Monoclonal antibody 6D11 for resisting DENV NS1 protein as well as preparation method and application of monoclonal antibody 6D11

Abstract

The invention provides a monoclonal antibody 6D11 and application thereof. The CDR amino acid sequence of a heavy chain of the monoclonal antibody 6D11 is shown as SEQ ID No.1, and the CDR amino acid sequence of a light chain of the monoclonal antibody 6D11 is shown as SEQ ID No.2. The invention also provides application of the monoclonal antibody 6D11 in preparation of drugs for treating dengue virus infection, and the monoclonal antibody 6D11 has the advantages of competitive binding of DENV1-4NS1 protein, inhibition of vascular leakage symptom, in vivo protection of DENV infection fatal injury and the like, and has potential huge treatment value of dengue virus resistance.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering technology, and in particular to a preparation method and application of a broad-spectrum monoclonal antibody D11 against NS1 protein capable of inhibiting vascular leakage symptoms caused by dengue virus infection. Background Art

[0002] Dengue virus (DENV) is a mosquito-borne flavivirus that can be divided into four serotypes (DV-1, DV-2, DV-3 and DV-4). Infection with the virus usually causes dengue fever and severe dengue fever, the latter of which can even lead to death in severe cases. In recent years, the number of severe dengue cases has increased significantly, bringing a heavy economic burden to the global health system. The prevention and control of severe dengue fever remains a global public health issue that needs to be addressed urgently. The clinical features of severe dengue fever can be manifested as severe bleeding, shock or dyspnea caused by severe plasma leakage. Severe dengue fever has obvious gastrointestinal symptoms and bleeding tendency.

[0003] One of the keys to preventing and treating severe dengue is dengue vaccination. Current dengue virus vaccine development is hampered by antibody-dependent enhancement (ADE), where cross-reactive antibodies against one serotype can enhance subsequent infection with a heterologous serotype. Therefore, stimulating stable and lasting immunity against all four DENV serotypes and reducing the impact of the ADE effect on the protective effect of viral vaccines are crucial to dengue vaccine development.

[0004] Currently, the NS1 protein has become a highly-regarded vaccine target for the prevention and treatment of DENV infection. NS1 is a highly conserved protein in dengue virus that is involved in viral replication and immune escape. Studies have shown that T cell immune responses to NS1 play a role in preventing dengue infection. In addition, DENV2 infection can be prevented by vaccination with DENV1, DENV3, or DENV4 NS1, while monoclonal antibodies against DENV2 effectively protect mice from infection with all serotypes of DENV. However, the screening of broad-spectrum antibodies against NS1 has been limited by the lack of natural hexameric antigen stimulation, the low efficiency of screening conserved epitopes due to immune weakness, and the limited popularity of structural analysis technology. Therefore, the NS1 antibodies reported so far are still insufficient in simultaneously inhibiting vascular leakage caused by four serotypes of dengue infection. To date, no broad-spectrum protective antibodies that can be used in the clinical treatment of severe dengue have been obtained. Summary of the invention

[0005] The purpose of the present invention is to provide a broad-spectrum protective antibody 6D11 against dengue virus NS1 protein and its preparation method and application. The broad-spectrum protective antibody of the present invention is a monoclonal antibody, which is produced by hybridoma cells. In addition, the antibody is a hybridoma cell line 6D11 obtained by fusion, screening, cloning, and stable passage of BALB / c mouse spleen cells and mouse myeloma cells SP2 / 0 sequentially immunized with DENV1-4 NS1 protein, and can stably secrete monoclonal antibody 6D11.

[0006] In the first aspect, the present invention provides a monoclonal antibody 6D11 against DENV1-4 NS1 protein, the subtype of the monoclonal antibody is IgG1, and it can specifically and broadly recognize DENV1-4 NS1 protein. The amino acid sequence of the heavy chain CDR of the antibody 6D11 is shown in SEQ ID No.1, and the amino acid sequence of the light chain CDR is shown in SEQ ID No.2.

[0007] SEQ ID No.1: Heavy chain CDR amino acid sequence

[0008] QVTLKESGPGILKPSQTLSLTCSFSGFSLSTSGVGVGWIRQPSGKGLEWLAHIWWDDDKFYNPSLKSQLTISKDSSRNQVFLKITSVDTADTATYYCARIYGNFSIFAMDYWGQGTSVTVSS.

[0009] SEQ ID No.2: Light chain CDR amino acid sequence

[0010] DIQMTQTTSSLSASLGDRVTISCSASQGISNYLNWYQQKPDGTVKLLIYYTSNLHSGVPSRFSGSGSGTDYSLTISNLEPEDIATYYCQQYSKLYTFGGGTKLEIKR.

