A monoclonal antibody 23E2 against the NP protein of influenza B virus and its application

Abstract

This invention provides a monoclonal antibody 23E2 against the NP protein of influenza B virus and its applications. The heavy chain variable regions CDR1, CDR2, and CDR3 of the monoclonal antibody 23E2 are shown in SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8, respectively; the light chain variable regions CDR1, CDR2, and CDR3 are shown in SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12, respectively. This antibody exhibits high affinity and good specificity.

Description

Technical Field

[0001] This invention relates to a monoclonal antibody 23E2 against the NP protein of influenza B virus and its applications, belonging to the field of antibody technology. Background Technology

[0002] Influenza B virus is a single-stranded RNA membrane virus belonging to the Orthomyxoviridae family. It is one of the common influenza viruses and can cause influenza B. Influenza B patients typically experience symptoms such as headache, muscle pain, cough, sore throat, fatigue, and nasal congestion. In addition, patients often experience a sudden onset of high fever, with body temperature reaching 39°C or even higher within a few hours.

[0003] Influenza B virus is divided into two lineages: the Yamagata lineage and the Victoria lineage. The nucleoprotein (NP) plays a crucial role in the replication and transcription of influenza B virus. It binds to single-stranded RNA to form a ribonucleoprotein complex, thereby protecting the RNA. Simultaneously, NP assists RNA polymerase in recognizing and binding RNA, promoting RNA replication and transcription. The NP protein is highly conserved in both the Yamagata and Victoria lineages and can serve as a target for in vitro diagnostic (IVD) detection.

[0004] Currently, influenza B virus detection suffers from poor specificity and low sensitivity, which urgently need to be addressed. Antibodies are the core raw material for influenza B virus detection, and the preparation of NP monoclonal antibodies with high specificity and high sensitivity will help promote the development of influenza B virus detection reagents. Summary of the Invention

[0005] This invention provides a monoclonal antibody 23E2 against the NP protein of influenza B virus and its application, which can effectively solve the above-mentioned problems.

[0006] A monoclonal antibody 23E2 against the NP protein of influenza B virus has the following sequences: heavy chain variable regions CDR1, CDR2, and CDR3, as shown in SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8, respectively; and light chain variable regions CDR1, CDR2, and CDR3, as shown in SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12, respectively.

[0007] In some embodiments, the heavy chain variable region sequence of the influenza B virus NP protein monoclonal antibody 23E2 is shown in SEQ ID NO:5, and the light chain variable region sequence is shown in SEQ ID NO:9.

[0008] In some embodiments, the heavy chain sequence of the influenza B virus NP protein monoclonal antibody 23E2 is shown in SEQ ID NO:3, and the light chain sequence is shown in SEQ ID NO:4.

[0009] A reagent for detecting the NP protein of influenza B virus, comprising the aforementioned monoclonal antibody 23E2 against the NP protein of influenza B virus.

[0010] A kit for detecting influenza B virus NP protein, comprising the aforementioned influenza B virus NP protein monoclonal antibody 23E2.

[0011] The application of the aforementioned monoclonal antibody 23E2 against the influenza B virus NP protein in the preparation of reagents for the diagnosis and prevention of influenza B.

[0012] A nucleic acid molecule encoding the heavy chain variable region and light chain variable region of the monoclonal antibody 23E2 of the influenza B virus NP protein.

[0013] An expression vector comprising the aforementioned nucleic acid molecule.

[0014] A host cell comprising the expression vector described above.

[0015] A non-disease diagnosis and treatment detection method for influenza B, using the aforementioned influenza B virus NP protein monoclonal antibody 23E2 for immune detection.

