4°C overnight. After blocking with 10% skim milk powder at 37°C for 2h, the plates were washed and 100 μl of Nipah virus N protein calibrators of different concentrations (0-100 ng / ml) were added. The plates were incubated at 37°C for 60 min. After washing, 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, the substrate was added to react for 15 min. After the stop solution was terminated, the absorbance was measured on an enzyme reader. The results are shown in Table 2. Finally, 4B3 was selected as the capture antibody of the kit and 4E9 was selected as the detection antibody for the detection of Nipah virus.

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

[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 CDRs 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 CDRs 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 CDRs 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 CDRs 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] 2μg / 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 100μl / well were used to coat the ELISA plate at 4℃ overnight. After discarding the liquid in the wells of the ELISA plate, block it with 10% skim milk powder at 37℃ incubator for 2h and then wash the plate. The antibody to be tested was diluted in a gradient, 100μl / well, and incubated at 37℃ incubator for 1h. After washing the plate, diluted goat anti-mouse IgG-HRP was added, 100μl / well, incubated at 37℃ incubator for 1h, and washed. After adding 100ul TMB colorimetric solution to each well for 15min, stop solution was added to stop. The OD450 value was read on the ELISA reader. The results showed that Nipah virus monoclonal antibodies 4B3 and 4E9 could specifically recognize Nipah virus antigen (NiV), and had no immune response to 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 monoclonal antibodies against Nipah virus N protein

[0075]

[0076] Example 6: Nipah virus chemiluminescent detection kit

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

[0078] Table 4: Nipah virus chemiluminescent detection kit

[0079]

[0080] Example 7: Sensitivity and specificity of Nipah virus chemiluminescent detection kit

[0081] Sensitivity of the kit: The positive control sample was diluted in a gradient manner and tested by chemiluminescence (CLIA) and ELISA, respectively. The results are shown in Table 5: Through analysis, it was found that the linear range of chemiluminescence detection was 12.5-4000pg / ml, while the linear range of ELISA was 250-4000pg / ml. The chemiluminescence method had a wider linear range and higher detection sensitivity than the ELISA method.

[0082] Table 5 - Sensitivity test results

[0083]

[0084]

[0085] Positive sample detection rate: The kit was used to detect 10 positive samples and 10 negative samples of Nipah virus N protein, and the detection results were consistent with the rate of 100%; the results are shown in Table 6.

[0086] Table 6- Positive sample test results

[0087] Sample No. 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 is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art can easily think of changes or substitutions within the technical scope disclosed by the present invention, which should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

Claims

1. A monoclonal antibody specific for Nipah virus N protein, characterized in that: The Nipah virus-specific monoclonal antibody is prepared by immunization with 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.

2. The monoclonal antibody specific for Nipah virus N protein according to claim 1, characterized in that: The monoclonal antibody comprises a VL domain and a VH domain; The VL domain includes CDR-L1 with an amino acid sequence as shown in SEQ ID No.3, CDR-L2 with an amino acid sequence as RMS, and CDR-L3 with an amino acid sequence as shown in SEQ ID No.4; 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.

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

8.

4. The monoclonal antibody binding to the HPV16 type E6 oncoprotein recombinant antigen according to claim 2, characterized in that: The VH domain has the amino acid sequence shown in SEQ ID No.

9.

5. The monoclonal antibody specific for Nipah virus N protein according to claim 1, characterized in that: The monoclonal antibody comprises a VL domain and a VH domain; The VL domain includes CDR-L1 with an amino acid sequence as shown in SEQ ID No.10, CDR-L2 with an amino acid sequence as LVS, and CDR-L3 with an amino acid sequence as shown in SEQ ID No.11; 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.

6. The monoclonal antibody specific for Nipah virus N protein according to claim 5, characterized in that: The VL domain has the amino acid sequence shown in SEQ ID No.

15.

7. The monoclonal antibody specific for Nipah virus N protein according to claim 5, characterized in that: The VH domain has the amino acid sequence shown in SEQ ID No.

16.

8. A kit comprising the monoclonal antibody according to any one of claims 1 to 7.

9. The kit according to claim 8, characterized in that In the kit, the antibody according to any one of claims 2 to 4 is used as a capture antibody, and the antibody according to any one of claims 5 to 7 is used as a detection antibody.

10. The kit according to claim 9, characterized in that The kit comprises immunomagnetic beads coupled to the capture antibody, Nipah virus N protein calibrator, alkaline phosphatase-labeled detection antibody and chemiluminescent substrate.

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