Monoclonal antibody for resisting novel bunyavirus NP protein as well as preparation method and application of monoclonal antibody

Abstract

The invention discloses a monoclonal antibody for resisting novel bunyavirus nucleocapsid protein (NP). The monoclonal antibody comprises a heavy chain variable region and a light chain variable region, the amino acid sequence of the heavy chain variable region is as shown in NO.1; the coding 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; and the coding DNA sequence of the light chain variable region is as shown in NO.4. The monoclonal antibody for resisting the novel bunyavirus NP protein can be specifically combined with the novel bunyavirus NP protein, and a basis is established for diagnosing virus infectious diseases through a genetic engineering method; the monoclonal antibody for resisting the novel bunyavirus NP protein has a specific antigen binding structural domain, can be combined with the novel bunyavirus NP protein in a targeted manner, and is used for antigen detection.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a monoclonal antibody against a novel bunyavirus nucleocapsid protein (NP) protein, and a preparation method and application thereof. Background Art

[0002] Sever fever with thrombocytopenia syndrome virus (SFTSV) is a new virus isolated and identified in recent years. The sever fever with thrombocytopenia syndrome (SFTS) caused by the virus infecting humans is characterized by fever, thrombocytopenia and leukopenia as the main clinical manifestations. Most cases have clinical symptoms such as fatigue, gastrointestinal symptoms, muscle aches and lymphadenopathy. Severe cases may suffer from shock, respiratory failure, disseminated intravascular coagulation and other multi-organ failure and die. SFTS is a new type of hemorrhagic fever, an acute infectious disease. The national average mortality rate of SFTS infection is 5.3%, and the mortality rate of the disease is as high as 30% in some endemic areas. Since the clinical manifestations of SFTS are non-specific, it is easy to misdiagnose, which delays the disease and leads to severe disease or even death. The genome of SFTSV is divided into three single-stranded negative-strand RNA fragments: large (L), medium (M), and small (S). The S segment has 1744 nucleotides and is a double-sense RNA segment that encodes the viral nucleocapsid protein (NP) and the viral nonstructural proteins (NSs). NP is the most abundant protein in SFTSV virus particles and infected cells. It protects viral RNA and participates in viral replication and viral assembly. NP has a highly conserved amino acid sequence and can be used as a protein or molecular marker for virus detection.

[0003] Currently, there is no effective vaccine or drug for SFTS. It is of great significance to develop a diagnostic kit for SFTS disease based on SFTSV-NP as an antigen, or to achieve early diagnosis of SFTS and control the clinical course of SFTSV infection through targeted therapy with anti-SFTSV-NP monoclonal antibodies. Summary of the invention

[0004] In view of this, the present invention provides a novel monoclonal antibody specific for the NP protein of Bunyavirus and its preparation method and application, which can realize the quantitative detection of recombinant NP antigen and NP protein in clinical patient serum by ELISA method. The specific scheme is as follows: In a first aspect, the present invention provides a monoclonal antibody against the 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 coding DNA sequence of 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 coding DNA sequence of the light chain variable region is shown in NO.4: ATGGACACGAGGGCCCCCACTCAGCTGCTGGGGCTCCTGCTGCTCTGGCTCCCAGGTGCCACATTTGCGCAAGTGCTGACCCAGACTCCATCCTCCGTGTCTGCAGTTGTGGGAGGCACAGTCACCATCAATTGCCAGGCCAGTCAGAATGTTGATAATAACAACTACTTAGCCTGGTATCACCACAAACCAGGGCAGCCT CCCAAGCTCCTGATCTACGATGCATCCAAATTGGCAGCTGGGGTCCCATCCCGGTTCGAAGGCAGTGGATCTGGGACACACTTCACTCTCACCATCAGCGGCGTGCAGTGTGACGATGCTGCCACTTACTATTGTCAAGCCACTTATTATAGTAGTGGACGGTATGGTACTTTCGGCGGAGGGACCGAGGTGGTGGTCAAA.