[0016] The beneficial effects of this invention are:

[0017] This invention provides a monoclonal antibody named 23E2, which possesses significant functional characteristics, accurately recognizing and binding to NP proteins with extremely high affinity. Specifically, this monoclonal antibody effectively recognizes NP proteins from both the Yamagata and Victoria lineages, demonstrating broad lineage coverage and thus exhibiting excellent broad-spectrum applicability. Furthermore, the 23E2 antibody demonstrates outstanding specificity; it does not cross-react with NP proteins of influenza A virus or many other viruses, ensuring high specificity in the detection process. Based on these superior characteristics, the 23E2 monoclonal antibody is highly suitable for the development of influenza B virus detection reagents, providing strong technical support for the rapid and accurate detection of influenza B virus, and possesses broad application prospects and significant clinical value. Attached Figure Description

[0018] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation of the scope. For those skilled in the art, other related drawings can be obtained from these drawings without creative effort.

[0019] Figure 1 The image shows the purification results of the NP protein.

[0020] Figure 2 This is a graph showing the detection results of the ELISA method. Detailed Implementation

[0021] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Therefore, the following detailed description of the embodiments of the present invention provided in the accompanying drawings is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments of the invention.

[0022] The raw materials used in the embodiments of the present invention are as follows:

[0023] BL21(DE3) was purchased from Tiangen Biotech (Beijing) Co., Ltd.; yeast powder was purchased from Solarbio Biotechnology Co., Ltd.; peptone was purchased from OXOID; IPTG and kanamycin were purchased from Sangon Biotech (Shanghai) Co., Ltd.; NaCl was purchased from Shanghai Husheng; Freund's complete adjuvant and Freund's incomplete adjuvant were purchased from Sigma; 6-8 week old SPF-grade Balb / c mice were purchased from Fuzhou Wu's Animal Experiment Center; RPMI 1640 basal culture medium was purchased from Shanghai Yuanpei Biotechnology Co., Ltd.; HAT culture medium was purchased from Sigma; DMEM serum-free culture medium was purchased from Shanghai Yuanpei Biotechnology Co., Ltd.; fetal bovine serum was purchased from Shanghai Xiaopeng Biotechnology Co., Ltd.; goat anti-mouse antibody was purchased from Sigma; influenza B virus Yamagata and Victoria strains, influenza A virus H1N1, influenza A virus H3N2, respiratory syncytial virus, SARS-CoV-2, Ebola virus, and rabies virus NP protein were purchased from Yiqiao Shenzhou Biotechnology Co., Ltd.; parainfluenza virus and adenovirus NP protein were purchased from Jiangsu Dongkang Biomedical Technology Co., Ltd.

[0024] Example 1: Preparation of 23E2 monoclonal antibody

[0025] 1. Preparation of recombinant NP protein

[0026] The amino acid sequence encoding the NP protein (SEQ ID NO:1) was obtained from NCBI: XCN40336.1. A His tag was added to the C-terminus, and the nucleotide sequence was optimized according to the codon preference of E. coli (SEQ ID NO:2). The sequence was then synthesized into the pET28a vector by Suzhou Genewiz.

[0027] SEQ ID NO:1

[0028] MSNMDIDGINTGTIDKTPEEITSGTSGTTRPIIRPATLAPPSNKRTRNPSPERATTSSSEDDVGRKAQKKQTPTEIKKSVYNMVVKLGEFYNQMMVKAGLNDDMERNLIQNAHAVERILLAATDDKKTEFQKKKNARDVKEG KEEIDHNKTGGTFYKMVRDDKTIYFSPIRITFLKEEVKTMYKTTMGSDGFSGLNHIMIGHSQMNDVCFQRSKALKRVGLDPSLISTFAGSTVPRRSGATGVAIKGGGTLVAEAIRFIGRAMADRGLLRDIKAKTAYEKILLN LKNKCSAPQQKALVDQVIGSRNPGIADIEDLTLLARSMVVVRPSVASKVVLPISIYAKIPQLGFNVEEYSMVGYEAMALYNMATPVSILRMGDDARDKSQLFFMSCFGAAYEDLRVLSALTGTEFKPRSALKCKGFHVPAK EQVEGMGAALMSIKLQFWAPMTRSGGNEAGGDGGSGQISCSPVFAVERPIALSKQAVRRMLSMNIEGRDADVKGNLLKMMNDSMAKKTSGNAFIGKKMFQISDKNKTNPIEIPIKQTIPNFFFGRDTAEDYDDLDYHHHHHH

[0029] SEQ ID NO:2

[0030]

[0031] The constructed pET28a-NP plasmid was transformed into BL21(DE3) competent cells. After transformation, the bacterial culture was plated on LB agar plates containing kanamycin resistance and then incubated overnight at 37°C.

