Monoclonal antibody against novel bunyavirus NP protein and preparation method and application thereof

Abstract

The application discloses a kind of anti-new bunyavirus nucleocapsid protein (NP) protein monoclonal antibody, including heavy chain variable region and light chain variable region;The amino acid sequence of the heavy chain variable region is as shown in NO.1;The encoding DNA sequence of the heavy chain variable region is as shown in NO.2;The amino acid sequence of the light chain variable region is as shown in NO.3;The encoding DNA sequence of the light chain variable region is as shown in NO.4.The anti-new bunyavirus NP protein monoclonal antibody of the application can be specifically combined with the NP protein of new bunyavirus, and establish foundation for diagnosing viral infectious disease by genetic engineering method;The anti-new bunyavirus NP protein monoclonal antibody described in the application has specific antigen binding domain, can target and combine the NP protein of new bunyavirus, for antigen detection.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a monoclonal antibody against novel Bunyavirus nucleocapsid protein (NP) and its preparation method and application. Background Technology

[0002] Severe fever with thrombocytopenia syndrome virus (SFTSV) is a novel virus isolated and identified in recent years. Infecting humans with this virus causes severe fever with thrombocytopenia syndrome (SFTS), characterized by fever, thrombocytopenia, and leukopenia. Most cases also present with fatigue, gastrointestinal symptoms, muscle aches, and lymphadenopathy. Severe cases can lead to shock, respiratory failure, disseminated intravascular coagulation, and multiple organ failure, resulting in death. SFTS is a newly emerging hemorrhagic fever, an acute infectious disease. The national average mortality rate for SFTS infection is 5.3%, but in some endemic areas, the mortality rate can reach as high as 30%. Because the clinical manifestations of SFTS are nonspecific, it is easily misdiagnosed, leading to delayed treatment, severe illness, and even death. The SFTSV genome consists of three single-stranded negative-sense RNA segments: large (L), medium (M), and small (S). The S fragment, consisting of 1744 nucleotides, is a ambiguous RNA fragment encoding the viral nucleocapsid protein (NP) and nonstructural proteins (NSs). The NP is the most abundant protein in SFTSV viral particles and infected cells, playing a protective role in viral RNA and participating in viral replication and assembly. The NP possesses a highly conserved amino acid sequence and can serve as a protein or molecular marker for viral detection.

[0003] Currently, there are no effective vaccines or drugs for SFTS. Developing diagnostic kits based on SFTSV-NP as an antigen for SFTS, or targeted therapy using anti-SFTSV-NP monoclonal antibodies to achieve early diagnosis of SFTS and control of the clinical progression of SFTSV infection, is of great significance. Summary of the Invention