[0005] In a second aspect, the present invention provides a method for preparing a monoclonal antibody against the novel bunyavirus NP protein, comprising the following steps: 1) Immunize rabbits with SFTSV NP protein as an immunogen, and kill the rabbits after they have made an immune response. Take out their spleens and isolate spleen cells; 2) Screening of single B cells with antigen specificity; 3) Subcloning the B cells to obtain the variable region coding sequences of the antibody heavy and light chains; 4) The obtained variable region coding sequence is recombined, transfected and purified to obtain monoclonal antibodies against the novel Bunyavirus NP protein.

[0006] In a third aspect, the present invention provides a use of the anti-SFTSV NP protein monoclonal antibody, wherein the monoclonal antibody is used to prepare a drug for treating or preventing human viral infection.

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

[0008] In a fourth aspect, the present invention provides a kit for detecting NP antigen, the kit comprising a monoclonal antibody against the novel bunyavirus NP protein, and a reagent for detecting the monoclonal antibody.

[0009] The beneficial effects of the present invention are as follows: 1. The present invention provides an anti-NP monoclonal antibody and its preparation method and application, which can specifically bind to NP antigen, and establish a basis for diagnosing viral infection through genetic engineering methods; 2. The anti-NP monoclonal antibody provided by the present invention can accurately measure the NP concentration in the serum and can quickly help doctors determine the immune status of patients; 3. The present invention provides a NP rabbit-derived monoclonal antibody, which enriches the types of antibodies; 4. The anti-NP monoclonal antibody of the present invention has a specific antigen binding domain, can target and bind to NP protein, and can also be used in immunohistochemistry, ELISA and other tests. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

[0012] Figure 2 This is a graph showing the results of ELISA in Example 3 of the present invention detecting that the antibody in LEM supernatant can specifically bind to SFTSV-NP protein.

[0013] Figure 3 This is a graph showing the results of the capture ELISA in Example 4 of the present invention detecting that the antibody in the LEM supernatant can specifically bind to the SFTSV-NP protein.

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

[0015] Figure 5 This is the immunofluorescence result of African green monkey kidney cells Vero cells in Example 6 of the present invention. DETAILED DESCRIPTION

[0016] In order to be able to understand the characteristics and technical contents of the present invention in more detail, the implementation of the present invention is described in detail below in conjunction with the accompanying drawings, which are only for reference and explanation purposes and are not used to limit the present invention. Those skilled in the art can make some non-essential improvements and adjustments to the present invention based on the above-mentioned content of the present invention. In the following examples, unless otherwise indicated, the reagents used can be obtained from commercial channels.

[0017] Example 1: Preparation of NP-specific monoclonal antibodies 1) Isolation and purification of antibodies: Use SFTSV NP protein (reference: Lee K, Choi MJ, Cho MH, Choi DO, Bhoo SH. Antibody production and characterization of the nucleoprotein ofsever fever with thrombocytopenia syndrome virus (SFTSV) for effective diagnosis of SFTSV. Virol J. 2023 Sep 7;20(1):206.) as an immunogen to stimulate New Zealand white rabbits for immunity. After the rabbits have an immune response and the serum antibody titer is evaluated, they are killed. Figure 1 : is a graph showing the serum titer test results of immunized rabbits in Example 1 of the present invention, as shown in Figure 1 As shown, rabbit 1 showed the highest antibody titer, and its spleen was isolated and obtained.

[0018] 2) Cultivate single B cells and screen positive clones: Use ELISA to detect specific antibodies in the supernatant of single B cells after culture. Coat the antigen SFTSV-NP protein on the ELISA plate, add the single B cell culture supernatant to incubate the antigen and antibody, and then use the anti-rabbit enzyme-labeled secondary antibody HRP Goat Anti-Rabbit IgG (H+L) (purchased from Jackson ImmunoResearch, catalog number: 111-035-045) for detection, and then select positive clones and store their cells in cell freezing solution.