[0032] Single colonies were picked and inoculated into 5 mL of LB medium containing 50 μg / mL kanamycin, and cultured at 37°C and 220 rpm for 2-3 hours. The bacterial culture was then transferred to 200 mL of LB medium containing 50 μg / mL kanamycin and cultured at 37°C and 220 rpm until the OD600 value reached approximately 0.8. IPTG was then added to a final concentration of 0.25 mM, and the target protein expression was induced for 4-5 hours before sampling.

[0033] Centrifuge the bacterial culture at 4°C and 12,000 rpm for 30 minutes and collect the bacterial pellet. Resuspend the bacterial cells in Buffer A (20 mM PB, pH 7.4) and sonicate them. After sonication, centrifuge again at 4°C and 12,000 rpm for 30 minutes and collect the supernatant.

[0034] The NiFF column was equilibrated with Buffer A (20 mM PB, pH 7.4), and then the cell supernatant was loaded onto the NiFF column for purification. After loading, the column was first rinsed with Buffer A to equilibrate it, then eluted with Buffer B (20 mM PB, 10 mM imidazole, pH 7.4) to remove contaminating proteins, and finally eluted with Buffer C (20 mM PB, 200 mM imidazole, pH 7.4) to remove the target protein.

[0035] The purified NP protein was subjected to ultrafiltration and concentration, and finally stored in PBS (pH 7.4). The purification results are as follows: Figure 1 As shown.

[0036] 2. Mouse immunization

[0037] The NP protein was thoroughly emulsified with an equal volume of Freund's complete adjuvant. Six- to eight-week-old SPF-grade Balb / c mice were selected and injected subcutaneously at multiple sites, with each mouse receiving 200 μg. Two weeks later, the antigen was emulsified with Freund's incomplete adjuvant and injected subcutaneously at multiple sites again, with each mouse receiving 100 μg, for two booster immunizations. Three days before fusion, an intraperitoneal injection was administered for a pulse immunization.

[0038] 3. Preparation of feeder cells

[0039] BALB / c mouse peritoneal macrophages were selected as feeder cells. One day before fusion, BALB / c mice were euthanized by cervical retraction and their entire bodies were disinfected by immersion in 75% alcohol. Under aseptic conditions, the abdominal skin was incised with scissors to expose the peritoneum in a laminar flow hood. 5 mL of RPMI 1640 basal culture medium was injected into the peritoneal cavity using a syringe, and the cavity was repeatedly flushed. The flushing fluid was then collected. The flushing fluid was centrifuged at 1000 rpm for 5 minutes, and the pellet was retained. The pellet was resuspended in RPMI 1640 complete culture medium containing HAT, and the cell concentration was adjusted to 1 × 10⁻⁶ cells / mL. 5 Cells / mL. Add 150 μL of cell suspension to each well of a 96-well plate and incubate overnight at 37°C and 5% CO2.

[0040] 4. Preparation of immune spleen cells

[0041] Three days after the last immunization of mice, the spleen was removed under sterile conditions, placed in a petri dish, and rinsed once with RPMI 1640 basal culture medium. The spleen was then ground and filtered through a nylon mesh in a small beaker to prepare a cell suspension. The cell suspension was centrifuged, the supernatant was discarded, and the cells were resuspended in RPMI 1640 basal culture medium. This process was repeated three times, and finally, cell counting was performed.

[0042] 5. Cell fusion

[0043] (1) Take 40 mL of HAT culture medium, 15 mL of DMEM serum-free culture medium and 1 mL of 50% PEG (M12 000) respectively, and place them in a 37°C water bath for preheating.

[0044] (2) Take mouse myeloma cells Sp2 / 0 (2 - 5 × 10⁻⁵) respectively. 7 (10) of the above-mentioned immune spleen cells 8 Add the suspension (number of cells) to a 50 mL centrifuge tube and mix well. Then add DMEM serum-free culture medium to a final volume of 40 mL. Centrifuge the tube for 10 minutes, discard the supernatant, and mix the contents of the tube again.

