Monoclonal antibody 23E2 of influenza B virus NP protein and application thereof

Abstract

The invention provides an influenza B virus NP protein monoclonal antibody 23E2 and application thereof, the sequences of heavy chain variable regions CDR1, CDR2 and CDR3 of the influenza B virus NP protein monoclonal antibody 23E2 are respectively shown as SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8; the sequences of the light chain variable regions CDR1, CDR2 and CDR3 are respectively as shown in SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12. The antibody is high in affinity and good in specificity.

Description

TECHNICAL FIELD

[0001] The application relates to an influenza B virus NP protein monoclonal antibody 23E2 and application thereof, and belongs to the technical field of antibodies. BACKGROUND

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

[0003] Influenza B virus is divided into Yamagata and Victoria lineages. Nucleoprotein (NP) plays a key role in the replication and transcription of influenza B virus, which can bind to single-stranded RNA to form a ribonucleoprotein complex to protect RNA. At the same time, NP can also help RNA polymerase to recognize and bind to RNA, and promote the replication and transcription of RNA. NP protein has high conservation in Yamagata and Victoria lineages, and can be used as a target for IVD detection.

[0004] At present, the detection of influenza B virus has the problems of poor specificity and low sensitivity, which need to be solved. Antibodies are the core raw materials for the detection of influenza B virus, and the preparation of NP monoclonal antibodies with high specificity and high sensitivity can help to promote the development of influenza B virus detection reagents. SUMMARY The application provides an influenza B virus NP protein monoclonal antibody 23E2 and application thereof, which can effectively solve the above problems.

[0005] The influenza B virus NP protein monoclonal antibody 23E2 has the sequence of the heavy chain variable region CDR1, CDR2 and CDR3 shown in SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8 respectively; and the sequence of the light chain variable region CDR1, CDR2 and CDR3 shown in SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12 respectively. In some embodiments, the influenza B virus NP protein monoclonal antibody 23E2 has the sequence of the heavy chain variable region shown in SEQ ID NO: 5, and the sequence of the light chain variable region shown in SEQ ID NO: 9.

[0006] In some embodiments, the influenza B virus NP protein monoclonal antibody 23E2 has the sequence of the heavy chain shown in SEQ ID NO: 3, and the sequence of the light chain shown in SEQ ID NO: 4.

[0007] A reagent for detecting NP protein of influenza B virus, comprising the NP protein of influenza B virus monoclonal antibody 23E2.

[0008] A kit for detecting NP protein of influenza B virus, comprising the NP protein of influenza B virus monoclonal antibody 23E2.

[0009] Use of the NP protein of influenza B virus monoclonal antibody 23E2 in the preparation of a reagent for diagnosing and preventing influenza B.

[0010] A nucleic acid molecule encoding the heavy chain variable region and the light chain variable region of the NP protein of influenza B virus monoclonal antibody 23E2.

[0011] An expression vector comprising the nucleic acid molecule.

[0012] A host cell comprising the expression vector.

[0013] A method for detecting non-disease diagnosis and treatment of influenza B, using the NP protein of influenza B virus monoclonal antibody 23E2 for immunodetection.

[0014] The beneficial effects of the present application are: The present application provides a monoclonal antibody named 23E2, which has significant functional characteristics, can accurately recognize and bind NP protein, and exhibits extremely high affinity in the binding process. Specifically, this monoclonal antibody can effectively recognize NP proteins of two different lineages, Yamagata and Victoria, showing its wide lineage coverage ability, thereby exhibiting good broad-spectrum in application. In addition, the 23E2 antibody also performs outstandingly in specificity, it does not cross-react with NP proteins of influenza A virus and other viruses, ensuring its high specificity in the detection process. Based on these excellent characteristics, the 23E2 monoclonal antibody is very suitable for the development of influenza B virus detection reagents, providing strong technical support for rapid and accurate detection of influenza B virus, and has broad application prospects and important clinical value. BRIEF DESCRIPTION OF DRAWINGS

[0015] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the following will briefly introduce the drawings needed to be used in the embodiments. It should be understood that the following drawings only show some embodiments of the present application, and therefore should not be regarded as a limitation on the scope, and for those skilled in the art, other related drawings can also be obtained without creative labor on the basis of these drawings.

[0016] Figure 1 Figure for NP protein purification results.