[0004] In view of this, the present invention provides a novel Bunyavirus NP protein-specific monoclonal antibody, its preparation method, and its application, which can achieve quantitative detection of recombinant NP antigen and NP protein in the serum of clinical patients using ELISA. The specific scheme is as follows: In a first aspect, the present invention provides a monoclonal antibody against a novel Bunyavirus NP protein, comprising a heavy chain variable region and a light chain variable region. The amino acid sequence of the heavy chain variable region is shown in NO.1: METGLRWLLLVAVLKGVQCQSLEESGGRLVTPGTPLTLTCTASGFSLSTYYMTWVRQAPGKGLEWIGVINTGGSTYYASWAKGRFTISRTSTTVELKIASPTTEDTATYFCARDSYESYGLAYDIWGPGTLVAVSS; The DNA sequence encoding the heavy chain variable region is shown in NO.2: ATGGAGACTGGGCTGCGCTGGCTTCTCCTGGTCGCTGTGCTCAAAGGTGTCCAGTGTCAGTCGCTGGAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGACACCCTGACACTCACCTGCACAGCCTCTGGATTCTCCCTCAGTACTTACTACATGACCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAATGGATCGGGGTC ATTAATACTGGTGGTAGCACATACTACGCGAGCTGGGCGAAAGGCCGATTCACCATCTCCAGAACCTCGACCACGGTGGAACTGAAAATCGCCAGTCCGACAACCGAGGACACGGCCACCTATTTCTGTGCCAGAGATAGTTACGAGAGCTATGGTCTTGCTTATGACATCTGGGGCCCAGGCACCCTGGTCGCCGTCTCCTCA; The amino acid sequence of the light chain variable region is shown in NO.3: MDTRAPTQLLGLLLLWLPGATFAQVLTQTPSSVSAVVGGTVTINCQASQNVDNNNYLAWYHHKPGQPPKLLIYDASKLAAGVPSRFEGSGSGTHFTLTISGVQCDDAATYYCQATYYSSGRYGTFGGGTEVVVK; The DNA sequence encoding the light chain variable region is shown in NO.4: ATGGACACGAGGGCCCCCACTCAGCTGCTGGGGCTCCTGCTGCTCTGGCTCCCAGGTGCCACATTTGCGCAAGTGCTGACCCAGACTCCATCCTCCGTGTCTGCAGTTGTGGGAGGCACAGTCACCATCAATTGCCAGGCCAGTCAGAATGTTGATAATAACAACTACTTAGCCTGGTATCACCACAAACCAGGGCAGCCT CCCAAGCTCCTGATCTACGATGCATCCAAATTGGCAGCTGGGGTCCCATCCCGGTTCGAAGGCAGTGGATCTGGGACACACTTCACTCTCACCATCAGCGGCGTGCAGTGTGACGATGCTGCCACTTACTATTGTCAAGCCACTTATTATAGTAGTGGACGGTATGGTACTTTCGGCGGAGGGACCGAGGTGGTGGTCAAA.

[0005] Secondly, the present invention provides a method for preparing a monoclonal antibody against a novel Bunyavirus NP protein, comprising the following steps: 1) Rabbits were immunized with SFTSV NP protein as an immunogen. After the rabbits made an immune response, they were sacrificed, their spleens were taken, and spleen cells were isolated. 2) Screening for antigen-specific single B cells; 3) Subcloning B cells to obtain the variable region coding sequences of the antibody heavy and light chains; 4) After recombination, transfection, and purification of the obtained variable region coding sequence, a monoclonal antibody against the novel Bunyavirus NP protein was obtained.

[0006] Thirdly, the present invention provides the application of the aforementioned anti-SFTSV NP protein monoclonal antibody, wherein the monoclonal antibody is used to prepare drugs for treating or preventing human viral infections.

[0007] Preferably, the monoclonal antibody is used for protein expression detection in Western blotting and / or immunofluorescence assays.

[0008] Fourthly, the present invention provides a kit for detecting NP antigen, the kit comprising a monoclonal antibody against novel Bunyavirus NP protein, and reagents for detecting said monoclonal antibody.

[0009] The beneficial effects of this invention are as follows: 1. This invention provides an anti-NP monoclonal antibody, its preparation method, and its application, which can specifically bind to NP antigens, laying the foundation for diagnosing viral infections through genetic engineering methods; 2. The anti-NP monoclonal antibody provided by this invention can accurately measure and detect the NP concentration in serum, and can quickly help doctors determine the patient's immune status; 3. This invention provides an NP rabbit-derived monoclonal antibody, enriching the types of antibodies; 4. The anti-NP monoclonal antibody described in this invention has a specific antigen-binding domain, which can target and bind to NP proteins, and can also be used in immunohistochemistry, ELISA and other experiments. Attached Figure Description

[0010] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0011] Figure 1 This is a graph showing the serum antibody titer test results of rabbits before and after immunization in Example 1 of the present invention.

[0012] Figure 2 This is a diagram showing the results of ELISA detection of the antibody in LEM supernatant specifically binding to SFTSV-NP protein in Example 3 of the present invention.

[0013] Figure 3 This is a diagram showing the results of the capture ELISA detection of LEM supernatant by the antibody in Example 4 of the present invention, which specifically binds to the SFTSV-NP protein.