[0045] (3) Place the centrifuge tube in water preheated to 37°C, add 0.7 mL of preheated 50% PEG solution to the tube, and let it stand for 90 seconds. Immediately add 15 mL of serum-free culture medium preheated to 37°C.

[0046] (4) Add DMEM serum-free culture medium to 40 mL, centrifuge for 10 minutes, and discard the supernatant. Add 40 mL of HAT culture medium containing 15%-20% fetal bovine serum. Mix the contents of the tube with a pipette and add 2 drops to each of the four wells of a 96-well cell culture plate containing feeder cells. Then, incubate the culture plates in a 37°C, 7% CO2 incubator.

[0047] 6. Selection, culture, and clone screening of hybridoma cells

[0048] On days 1, 3, 5, and 7 after cell fusion, the cells were cultured in the aforementioned HAT medium, and the medium was changed to select true hybrid cells. Subsequently, three rounds of subcloning were performed to screen for single clones that could specifically recognize NP, ultimately obtaining the hybridoma cell line 23E2.

[0049] 7. Expression and purification of ascites fluid

[0050] Balb / c mice were injected intraperitoneally with 0.5 mL of liquid paraffin. Ten days later, the selected hybridoma cell lines (1×10⁻⁶) were... 6 (Number of mice) were inoculated intraperitoneally into Balb / c mice. Approximately 10 days later, the mice's abdomens began to swell. At this point, the mice were euthanized by cervical dislocation, disinfected by immersion in 75% alcohol for 5 minutes, and ascites fluid was extracted once. The ascites fluid was purified using a Protein A affinity column to obtain the monoclonal antibody 23E2.

[0051] The sequence of monoclonal antibody 23E2, after sequencing, is as follows:

[0052] Heavy chain:

[0053] QVNIQNSGTEIAKPGSSVKFTCRASGYTWTKFTQWWIYERPVQGIEWIFTDFGTVWKEWQERFWEKATLSAERSGATAYYQLNSVMSEDGLSYCARHSFAFLSTESWGNGM TVTVASAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCK PCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKT KGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPG (SEQ IDNO:3)

[0054] Light chain:

[0055] DIFLSESPAIHSSSPGDKVSMTCGGATAFTWIKWYNQRSGTSFKRWIYEHGRVGTGIPVKFSASASGTSYTLTISSWDAEDAGTYYCDNYTGQVFSFGGSTKIEIKRA DAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC (SEQ IDNO:4)

[0056] Heavy chain variable region: QVNIQNSGTEIAKPGSSVKFTCRASGYTWTKFTQWWIYERPVQGIEWIFTDFGTVWKEWQERFWEKATLSAERSGATAYYQLNSVMSEDGLSYCARHSFAFLSTESWGNGMTVTVAS (SEQ ID NO: 5)

[0057] Heavy chain variable region CDR1: KFTQW (SEQ ID NO:6)

[0058] Heavy chain variable region CDR2: TDFGTVWKEWQERFWE (SEQ ID NO:7)

[0059] Heavy chain variable region CDR3: HSFAFLSTES (SEQ ID NO:8)

[0060] Light chain variable region:

[0061] DIFLSESPAIHSSSPGDKVSMTCGGATAFTWIKWYNQRSGTSFKRWIYEHGRVGTGIPVKFSASASGTSYTLTISSWDAEDAGTYYCDNYTGQVFSFGGSTKIEIK (SEQ ID NO: 9)

[0062] Light chain variable region CDR1: GGATAFTWIK (SEQ ID NO:10)

[0063] Light chain variable region CDR2: EHGRVGT (SEQ ID NO:11)

[0064] Light chain variable region CDR3: DNYTGQVFS (SEQ ID NO:12)

[0065] Example 2: Determining the detection linear range, sensitivity, and specificity using ELISA

[0066] The NP protein was dissolved in 20 mM PB pH 7.4 coating buffer at a concentration of 100 ng / mL and serially diluted 3-fold until a final concentration of 0.005 ng / mL was achieved. 100 μL of the diluted NP protein solution was added to the corresponding well, along with 100 μL of 100 ng / mL NP protein solution of other viruses such as influenza A H1N1 and H3N2. Wells containing only 20 mM PB pH 7.4 were used as negative controls (NC). The mixture was incubated overnight at 4°C.