[0019] 3) B cell subcloning: RNA extraction kit RNeasy mini Kit (purchased from Qiagen, catalog number: 74104) was used to extract RNA from B cells secreting IP-10 antibody. RT-PCR reaction was performed using SuperScript III One-Step RT-PCR Systemwith Platinum Taq DNA Polymerase (purchased from Invitrogen, catalog number: 12574026), and RNA of specific B cells was reverse transcribed into cDNA, and then full-length fragments encoding antibody heavy and light chains were amplified respectively.

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

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

[0022] Example 2: Full-length gene sequencing of the heavy and light chains of the anti-SFTSV-NP protein monoclonal antibody DT-BunyaNPmAb and antibody recombinant production (1) The gene fragments recovered in Example 1 were subjected to full-length gene sequencing to finally obtain unique nucleotide / protein sequences, and the sequence information was 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 variable region of the DT-BunyaNPmAb light chain (NO. 3): MDTRAPTQLLGLLLLWLPGATFAQVLTQTPSSVSAVVGGTVTINCQASQNVDNNNYLAWYHHKPGQPPKLLIYDASKLAAGVPSRFEGSGSGTHFTLTISGVQCDDAATYYCQATYYSSGRYGTFGGGTEVVVK。

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

[0026] (2) Production and purification of anti-SFTSV-NP antibodies Antibodies are produced by the Linear Expression Module (LEM). First, an expression plasmid is constructed, and then the LEM stage antibody is prepared. In the LEM stage, the uniquely cloned heavy and light chains are subcloned into independent expression vectors, transfected in mammalian cells HEK293F, and rabbit monoclonal antibodies are recombinantly expressed by HEK293F cells to obtain LEM culture supernatant containing rabbit monoclonal antibodies.

[0027] Example 3: ELISA to detect the binding ability of LEM supernatant antibodies to NP protein For the recombinant rabbit monoclonal antibody LEM culture supernatant obtained in Example 2, the LEM supernatant contains monoclonal antibody IgG expressed and secreted by HEK293F cells. The antibody IgG against the specific antigen can bind to the antigen SFTS-NP, and the SFTS-NP antigen can be detected in different scenarios such as diagnostic reagents. The present invention uses ELISA to detect the binding function of antigen and antibody. When the amount of antigen is the same, the LEM supernatant is gradiently diluted. The higher the OD reading of the conventional ELISA and capture ELISA used in the present invention, the higher the affinity of the antibody binding to the antigen.

[0028] The routine ELISA test steps are as follows: 1) ELISA screening source: SFTS-NP, 1 μg / ml, 100 μl / well, incubate at 37℃ for 1h; 2) Blocking: ELISA blocking solution, 200 μl / well, incubate at 37°C for 1 h; 3) Primary antibody: LEM supernatant was diluted 1:1 as the starting concentration, 3-fold serial dilutions were performed for 10 concentrations, 100 μl / well, incubated at 37°C for 1 h; 4) Secondary antibody: HRP Goat Anti-Rabbit IgG (H+L) (purchased from Jackson ImmunoResearch, catalog number: 111-035-045), 1:20000, 100 μl / well, incubate at 37°C for 1 hour; 5) Color development: TMB, 80 μl / well, color development at room temperature in the dark for 3 min; 6) Termination: H2SO4 stop solution, 40 μl / well.

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

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

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

[0032] Example 5: Detection of NP protein expression in SFTSV-infected cells by Western Blot The present invention infects human cervical cancer tumor cells (Hela cells) with different virus doses of SFTSV (MOI=0, 0.5, 1, 2), and performs Western-Blot detection using SFTSV-NP antibody after different time periods (0, 2, 4, 6, 12, 24 hours). Figure 4 This is the Western-Blot result of this example. In the figure, GAPDH (purchased from ABclonal, item number: AC002) is a commonly used WB internal reference protein, and its expression is usually relatively stable, and is often used to analyze relative protein expression levels. It was found that with the increase in the concentration of the antigen SFTSV-NP protein, the SFTSV NP protein band also showed a significant deepening trend; when the dose of the infected virus added, that is, the same SFTSV MOI, the SFTSV NP protein band deepened with the extension of the treatment time. The above results show that the SFTSV-NP antibody can be used for Western Blot to detect SFTSV NP protein.