[0014] Figure 4 This is the Western-Blot result of HeLa cells, human cervical cancer tumor cells, in Example 5 of the present invention.

[0015] Figure 5 The results of immunofluorescence of Vero cells (kidney cells of African green monkeys) in Example 6 of this invention are shown. Detailed Implementation

[0016] To provide a more detailed understanding of the features and technical content of this invention, the implementation of the invention will be described in detail below with reference to the accompanying drawings. The drawings are for illustrative purposes only and are not intended to limit the invention. Those skilled in the art can make some non-essential improvements and adjustments to this invention based on the above description. In the following embodiments, unless otherwise specified, all reagents used are commercially available.

[0017] Example 1: Preparation of NP-specific monoclonal antibodies 1) Isolation and purification of antibodies: New Zealand white rabbits were immunized with SFTSV NP protein (Reference: Lee K, Choi MJ, Cho MH, Choi DO, Bhoo SH. Antibody production and characterization of the nucleoprotein of sever fever with thrombocytopenia syndrome virus (SFTSV) for effective diagnosis of SFTSV. Virol J. 2023 Sep 7;20(1):206.) as an immunogen. After the rabbits showed an immune response and the serum antibody titer was assessed, they were euthanized. Figure 1 This is a graph showing the serum titer detection results of immunized rabbits in Example 1 of the present invention. Figure 1 As shown, rabbit 1 exhibited the highest antibody titer, and its spleen was obtained by separation.

[0018] 2) Culture B cells and screen for positive clones: Specific antibodies were detected in the supernatant of cultured B cells using ELISA. The antigen SFTSV-NP protein was coated on an enzyme-labeled plate, and the B cell culture supernatant was added for antigen-antibody incubation. Then, the cells were detected using the enzyme-labeled secondary antibody HRP Goat Anti-Rabbit IgG (H+L) (purchased from Jackson Immuno Research, catalog number: 111-035-045) to select positive clones and preserve them in cell cryopreservation medium.

[0019] 3) B-cell subcloning: RNA was extracted from B cells secreting the IP-10 antibody using the RNeasy mini Kit (Qiagen, catalog number 74104). RT-PCR was performed using a SuperScript III One-Step RT-PCR System with Platinum Taq DNA Polymerase (Invitrogen, catalog number 12574026) to reverse transcribe the specific B-cell RNA into cDNA, which was then amplified to encode the full-length fragments encoding the antibody heavy and light chains.

[0020] The primer sequences for heavy chain RT-PCR are as follows: RHC1: 5'-CCGTCCAAGCTTATGGAGACTGGGCTGCGCTGGC-3'; RHC2: 5'-CAACAAGGATCCCTATTTACCCGGAGAGCGGGAG-3'; The primer sequences for light chain RT-PCR are as follows: RLC1: 5'-CCGTCCAAGCTTATGGACACGAGGGCCCCACTC-3'; RLC2: 5'-CAACAAGGATCCCTAACAGTCACCCCTATTGAAGC-3.

[0021] The reaction conditions were 50℃ for 30 min, 94℃ for 2 min, followed by 35 cycles of (94℃ for 30 s, 57℃ for 30 s, 68℃ for 1 min), with an extension at 68℃ for 5 min and 4℃ for 5 min. After PCR amplification, the PCR products were purified by agarose gel electrophoresis.

[0022] Example 2: Sequencing of the full-length heavy and light chains of the monoclonal antibody DT-BunyaNPmAb encoding the anti-SFTSV-NP protein and production of the antibody via recombinant DNA. (1) The gene fragments recovered in Example 1 were subjected to full-length gene sequencing, and unique nucleotide / protein sequences were finally obtained. The sequence information is as follows: DT-BunyaNPmAb heavy chain variable region amino acid sequence (NO.1): METGLRWLLLVAVLKGVQCQSLEESGGRLVTPGTPLTLTCTASGFSLSTYYMTWVRQAPGKGLEWIGVINTGGSTYYASWAKGRFTISRTSTTVELKIASPTTEDTATYFCARDSYESYGLAYDIWGPGTLVAVSS.