[0011] SEQ ID No.3: Heavy chain CDR nucleotide sequence

[0012] CAAGTTACTCTAAAAGAGTCTGGCCCTGGGATATTGAAGCCCTCACAGACCCTCAGTCTGACTTGTTCTTTCTCTGGGTTTTCACTGAGCACTTCTGGTGTGGGTGTAGGCTGGATTCGTCAGCCTTCAGGGAAGGGTCTGGAGTGGCTGGCACACATTTGGTGGGATGATGATAAATTCTAT AACCCATCCCTGAAGAGCCAGCTCACAATCTCCAAGGATTCCTCCAGAAACCAGGTTTTCCTCAAGATCACCAGTGTGGACACTGCAGATACTGCCACTTACTACTGTGCTCGAATATATGGTAACTTTTCTATCTTTGCTATGGACTACTGGGGTCAAGGCACCTCAGTCACCGTCTCCTCA.

[0013] SEQ ID No.4: Light chain CDR nucleotide sequence

[0014] GATATCCAGATGACACAAACTACATCCTCCCTGTCTGCCTCTCTGGGAGACAGAGTCACCATCAGTTGCAGTGCAAGTCAGGGCATTAGCAATTATTTAAACTGGTATCAGCAGAAACCAGATGGAACTGTTAAACTCCTGATCTATTACACATCAAATTT ACACTCAGGAGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGGACAGATTATTCTCTCACCATCAGCAACCTGGAACCTGAAGATATTGCCACTTACTATTGTCAGCAGTATAGTAAGTTGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAACGG.

[0015] In some embodiments, the monoclonal antibody 6D11 is a murine antibody.

[0016] In some of these embodiments, the subtype of the monoclonal antibody 6D11 is IgG1.

[0017] In the second aspect, the present invention proposes a method for screening monoclonal antibody 6D11 against DENV1-4 NS1 protein based on sequential immunization strategy, inducing immune response of conserved epitopes by sequential immunization with four serotypes of DENV NS1 protein, and improving the induction efficiency of broad-spectrum cross-reactive antibodies. Through this method, a monoclonal antibody 6D11 capable of broadly binding to four serotypes of DENV NS1 was screened and identified.

[0018] A method for preparing the above-mentioned monoclonal antibody 6D11 against DENV1-4 NS1 protein, the preparation method comprising the following steps:

[0019] (1) Animal immunization: A sequential immunization strategy was used to immunize BALB / c mice with serotypes DV-1, DV-2, DV-3, and DV-4, respectively;

[0020] (2) Preparation of hybrid cells: Cultivating mouse myeloma cells SP2 / 0 and maintaining them in a good growth state;

[0021] Using polyethylene glycol-mediated cell fusion method, the spleen lymphocytes of the mouse in step (1) are obtained, mixed with the above-mentioned SP2 / 0 cells and centrifuged to mediate cell fusion, and the fused cells are cultured under appropriate conditions to obtain hybrid cells;

[0022] (3) Screening of hybridoma cells: the hybridoma cells are cultured in a HAT selective medium for screening cells deficient in hypoxanthine phosphoribosyltransferase (HPRT) or thymidine kinase (TK) activity, and positive clones are obtained by screening;

[0023] (4) Cloning of hybridoma cells: selecting the culture well with the highest antibody titer and the growth of a single clone cell, performing limiting dilution again, performing limiting dilution for more than 4 times in succession, and continuously passage for more than 20 generations to obtain a single specific hybridoma cell line 6D11 that stably and efficiently expresses the monoclonal antibody 6D11; the amino acid sequences of the heavy chain CDR and light chain CDR of the monoclonal antibody 6D11 are shown in SEQ ID No. 1 and SEQ ID No. 2, respectively;

[0024] (5) Production of monoclonal antibodies using BALB / c mice: 8-10 week old BALB / c mice were selected, and an appropriate amount of Freund's incomplete adjuvant was injected intraperitoneally one week later. Then, hybridoma cells 6D11 were inoculated in PBS buffer solution again in the abdomen. When the abdomen of the mouse was obviously swollen and before the mouse was on the verge of death, the ascites was collected and centrifuged to measure the antibody titer;

[0025] (6) Purify the monoclonal antibody in the mouse ascites.

[0026] In a third aspect, the present invention provides a monoclonal antibody hybridoma line targeting DENV1-4 NS1 protein, namely 6D11. The cell line was frozen in liquid nitrogen, grew well after recovery, and the antibody secretion did not decline after continuous culture for more than 20 generations.