[0017] Figure 2 Figure for detection results of ELISA method. DETAILED DESCRIPTION

[0018] In order to make the purposes, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be described clearly and completely below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative labor fall within the scope of protection of the present application. Therefore, the following detailed description of the embodiments of the present application provided in the drawings is not intended to limit the scope of the claimed present application, but only represents selected embodiments of the present application.

[0019] The raw materials used in the embodiments of the present application are as follows: BL21 (DE3) was purchased from Tiangene Biochem (Beijing) Co., Ltd.; yeast powder was purchased from Solabio Biotechnology Co., Ltd.; proteose peptone was purchased from OXOID company; IPTG and kanamycin were purchased from Shengong Bioengineering (Shanghai) Co., Ltd.; NaCl was purchased from Shanghai Huishi; Freund's complete adjuvant and Freund's incomplete adjuvant were purchased from Sigma; 6-8 week old SPF Balb / c mice were purchased from Fuzhou Wu's Animal Experimental Center; RPMI 1640 basic culture medium was purchased from Shanghai Yunduo Biotechnology Co., Ltd.; HAT culture medium was purchased from Sigma; DMEM serum-free culture medium was purchased from Shanghai Yunduo Biotechnology Co., Ltd.; fetal bovine serum was purchased from Shanghai Xiaopeng Biological Technology Co., Ltd.; goat anti-mouse antibody was purchased from Sigma; influenza B virus Yamagata and Victoria, influenza A virus H1N1, influenza A virus H3N2, respiratory syncytial virus, new coronavirus, Ebola virus, and rabies virus NP proteins were purchased from Yiqiao God of the World Biological Technology Co., Ltd.; parainfluenza virus and adenovirus NP proteins were purchased from Jiangsu Eastland Biomedicine Technology Co., Ltd. Example 1: Preparation of 23E2 monoclonal antibody 1. Preparation of recombinant NP protein The amino acid sequence (SEQ ID NO: 1) encoding the NP protein is derived from NCBI: XCN40336.1, a His tag is added at the C-terminus, the nucleotide sequence (SEQ ID NO: 2) is optimized according to the codon bias of Escherichia coli, and the sequence is synthesized by Suzhou Jinyuizhi into a pET28a vector.

[0020] SEQ ID NO: 1 MSNMDIDGINTGTIDKTPEEITSGTSGTTRPIIRPATLAPPSNKRTRNPSPERATTSSEDDVGRKAQKKQTPTEIKKSVYNMVVKLGEFYNQMMVKAGLNDDMERNLIQNAHAVERILLAATDDKKTEFQKKKNARDVKEGKEEIDHNKTGGTFYKMVRDDKTIYFSPIRITFLKEEVKTMYKTTMGSDGFSGLNHIMIGHSQMNDVCFQRSKALKRVGLDPSLISTFAGSTVPRRSGATGVAIKGGGTLVAEAIRFIGRAMADRGLLRDIKAKTAYEKILLNLKNKCSAPQQKALVDQVIGSRNPGIADIEDLTLLARSMVVVRPSVASKVVLPISIYAKIPQLGFNVEEYSMVGYEAMALYNMATPVSILRMGDDARDKSQLFFMSCFGAAYEDLRVLSALTGTEFKPRSALKCKGFHVPAKEQVEGMGAALMSIKLQFWAPMTRSGGNEAGGDGGSGQISCSPVFAVERPIALSKQAVRRMLSMNIEGRDADVKGNLLKMMNDSMAKKTSGNAFIGKKMFQISDKNKTNPIEIPIKQTIPNFFFGRDTAEDYDDLDYHHHHHH SEQ ID NO: 2 The constructed pET28a-NP plasmid was transformed into BL21(DE3) competent cells. After transformation, the bacterial solution was spread on LB plates containing kanamycin resistance, and then placed in a 37°C incubator for overnight culture.

[0021] A single colony was picked and inoculated into 5 mL of LB medium containing 50 μg / mL kanamycin, and cultured at 37°C, 220 rpm for 2-3 hours. Then, the bacterial solution was transferred to 200 mL of LB medium containing 50 μg / mL kanamycin, and continued to be cultured at 37°C, 220 rpm until the OD600 value reached about 0.8. Then IPTG was added to a final concentration of 0.25 mM, and the target protein expression was induced for 4-5 hours before sampling.

[0022] The bacterial solution was centrifuged at 4°C, 12000 rpm for 30 minutes to collect the bacterial pellet. The bacterial pellet was resuspended with Buffer A (20 mM PB, pH 7.4) and broken using an ultrasonic disrupter. After breaking, the supernatant was collected by centrifugation at 4°C, 12000 rpm for 30 minutes.

