Influenza B virus NP protein monoclonal antibody 10G11 and application thereof

Abstract

The invention provides an influenza B virus NP protein monoclonal antibody 10G11 and application thereof, the sequences of heavy chain variable regions CDR1, CDR2 and CDR3 of the influenza B virus NP protein monoclonal antibody 10G11 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 10G11 and application thereof, and belongs to the technical field of antibodies. BACKGROUND

[0002] Influenza B virus is a single-stranded RNA membrane virus of the Orthomyxoviridae family, and is one of common influenza viruses, which can cause influenza B. Influenza B patients usually have symptoms such as headache, myalgia, cough, sore throat, fatigue, nasal congestion, and the like, in addition, the patients often have acute 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 two lineages. Nucleoprotein (nucleoprotein, NP) plays an important role in the replication and transcription process of influenza B virus, which can bind to single-stranded RNA to form a ribonucleoprotein complex to protect RNA. NP can also help RNA polymerase to recognize and bind to RNA, and promote the replication and transcription of RNA. NP protein is highly conserved in Yamagata and Victoria, and can be used as a target for detection of influenza B virus.

[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 is helpful for the development of influenza B virus detection reagents. SUMMARY The application provides an influenza B virus NP protein monoclonal antibody 10G11 and application thereof, which can effectively solve the above problems.

[0005] An influenza B virus NP protein monoclonal antibody 10G11, the sequences of CDR1, CDR2 and CDR3 of the heavy chain variable region are respectively shown in SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8; the sequences of CDR1, CDR2 and CDR3 of the light chain variable region are respectively shown in SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12. In some embodiments, the influenza B virus NP protein monoclonal antibody 10G11 has a heavy chain variable region sequence shown in SEQ ID NO: 5 and a light chain variable region sequence shown in SEQ ID NO: 9.

[0006] In some embodiments, the influenza B virus NP protein monoclonal antibody 10G11 has a heavy chain sequence shown in SEQ ID NO: 3 and a light chain sequence shown in SEQ ID NO: 4.

[0007] A reagent for detecting NP protein of influenza B virus, comprising the NP protein monoclonal antibody 10G11 of the influenza B virus.

[0008] A kit for detecting NP protein of influenza B virus, comprising the NP protein monoclonal antibody 10G11 of the influenza B virus.

[0009] Use of the NP protein monoclonal antibody 10G11 of the influenza B virus 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 monoclonal antibody 10G11 of the influenza B virus.

[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 monoclonal antibody 10G11 of the influenza B virus for immunodetection.

[0014] The beneficial effects of the present application are: The present application provides a monoclonal antibody named 10G11, which has significant functional characteristics, can accurately recognize and bind NP protein, and has extremely high affinity in the binding process, ensuring the close binding between the antibody and the target protein. Further, the antibody can not only recognize Yamagata system NP protein, but also effectively recognize Victoria system NP protein, covering the NP protein recognition needs of the two important lineages. In addition, the 10G11 monoclonal antibody performs particularly well in specificity and does not cross-react with NP protein of influenza A virus and other viruses, thereby ensuring its high specificity in application. Based on these excellent characteristics, the 10G11 monoclonal antibody has a wide application prospect, and is particularly suitable for the development of influenza B virus detection reagents, providing strong technical support for rapid and accurate detection of influenza B. 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 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. 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 the purification result of NP protein.

[0017] Figure 2 Linear graph for detecting 10G11 on NP protein by 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, rabies virus NP protein were purchased from Yiqiao God of the World Biological Technology Co., Ltd.; parainfluenza virus, adenovirus NP protein were purchased from Jiangsu Eastland Biomedicine Technology Co., Ltd.

[0020] Example 1: Preparation of 10G11 monoclonal antibody 1. Preparation of recombinant NP protein The amino acid sequence (SEQ ID NO: 1) encoding 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 pET28a vector.