[0027] In a fourth aspect, the present invention provides the potential antiviral therapeutic value and application of the monoclonal antibody 6D11.

[0028] Use of any of the above monoclonal antibodies 6D11 in the preparation of a drug or vaccine for preventing or treating dengue virus infection.

[0029] Use of any of the above monoclonal antibodies 6D11 in the preparation of a drug or vaccine for inhibiting or treating vascular leakage caused by dengue virus infection.

[0030] Use of any of the above monoclonal antibodies 6D11 in the preparation of drugs or vaccines for inhibiting or treating dengue fever.

[0031] In some of these embodiments, the dengue fever is severe dengue fever.

[0032] The monoclonal antibody 6D11 of the present invention has the ability to competitively bind to DENV1-4 NS1 protein and can inhibit vascular leakage symptoms, while providing protection against fatal damage caused by DENV infection in vivo.

[0033] The anti-DENV NS1 protein monoclonal antibodies described in the present invention are capable of treating at least two, three or four dengue virus serotypes. Therefore, in certain embodiments, the antibody molecules are administered to patients infected with dengue virus or at risk of infection with dengue virus, when no test is performed to determine the serotype of the dengue virus, for example, the serotype of the dengue virus may be unknown, in some embodiments of which the dengue virus has serotype DV-1, DV-2, DV-3 or DV-4.

[0034] In a fifth aspect, the present invention provides a pharmaceutical composition for treating dengue virus infection, comprising any one of the above-mentioned monoclonal antibodies 6D11, and a pharmaceutically acceptable carrier.

[0035] In some embodiments, the drug is used to treat severe dengue fever caused by dengue virus infection.

[0036] In some embodiments, the pharmaceutical composition is a drug preparation or a vaccine.

[0037] In a sixth aspect, the present invention provides a method for neutralizing dengue virus, comprising contacting the dengue virus with monoclonal antibody 6D11.

[0038] In a seventh aspect, the present invention provides a nucleotide encoding the variable region of the antibody heavy chain and / or light chain of the anti-monoclonal antibody 6D11.

[0039] In some of the embodiments, the CDR nucleotide sequence of the heavy chain of the monoclonal antibody 6D11 is shown as SEQ ID No.3, and the CDR nucleotide sequence of the light chain is shown as SEQ ID No.4.

[0040] The monoclonal antibody 6D11 obtained by screening of the present invention has a broad-spectrum binding ability to 4 serotypes (DV-1, DV-2, DV-3 or DV-4) DENV1-4 NS1 protein (especially better inhibitory effect on DENV2 and DENV3 NS1 protein), and in an in vitro cell model, it competitively inhibits the binding of NS1 protein to target cells and effectively inhibits the biological function of NS1 protein. The antibody 6D11 can delay the occurrence and progression of mouse symptoms in the AG129 mouse model and protect 25% of mice from the attack of a lethal dose of DENV2. Therefore, the monoclonal antibody 6D11 of the present invention has potential antiviral uses in the design of dengue vaccine candidates, screening of therapeutic drugs, or preparation of antiviral therapeutic drugs for DENV1-4 viral infectious diseases.

[0041] The monoclonal antibody 6D11 against DENV NS1 protein of the present invention has the performance of competitively binding to DENV1-4 NS1 protein, inhibiting vascular leakage symptoms and protecting fatal damage of DENV infection in vivo, and has potential antiviral therapeutic value. The monoclonal antibody 6D11 developed by the present invention has important significance and application potential for the prevention and treatment of dengue infection (including severe dengue fever). BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 It is the technical roadmap for the preparation of the broad-spectrum protective monoclonal antibody 6D11.

[0043] Figure 2 This is the result of SDS-PAGE to identify the purity of the antibody.

[0044] Figure 3 This is the result of ELISA identification of the binding curve of monoclonal antibody 6D11 and DENV1-4 NS1 protein.

[0045] Figure 4 This is a flow cytometry validation result of monoclonal antibody 6D11 competitively inhibiting the binding of NS1 protein to target cells.

[0046] Figure 5 This is a diagram showing the results of validating the ability of monoclonal antibody 6D11 to protect against lethal damage caused by vascular leakage caused by DENV infection in the AG129 mouse model. DETAILED DESCRIPTION

[0047] In order to facilitate the understanding of the present invention, the present invention will be described more fully below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0048] The experimental methods in the following examples without specifying specific conditions are usually carried out according to conventional conditions, such as the fourth edition of Molecular Cloning: A Laboratory Manual edited by Green and Sambrook, published in 2013, or according to the conditions recommended by the manufacturer. The various commonly used chemical reagents used in the examples are all commercially available products.