[0023] DT-BunyaNPmAb heavy chain variable region DNA sequence (NO.2): ATGGAGACTGGGCTGCGCTGGCTTCTCCTGGTCGCTGTGCTCAAAGGTGTCCAGTGTCAGTCGCTGGAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGACACCCCTGACACTCACCTGCACAGCCTCTGGATTCTCCCTCAGTACTTACTACATGACCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAATGGATCGGGGTCATTAATACTGGTGGTAGCACATACTACGCGAGCTGGGCGAAAGGCCGATTCACCATCTCCAGAACCTCGACCACGGTGGAACTGAAAATCGCCAGTCCGACAACCGAGGACACGGCCACCTATTTCTGTGCCAGAGATAGTTACGAGAGCTATGGTCTTGCTTATGACATCTGGGGCCCAGGCACCCTGGTCGCCGTCTCCTCA.

[0024] Amino acid sequence of the light chain variable region of DT-BunyaNP mAb (NO.3): MDTRAPTQLLGLLLLWLPGATFAQVLTQTPSSVSAVVGGTVTINCQASQNVDNNNYLAWYHHKPGQPPKLLIYDASKLAAGVPSRFEGSGSGTHFTLTISGVQCDDAATYYCQATYYSSGRYGTFGGGTEVVVK.

[0025] DNA sequence of the light chain variable region of DT-BunyaNP mAb (NO.4): ATGGACACGAGGGCCCCCACTCAGCTGCTGGGGCTCCTGCTGCTCTGGCTCCCAGGTGCCACATTTGCGCAAGTGCTGACCCAGACTCCATCCTCCGTGTCTGCAGTTGTGGGAGGCACAGTCACCATCAATTGCCAGGCCAGTCAGAATGTTGATAATAACAACTACTTAGCCTGGTATCACCACAAACCAGGGCAGCCT CCCAAGCTCCTGATCTACGATGCATCCAAATTGGCAGCTGGGGTCCCATCCCGGTTCGAAGGCAGTGGATCTGGGACACACTTCACTCTCACCATCAGCGGCGTGCAGTGTGACGATGCTGCCACTTACTATTGTCAAGCCACTTATTATAGTAGTGGACGGTATGGTACTTTCGGCGGAGGGACCGAGGTGGTGGTCAAA.

[0026] (2) Production and purification of anti-SFTSV-NP antibody Antibody production using the Linear Expression Module (LEM) involves first constructing an expression plasmid, followed by antibody preparation during the LEM stage. In the LEM stage, the heavy and light chains of the subclonal unique clone are introduced into separate expression vectors, which are then transfected into HEK293F mammalian cells. Rabbit monoclonal antibodies are recombinantly expressed in HEK293F cells, yielding the LEM culture supernatant containing the rabbit monoclonal antibody.

[0027] Example 3: ELISA detection of the binding ability of LEM supernatant antibody to NP protein The recombinant rabbit monoclonal antibody LEM culture supernatant obtained in Example 2 contains monoclonal antibody IgG expressed and secreted by HEK293F cells. This IgG antibody, targeting a specific antigen, can bind to the SFTS-NP antigen, allowing detection of the SFTS-NP antigen in various scenarios, including diagnostic reagents. This invention utilizes ELISA to detect the antigen-antibody binding function. With the same amount of antigen, the LEM supernatant is serially diluted. Higher OD readings from both the conventional ELISA and capture ELISA used in this invention indicate higher antibody affinity for the antigen.