[0023] 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 washed with Buffer A to balance the column, then the impurities were eluted with Buffer B (20 mM PB, 10 mM imidazole, pH 7.4), and finally the target protein was eluted with Buffer C (20 mM PB, 200 mM imidazole, pH 7.4).

[0024] The purified NP protein was ultrafiltrated and concentrated, and finally stored in PBS (pH 7.4). The purification results are shown in Figure 1 . 2. Mouse Immunization The NP protein was emulsified with an equal volume of Freund's complete adjuvant. SPF grade Balb / c mice of 6-8 weeks old were selected and injected subcutaneously with 200 μg of antigen at multiple sites. Two weeks later, the antigen was emulsified with Freund's incomplete adjuvant and injected subcutaneously again, with 100 μg per mouse, for 2 times of booster immunization. Three days before fusion, intraperitoneal injection was performed for challenge immunization.

[0025] 3. Preparation of feeder cells BALB / c mouse peritoneal macrophage was selected as the feeder cell. One day before fusion, BALB / c mouse was killed by cervical dislocation, and its whole body was sterilized by 75% alcohol. In the clean bench, the abdominal skin was cut open with scissors to expose the peritoneum according to the aseptic operation standard. 5 mL of RPMI 1640 basic culture solution was injected into the abdominal cavity using a syringe to repeatedly rinse the abdominal cavity, and then the rinsing solution was recovered. The rinsing solution was centrifuged at 1000 rpm for 5 minutes, and the precipitate was reserved. The precipitate was resuspended in RPMI 1640 complete culture solution containing HAT, and the cell concentration was adjusted to 1 x 10 5 The cell suspension was added to a 96-well plate, 150 μL was added to each well, and the plate was incubated in a 37°C, 5% CO2 environment overnight.

[0026] 4. Preparation of immune spleen cells Three days after the last immunization of the mouse, the spleen was taken out under sterile conditions, placed in a dish, and rinsed once with RPMI 1640 basic culture solution. Then the spleen was ground and filtered on a nylon mesh in a small beaker to prepare a cell suspension. The cell suspension was centrifuged, and the supernatant was discarded. The cells were resuspended in RPMI 1640 basic culture solution, and this operation was repeated three times. Finally, the cells were counted.

[0027] 5. Cell fusion (1) 40 mL of HAT culture solution, 15 mL of DMEM serum-free culture solution, and 1 mL of 50% PEG (M12 000) were taken respectively and preheated in a 37°C water bath.

[0028] (2) Mouse myeloma cells Sp2 / 0 (2 - 5 x 10 7 cells) and the above immune spleen cell (10 8 mL) suspension were taken respectively, mixed in a 50 mL centrifuge tube, and DMEM serum-free culture solution was added to 40 mL. The centrifuge tube was centrifuged for 10 minutes, the supernatant was poured out, and the tube contents were mixed again.

[0029] (3) The centrifuge tube was placed in a 37°C preheated water, 0.7 mL of preheated 50% PEG solution was added to the tube, and it was left for 90 seconds. Immediately, 15 mL of 37°C serum-free culture solution was added dropwise.

[0030] (4) DMEM serum-free culture solution was added to 40 mL, centrifuged for 10 minutes, and the supernatant was poured out. 40 mL of HAT culture solution containing 15% - 20% fetal bovine serum was added. The tube contents were mixed with a pipette and added dropwise to the small holes of the four 96-well cell culture plates containing feeder cells, 2 drops were added to each well, and then the culture plates were incubated in a 37°C, 7% CO2 incubator.

[0031] 6. Selection and cloning of hybridoma cells After cell fusion, the HAT medium was used to replace the culture medium on the 1st, 3rd, 5th, and 7th days to select the true hybrid cells. Three rounds of subcloning were then performed to screen for monoclonal cells that specifically recognize NP, and the hybridoma cell line 23E2 was ultimately obtained.