[0021] SEQ ID NO: 1 MSNMDIDGINTGTIDKTPEEITSGTSGTTRPIIRPATLAPPSNKRTRNPSPERATTSSEDDV GRKAQKKQTPTEIKKSVYNMVVKLGEFYNQMMVKAGLNDDMERNLIQNAHAVERILLAATD DKKTEFQKKKNARDVKEGKEEIDHNKTGGTFYKMVRDDKTIYFSPIRITFLKEEVKTMYKTT MGSDGFSGLNHIMIGHSQMNDVCFQRSKALKRVGLDPSLISTFAGSTVPRRSGATGVAIK GGGTLVAEAIRFIGRAMADRGLLRDIKAKTAYEKILLNLKNKCSAPQQKALVDQVIGSRN PG IADIEDLTLLARSMVVVRPSVASKVVLPISIYAKIPQLGFNVEEYSMVGYEAMALYN MATPVSILRMGDDARDKSQLFFMSCFGAAYEDLRVLSALTGTEFKPRSALKCKGFHVPAKE QVEGMGAALMSIKLQFWAPMTRSGGNEAGGDGGSGQISCSPVFAVERPIALSKQAVRRML SMNIEGRDADVKGNLLKMMNDSMAKKTSGNAFIGKKMFQISDKNKTNPIEIPIKQTIPNFF FGRDTAEDYDDLDYHHHHHH SEQ ID NO: 2 The constructed pET28a-NP plasmid was transformed into BL21(DE3). After transformation, it was coated on LB plate containing kanamycin resistance, and then placed in 37°C incubator for overnight culture.

[0022] The colony was picked into 5 mL LB medium containing 50 μg / mL kanamycin, and cultured in a 37°C, 220 rpm shaker for 2-3 hours. Then, it was transferred to 200 mL LB medium containing 50 μg / mL kanamycin, and continued to be cultured in a 37°C, 220 rpm shaker until the OD600 was about 0.8. IPTG was added to a final concentration of 0.25 mM, and the sample was taken after 4-5 hours of induction.

[0023] Centrifuged at 4°C, 12000 rpm for 30 minutes to collect the bacterial pellet. Resuspend the bacteria with Buffer A (20 mM PB, pH 7.4), and use an ultrasonic disrupter for disruption. After disruption, centrifuged again at 4°C, 12000 rpm for 30 minutes to collect the supernatant.

[0024] 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 washed with Buffer A, and 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). The purified NP protein was ultrafiltrated and concentrated, and 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, and 6-8 week old SPF Balb / c mice were selected for subcutaneous multi-point injection, with each mouse injected with 200 μg. After 2 weeks, the antigen was emulsified with Freund's incomplete adjuvant, and subcutaneous multi-point injection was performed again, with each mouse injected with 100 μg. The mice were injected intraperitoneally 3 days before fusion to boost the immune response.

[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 then the whole body was treated with 75% alcohol. In the clean bench, the abdominal skin was cut with scissors to expose the peritoneum. 5 mL of RPMI 1640 basic medium was injected into the abdominal cavity with a syringe, and then the washing liquid was recovered after repeated washing. The washing liquid was centrifuged at 1000 rpm for 5 minutes, and the precipitate was retained. The precipitate was resuspended with RPMI 1640 complete medium containing HAT, and the cell concentration was adjusted to 1 x 10 5 9 / mL, 150 μL was added to each well of a 96-well plate, and the plate was incubated at 37°C in a 5% CO2 environment overnight.

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

[0027] 5. Cell fusion (1) 40 mL of HAT medium, 15 mL of DMEM serum-free medium, and 1 mL of 50% PEG (M12 000) were taken separately 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 suspension (10 8 cells) were mixed in a 50 mL centrifuge tube, and DMEM serum-free medium was added to 40 mL. After centrifugation for 10 minutes, the supernatant was discarded and the mixture was mixed again.

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

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

[0031] 6. Selection and culture of hybridoma cells and cloning screening After cell fusion, on the 1st, 3rd, 5th, and 7th day, the HAT culture solution described above is used to replace the culture solution to select the true hybrid cells. Three rounds of subcloning are then performed to screen the monoclonal cells capable of specifically recognizing the NP protein, and finally the hybridoma cell strain 10G11 is obtained.