[0028] The standard ELISA testing steps are as follows: 1) ELISA screening precursor: SFTS-NP, 1 μg / ml, 100 μl / well, incubated at 37℃ for 1 h; 2) Blocking: ELISA blocking solution, 200 μl / well, incubate at 37℃ for 1 h; 3) Primary antibody: LEM supernatant was diluted 1:1 to the starting concentration, followed by 10 concentration gradients of 3-fold serial dilution, 100 μl / well, and incubated at 37℃ for 1 h; 4) Secondary antibody: HRP Goat Anti-Rabbit IgG (H+L) (purchased from Jackson Immuno Research, catalog number: 111-035-045), 1:20000, 100 μl / well, incubated at 37℃ for 1 h; 5) Color development: TMB, 80 μl / well, develop color at room temperature in the dark for 3 min; 6) Termination: H2SO4 termination solution, 40 μl / well.

[0029] ELISA test results as follows Figure 2 As shown, DT-BunyaNPmAb is the monoclonal antibody against SFTS-NP protein in this invention. The higher the antibody concentration in the supernatant, the higher the OD reading. As the supernatant is diluted, the OD reading shows a decreasing trend. Figure 2 The results showed that the monoclonal antibody in the LEM supernatant expressed by mammalian cells HEK293F could specifically bind to the SFTS-NP protein.

[0030] Example 4: Capture ELISA to detect the binding ability of antibodies to NP protein The steps for capturing an ELISA test are as follows: 1) Coating: Anti-Rabbit IgG Fc fragment, 1 μg / mL, 100 μl / well, coating (96-well plate), incubated at 37℃ for 1 h; 2) Blocking: ELISA blocking solution 200 μl / well, incubate at 37℃ for 1 h; 3) Capture antibody: LEM supernatant diluted 1:1, 100 μl / well, incubated at 37℃ for 1 h; 4) Primary antibody: SFTSV NP protein Nucleoprotein, starting with 1 μg / ml, 3-fold gradient, 10 concentration gradients, 100 μl / well, incubated at 37℃ for 1 h; 5) Secondary antibody: Serum samples from clinical SFTS patients, diluted 1:200, 100 μl / well, incubated at 37℃ for 1 h; Goatanti human IgG HRP 1:20000, 100 μl / well, incubated at 37℃ for 1 h; 6) Color development: TMB, 80 μl / well, develop color at room temperature in the dark for 3 min; 7) Termination: H2SO4, 40 μl / well.

[0031] Capture ELISA test results as follows Figure 3 As shown, the results indicate that the monoclonal antibody expressed in the LEM supernatant of mammalian cells HEK293F can specifically bind to the SFTS-NP protein. In the capture ELISA, the OD reading showed a significant increasing trend with the increase of the concentration of the antigen SFTS-NP protein, indicating that the monoclonal antibody has a high affinity for the SFTS-NP antigen.

[0032] Example 5: Western blotting detection of NP protein expression in SFTSV-infected cells This invention involves infecting human cervical cancer tumor cells (HeLa cells) with different viral doses of SFTSV (MOI=0, 0.5, 1, 2) and performing Western-Blot detection using SFTSV-NP antibody after different time periods (0, 2, 4, 6, 12, 24 hours). Figure 4 This is the Western blotting result of this embodiment. GAPDH (purchased from ABclonal, catalog number: AC002) is a commonly used WB internal control protein, whose expression is usually relatively stable and is often used to analyze relative protein expression levels. It was found that as the concentration of the SFTSV-NP antigen protein increased, the SFTSV NP protein band also showed a significant deepening trend; when the viral load (i.e., the SFTSV MOI) was the same, the SFTSV NP protein band deepened with prolonged treatment time. These results indicate that the SFTSV-NP antibody can be used for Western blotting to detect SFTSV NP protein.