[0033] Example 6: Immunofluorescence detection of NP protein expression in SFTSV-infected cells The present invention infects African green monkey kidney Vero cells with SFTSV virus, and the Vero cells are plated in a 96-well flat-bottom plate, and the culture medium is 10% FBS+89% DMEM+1% double antibody. SFTSV virus (MOI=1) infects Vero cells, and samples are collected and processed after 48 hours. 100μL of paraformaldehyde is added to each well and fixed at room temperature for 40 minutes. Then wash with PBS, add 100µL 0.5% Triton to each well, incubate at room temperature for 10 minutes, and wash with PBS; add 100µL 4% BSA to each well, block at room temperature for 1 hour, and wash three times with PBS; add 100µL SFTSV NP antibody to each well, incubate at room temperature in the dark for 1.5 hours, and then wash with PBS; add 100µL Alexa Fluor 488-labeled goat anti-rabbit IgG (purchased from abcam, catalog number: ab150077) to each well, incubate at room temperature in the dark for 1.5 hours, and wash three times with PBS; add 50µL membrane permeable fluorescent dye Hoechst (purchased from Thermo Scientific, catalog number: 62249) to each well, incubate at room temperature in the dark for 10 minutes, wash with PBS, and take pictures using ThunderImager high-throughput and high-resolution fluorescence imager.

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

Claims

1. A monoclonal antibody against the NP protein of a novel Bunyavirus, characterized in that: including 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 coding DNA sequence of 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 coding DNA sequence of the light chain variable region is shown in NO.4: ATGGACACGAGGGCCCCCACTCAGCTGCTGGGGCTCCTGCTGCTCTGGCTCCCAGGTGCCACATTTGCGCAAGTGCTGACCCAGACTCCATCCTCCGTGTCTGCAGTTGTGGGAGGCACAGTCACCATCAATTGCCAGGCCAGTCAGAATGTTGATAATAACAACTACTTAGCCTGGTATCACCACAAACCAGGGCAGCCT CCCAAGCTCCTGATCTACGATGCATCCAAATTGGCAGCTGGGGTCCCATCCCGGTTCGAAGGCAGTGGATCTGGGACACACTTCACTCTCACCATCAGCGGCGTGCAGTGTGACGATGCTGCCACTTACTATTGTCAAGCCACTTATTATAGTAGTGGACGGTATGGTACTTTCGGCGGAGGGACCGAGGTGGTGGTCAAA.

2. A method for preparing a monoclonal antibody against a novel bunyavirus NP protein, characterized in that: The following steps are involved: 1) Immunize rabbits with SFTSV NP protein as an immunogen, and kill the rabbits after they have made an immune response. Take out their spleens and isolate spleen cells; 2) Screening of single B cells with antigen specificity; 3) Subcloning the B cells to obtain the variable region coding sequences of the antibody heavy and light chains; 4) The obtained variable region coding sequence is recombined, transfected and purified to obtain monoclonal antibodies against the novel Bunyavirus NP protein.

3. The use of a monoclonal antibody against the NP protein of a novel bunyavirus according to claim 1, characterized in that: The monoclonal antibody is used for preparing medicine for treating or preventing human viral infection.

4. The use of a monoclonal antibody against the NP protein of a novel bunyavirus according to claim 3, characterized in that: The monoclonal antibody is used for protein expression in Western Blot and / or immunofluorescence detection.

5. A kit for detecting NP antigen, characterized in that: The kit comprises a monoclonal antibody against the novel bunyavirus NP protein and a reagent for detecting the monoclonal antibody.

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