[0067] Aspirate the liquid from the wells and wash three times with 300 μL of wash buffer. Add 300 μL of blocking buffer (prepared with PBST from 5% skim milk powder) to each well and incubate at 37°C for 1 h. Aspirate the liquid from the wells and wash three more times with 300 μL of wash buffer. Then add 100 μL of 1 μg / ml detection antibody 23E2 to each well.

[0068] Aspirate the liquid from the wells, wash three times with 300 μL of washing buffer, then add 100 μL of HRP-labeled rabbit anti-mouse antibody (diluted 1:5000 with PBST) to each well and incubate at 37°C for 30 min. Aspirate the liquid from the wells, and wash five times with 300 μL of washing buffer.

[0069] Add 100 μL of chromogenic solution to each well and incubate at 37°C for 10 min. Then add 50 μL of 2 mol / L H2SO4 stop solution. Within 20 min after adding the stop solution, read the OD450 value on a microplate reader.

[0070] The detailed test results are shown in Tables 1 and 2. Figure 2 Within the concentration range of 0.015-100 ng / mL, the 23E2 antibody exhibited a significant linear relationship with NP proteins, demonstrating its detection sensitivity and accuracy within this concentration range. Furthermore, the 23E2 antibody possesses the ability to simultaneously recognize NP proteins from both the Yamagata and Victoria lineages, indicating its broad applicability in influenza virus detection. Notably, for NP proteins of influenza A (H1N1) and several other different viral types, the readings of the 23E2 antibody were very close to those of the negative control (NC), meaning that there was no cross-reactivity between the 23E2 antibody and the NP proteins of these viruses, further confirming its strong specificity and ability to accurately identify target proteins in complex samples.

[0071] Table 1

[0072]

[0073] Table 2

[0074]

[0075] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the invention by those skilled in the art. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the invention should be included within the scope of protection of the invention.

Claims

1. A monoclonal antibody 23E2 against the NP protein of influenza B virus, characterized in that, The sequences of the heavy chain variable regions CDR1, CDR2, and CDR3 are shown in SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8, respectively; the sequences of the light chain variable regions CDR1, CDR2, and CDR3 are shown in SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12, respectively.

2. The influenza B virus NP protein monoclonal antibody 23E2 according to claim 1, characterized in that, The heavy chain variable region sequence is shown in SEQ ID NO:5, and the light chain variable region sequence is shown in SEQ ID NO:

9.

3. The influenza B virus NP protein monoclonal antibody 23E2 according to claim 1, characterized in that, Its heavy chain sequence is shown in SEQ ID NO:3, and its light chain sequence is shown in SEQ ID NO:

4.

4. A reagent for detecting the NP protein of influenza B virus, characterized in that, Including the influenza B virus NP protein monoclonal antibody 23E2 as described in any one of claims 1 to 3.

5. A kit for detecting the NP protein of influenza B virus, characterized in that, Including the influenza B virus NP protein monoclonal antibody 23E2 as described in any one of claims 1 to 3.

6. The use of the monoclonal antibody 23E2 against the influenza B virus NP protein according to any one of claims 1 to 3 in the preparation of a reagent for diagnosing influenza B.

7. A nucleic acid molecule encoding the heavy chain variable region and the light chain variable region of the monoclonal antibody 23E2 against the influenza B virus NP protein as described in any one of claims 1 to 3.

8. An expression vector comprising the nucleic acid molecule of claim 7.

9. A host cell comprising the expression vector of claim 8.

10. A detection method for non-disease diagnosis and treatment of influenza B, characterized in that, Immunological detection was performed using the monoclonal antibody 23E2 of the influenza B virus NP protein as described in any one of claims 1 to 3.

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