[0033] Example 6: Immunofluorescence detection of NP protein expression in SFTSV-infected cells This invention uses SFTSV virus to infect Vero cells, the kidney cells of African green monkeys. Vero cells are seeded in 96-well plates with a culture medium of 10% FBS + 89% DMEM + 1% penicillin-dextrose antibody. SFTSV virus (MOI=1) is used to infect Vero cells, and samples are collected and processed after 48 hours. 100 µL of paraformaldehyde is added to each well, and the cells are fixed at room temperature for 40 minutes. The cells were then washed with PBS. 100 µL of 0.5% Triton was added to each well, and the cells were incubated at room temperature for 10 minutes, followed by another wash with PBS. 100 µL of 4% BSA was added to each well, and the cells were blocked at room temperature for 1 hour, followed by three washes with PBS. 100 µL of SFTSV NP antibody was added to each well, and the cells were incubated at room temperature in the dark for 1.5 hours, followed by another wash with PBS. 100 µL of Alexa Fluor 488-labeled goat anti-rabbit IgG (purchased from abcam, catalog number: ab150077) was added to each well, and the cells were incubated at room temperature in the dark for 1.5 hours, followed by three washes with PBS. 50 µL of the membrane permeability fluorescent dye Hoechst (purchased from Thermo Scientific, catalog number: 62249) was added to each well, and the cells were incubated at room temperature in the dark for 10 minutes, followed by another wash with PBS. Images were then taken using a ThunderImager high-throughput high-resolution fluorescence imager.

[0034] The above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A monoclonal antibody against a novel Bunyavirus NP protein, characterized in that, Includes variable regions of heavy chains and variable regions of light chains; The amino acid sequence of the heavy chain variable region is shown in NO.1: METGLRWLLLVAVLKGVQCQSLEESGGRLVTPGTPLTLTCTASGFSLSTYYMTWVRQAPGKGLEWIGVINTGGSTYYASWAKGRFTISRTSTTVELKIASPTTEDTATYFCARDSYESYGLAYDIWGPGTLVAVSS; The DNA sequence encoding the heavy chain variable region is shown in NO.2: ATGGAGACTGGGCTGCGCTGGCTTCTCCTGGTCGCTGTGCTCAAAGGTGTCCAGTGTCAGTCGCTGGAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGACACCCTGACACTCACCTGCACAGCCTCTGGATTCTCCCTCAGTACTTACTACATGACCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAATGGATCGGGGTC ATTAATACTGGTGGTAGCACATACTACGCGAGCTGGGCGAAAGGCCGATTCACCATCTCCAGAACCTCGACCACGGTGGAACTGAAAATCGCCAGTCCGACAACCGAGGACACGGCCACCTATTTCTGTGCCAGAGATAGTTACGAGAGCTATGGTCTTGCTTATGACATCTGGGGCCCAGGCACCCTGGTCGCCGTCTCCTCA; The amino acid sequence of the light chain variable region is shown in NO.3: MDTRAPTQLLGLLLLWLPGATFAQVLTQTPSSVSAVVGGTVTINCQASQNVDNNNYLAWYHHKPGQPPKLLIYDASKLAAGVPSRFEGSGSGTHFTLTISGVQCDDAATYYCQATYYSSGRYGTFGGGTEVVVK; The DNA sequence encoding the light chain variable region is shown in NO.4: ATGGACACGAGGGCCCCCACTCAGCTGCTGGGGCTCCTGCTGCTCTGGCTCCCAGGTGCCACATTTGCGCAAGTGCTGACCCAGACTCCATCCTCCGTGTCTGCAGTTGTGGGAGGCACAGTCACCATCAATTGCCAGGCCAGTCAGAATGTTGATAATAACAACTACTTAGCCTGGTATCACCACAAACCAGGGCAGCCT CCCAAGCTCCTGATCTACGATGCATCCAAATTGGCAGCTGGGGTCCCATCCCGGTTCGAAGGCAGTGGATCTGGGACACACTTCACTCTCACCATCAGCGGCGTGCAGTGTGACGATGCTGCCACTTACTATTGTCAAGCCACTTATTATAGTAGTGGACGGTATGGTACTTTCGGCGGAGGGACCGAGGTGGTGGTCAAA.

2. A kit for detecting NP antigen, characterized in that, The kit includes a monoclonal antibody against the novel Bunyavirus NP protein as described in claim 1, and reagents for detecting the monoclonal antibody.

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