[0032] 7. Ascites expression and purification The hybridoma cell line (1 x 10 6 After about 10 days, the mouse abdomen began to swell. At this time, the mouse was sacrificed by cervical dislocation, soaked in 75% alcohol for 5 minutes, and ascites was extracted once. The ascites was purified using a Protein A affinity column to obtain the monoclonal antibody 23E2. After sequencing, the sequence of the monoclonal antibody 23E2 was as follows: Heavy chain: QVNIQNSGTEIAKPGSSVKFTCRASGYTWTKFTQWWIYERPVQGIEWIFTDFGTVWKEWQERFWEKATLSAERSGATAYYQLNSVMSEDGLSYCARHSFAFLSTESWGNGMTVTVASAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPG (SEQ ID NO: 3) Light chain: DIFLSESPAIHSSSPGDKVSMTCGGATAFTWIKWYNQRSGTSFKRWIYEHGRVGTGIPVKFSASASGTSYTLTISSWDAEDAGTYYCDNYTGQVFSFGGSTKIEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC (SEQ ID NO: 4) Heavy chain variable region: QVNIQNSGTEIAKPGSSVKFTCRASGYTWTKFTQWWIYERPVQGIEWIFTDFGTVWKEWQERFWEKATLSAERSGATAYYQLNSVMSEDGLSYCARHSFAFLSTESWGNGMTVTVAS (SEQ ID NO: 5) Heavy chain variable region CDR1 : KFTQW (SEQ ID NO: 6) Heavy chain variable region CDR2: TDFGTVWKEWQERFWE (SEQ ID NO: 7) Heavy chain variable region CDR3: HSFAFLSTES (SEQ ID NO: 8) Light chain variable region: DIFLSESPAIHSSSPGDKVSMTCGGATAFTWIKWYNQRSGTSFKRWIYEHGRVGTGIPVKFSASASGTSYTLTISSWDAEDAGTYYCDNYTGQVFSFGGSTKIEIK (SEQ ID NO: 9) Light chain variable region CDR1 : GGATAFTWIK (SEQ ID NO: 10) Light chain variable region CDR2: EHGRVGT (SEQ ID NO: 11) Light chain variable region CDR3: DNYTGQVFS (SEQ ID NO: 12) Example 2: Determination of the linear range, sensitivity and specificity by ELISA NP protein was dissolved in coating solution of 20 mM PB pH 7.4 at a concentration of 100 ng / mL, and was diluted by 3-fold gradient until the final concentration reached 0.005 ng / mL. 100 μL of the above diluted NP protein solution was added to the corresponding wells, and 100 μL of 100 ng / mL NP protein solution of influenza A H1N1, H3N2 and other viruses was added to the corresponding wells, and the wells with only 20 mM PB pH 7.4 were used as negative controls (NC) and incubated at 4°C overnight.

[0033] The liquid in the wells was aspirated, and the wells were washed 3 times with 300 μL of washing buffer. 300 μL of blocking solution (prepared by 5% skim milk powder with PBST) was added to each well, and incubated at 37°C for 1 h. The liquid in the wells was aspirated, and the wells were washed 3 times with 300 μL of washing buffer, and then 100 μL of 1 μg / ml detection antibody 23E2 was added to each well.

[0034] The liquid in the wells was aspirated, and the wells were washed 3 times with 300 μL of washing buffer, and then 100 μL of HRP-labeled rabbit anti-mouse antibody (diluted 1:5 000 with PBST) was added to each well and incubated at 37°C for 30 min. The liquid in the wells was aspirated, and the wells were washed 5 times with 300 μL of washing buffer.

[0035] 100 μL of color developing solution was added to each well, and after color development at 37°C for 10 min, 50 μL of 2 mol / L H2SO4 stop solution was added. Within 20 min after the addition of the stop solution, the OD450 value was read on a microplate reader.

[0036] The specific results of the detection are shown in detail in Tables 1, 2 and Figure 2 Within the concentration range of 0.015-100 ng / mL, the 23E2 antibody showed a significant linear relationship with NP protein, showing its detection sensitivity and accuracy within this concentration range. In addition, the 23E2 antibody has the ability to recognize NP proteins of both Yamagata and Victoria lineages, which indicates its wide applicability in influenza virus detection. It is worth noting that for influenza A H1N1 and other various types of viral NP proteins, the 23E2 antibody has a reading very close to the negative control (NC), which means that there is no cross-reaction between the 23E2 antibody and the NP proteins of these viruses, further confirming its strong specificity and ability to accurately recognize target proteins in complex samples.

[0037] Table 1

[0038] Table 2

[0039] The above description is only the preferred embodiment of the present application and is not intended to limit the present application. The present application can be variously changed and modified by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the scope of the present application.

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, Includes 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, Includes 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 of the influenza B virus NP protein as described in any one of claims 1 to 3 in the preparation of reagents for the diagnosis and prevention of 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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