[0032] 7. Ascites expression and purification The hybridoma cell strain 10G11 screened out is inoculated into the abdominal cavity of a Balb / c mouse at a concentration of 1 x 10 6 About 10 days later, the mouse abdomen starts to swell, and the mouse is killed by cervical dislocation and soaked in 75% alcohol for disinfection for 5 minutes, and ascites extraction is performed once. The ascites is purified using a Protein A affinity column to obtain the monoclonal antibody 10G11. After testing, the sequence of the monoclonal antibody 10G11 is as follows: Heavy chain: QVNIQNSGTEIAKPGGSVKFTCRASGYTWTKFTVWWIYERPAQGIEWIFTDFGSVWKEYQERLWEKATLSADRSGATAYYQLNSVMSEDGISYCARHSWAFLSTETWGNGTTVTVASAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPG (SEQ ID NO: 3) Heavy chain: QVNIQNSGTEIAKPGGSVKFTCRASGYTWTKFTVWWIYERPAQGIEWIFTDFGSVWKEYQERLWEKATLSADRSGATAYYQLNSVMSEDGISYCARHSWAFLSTETWGNGTTVTVAS (SEQ ID NO: 5) Heavy chain variable region: QVNIQNSGTEIAKPGGSVKFTCRASGYTWTKFTVWWIYERPAQGIEWIFTDFGSVWKEYQERLWEKATLSADRSGATAYYQLNSVMSEDGISYCARHSWAFLSTETWGNGTTVTVAS (SEQ ID NO: 5) Heavy chain variable region CDR1 : KFTVW (SEQ ID NO: 6) Heavy chain variable region CDR2: TDFGSVWKEYQERLWE (SEQ ID NO: 7) Heavy chain variable region CDR3: HSWAFLSTET (SEQ ID NO: 8) Light chain variable region: DIFLSNSPAIHSGSPGDKVSMTCGGASAFTWLKWYNQHSGTSVKRWIYESGRVGTGIPVKFSASSSGTSYTLTISSHDAEDAGTYYCENYTGQVWSFGGATKIEIK (SEQ ID NO: 9) Light chain variable region CDR1 : GGASAFTWLK (SEQ ID NO: 10) Light chain variable region CDR2: ESGRVGT (SEQ ID NO: 11) Light chain variable region CDR3: ENYTGQVWS (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 into the corresponding wells, and 100 μL of NP protein solution of influenza A H1N1, H3N2 and other viruses at a concentration of 100 ng / mL was added into the corresponding wells. Wells with only 20 mM PB pH 7.4 were used as negative controls (NC), and were 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 (5% skim milk powder prepared with PBST) was added into each well, and the wells were incubated at 37°C for 1 hour. The liquid in the wells was aspirated, and the wells were washed 3 times with 300 μL of washing buffer. 100 μL of detection antibody 10G11 at a concentration of 1 μg / mL was added into each well.

[0034] The liquid in the wells was aspirated, and the wells were washed 3 times with 300 μL of washing buffer. 100 μL of HRP-labeled rabbit anti-mouse antibody (1:5 000 diluted with PBST) was added into each well, and the wells were incubated at 37°C for 30 minutes. 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 into each well, and after color development at 37°C for 10 minutes, 50 μL of 2 mol / L H2SO4 stop solution was added. Within 20 minutes after the addition of the stop solution, OD450 values were read on an enzyme marker.

[0036] The detection results are shown in Tables 1, 2 and Figure 2 10G11 showed good linearity to NP protein in the range of 0.015-100 ng / mL; 10G11 could simultaneously recognize NP proteins of Yamagata and Victoria systems; the read values of NP proteins of influenza A H1N1 and other viruses were close to the negative control (NC), i.e., 10G11 did not have cross-reaction with the NP proteins of these viruses, and had strong specificity. Table 1

[0037] Table 2

[0038] The above only describes preferred embodiments of the present application and is not intended to limit the present application. The present application can have various modifications and changes for those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. An influenza B virus NP protein monoclonal antibody 10G11, characterized in that, the sequence of CDR1, CDR2, CDR3 of the heavy chain variable region is respectively shown as SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8; the sequence of CDR1, CDR2, CDR3 of the light chain variable region is respectively shown as SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO:

12.

2. The NP protein monoclonal antibody 10G11 of influenza B virus according to claim 1, characterized by, the sequence of the heavy chain variable region is shown as SEQ ID NO: 5, and the sequence of the light chain variable region is shown as SEQ ID NO:

9.

3. The NP protein monoclonal antibody 10G11 of influenza B virus according to claim 1, characterized by, the sequence of the heavy chain is shown as SEQ ID NO: 3, and the sequence of the light chain is shown as SEQ ID NO:

4.

4. A reagent for detecting NP protein of influenza B virus, characterized by comprising an antibody which specifically binds to the NP protein of influenza B virus. The influenza B virus NP protein monoclonal antibody 10G11 according to any one of claims 1 to 3.

5. A kit for detecting NP protein of influenza B virus, characterized by comprising the antibody according to claim 1 or 2. The influenza B virus NP protein monoclonal antibody 10G11 according to any one of claims 1 to 3.

6. Use of the influenza B virus NP protein monoclonal antibody 10G11 according to any one of claims 1 to 3 in the preparation of a reagent for diagnosing or preventing influenza B.

7. A nucleic acid molecule encoding the heavy chain variable region and the light chain variable region of the influenza B virus NP protein monoclonal antibody 10G11 according to any one of claims 1 to 3.

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

9. A host cell comprising the expression vector according to claim 8.

10. A method for detecting non-disease diagnostic treatment of influenza B, characterized by, The influenza B virus NP protein monoclonal antibody 10G11 according to any one of claims 1 to 3 is used for immunodetection.

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