[0049] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art to which the present invention belongs. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used in the present invention includes any and all combinations of one or more of the related listed items.

[0050] CDR: Ig, complementarity-determining region. Complementarity determining region of immunoglobulin molecule: The hypervariable regions within the variable regions of the heavy and light chains of antibodies (Ab) constitute the antigen (Ag) binding sites of antibody molecules. Because the antigen binding sites are complementary to the antigen epitope structure, the hypervariable regions are also called the complementarity determining regions of antibody molecules.

[0051] The present invention is further described in detail below with reference to specific embodiments.

[0052] Example 1: Preparation of monoclonal antibodies

[0053] The technical roadmap for the preparation of monoclonal antibody 6D11 can be found in Figure 1 .

[0054] 1.1 Immunization of mice: Mice were immunized using a sequential immunization strategy. DENV1 NS1 protein was emulsified with Freund's complete adjuvant for basic immunization, followed by immunization with DENV2 NS1 protein, DENV3 NS1 protein, and DENV4 NS1 protein emulsified with Freund's incomplete adjuvant in serotype order, with an immunization interval of 2 weeks. The control group used PBS instead of antigen for the same immunization procedure. In this experiment, 10 female BALB / c mice aged 4-6 weeks were selected and randomly divided into two groups, namely the sequential immunization group and the blank control group. Basic immunization was performed first, and booster immunization was performed 2 weeks after basic immunization. The interval between booster immunizations was 2 weeks.

[0055] 1.2 Serum titer determination

[0056] 7 days after the last immunization of mice, the mouse serum was collected and the titer of the immune mouse serum was determined by sampling indirect ELISA. Blood was collected from the tail vein, centrifuged at room temperature for 2 hours (10000rpm, 10min), and the upper clear liquid was mouse serum. The DENV1-4 NS1 protein was diluted to 1μg / mL with PBS, added to a 96-well ELISA plate at 100μL / well, and coated overnight at 4°C. After washing the plate 3 times with PBST, 5% skim milk powder solution was added, 300μL / well, blocked at 37°C for 2h, and washed 3 times with PBST. Take 1μL of mouse serum, dilute it to 1mL with PBS, dilute 12 times with 2-fold gradients, and then add 100μL / well to the corresponding wells of the ELISA plate, and incubate at 37°C for 60min. The negative control is PBS immunized mouse serum, and the PBS solution is used as a blank control. Then, the serum sample was discarded and the plate was washed 3 times as above. Add 100 μL / well PBS diluted 5000 times HRP-labeled goat anti-mouse secondary antibody solution, incubate at 37°C for 60 min. Discard the solution and wash 5 times with PBST. After tax, add TMB substrate for color development, 100 μL / well, color development at room temperature for 10 min. Add 50 μL / well 2M concentrated sulfuric acid to stop color development, and use a microplate reader to read the light absorbance of each well at OD450.

[0057] Example 2: Cell fusion and hybridoma cell screening

[0058] 2.1 Fusion of mouse spleen cells and myeloma cells SP2 / 0

[0059] A sterile spleen cell suspension (10 8 cells) and myeloma cells SP2 / 0 (2×10 7Cells) were added to a new 50mL centrifuge tube at the same time, mixed and refilled to 30mL, centrifuged at 1000rpm for 5min, and the supernatant was discarded. At 37°C, 1mL of preheated 50% PEG solution was slowly added within 1min and allowed to act for 90s. Immediately thereafter, 15mL of 37°C preheated 1640 culture medium was added dropwise, specifically 1mL was added in the first 30s, 3mL was added in the last 30s, and 11mL was added in the last 1min to dilute PEG and terminate its fusion. 50mL of preheated 1×HAT 1640 culture medium was added at 37°C, mixed and added dropwise with an elbow dropper to a 96-well cell culture plate that had been paved with feeder cells the day before, 100ul / well, and then placed in a 37°C, 5% CO2 incubator for culture.

[0060] 2.2 Screening of hybridoma cells

[0061] The specific antibody titer was detected by indirect ELISA, and the fusion cell lines with higher titer and single mass growth were selected for cloning. The cloning culture of hybridoma cells was carried out according to the limiting dilution method, and the cells of the fusion cell wells with positive antibody detection were counted. Take 150-200 cells and use 1640 complete medium to prepare a cell suspension and inoculate it into a 96-well culture plate with feeder cells, 100 μL per well, observe the cell growth, and detect the antibody level in the supernatant. Select the culture well with the highest antibody titer and single clone cell growth, and perform limiting dilution again. Perform limiting dilution for more than 4 times until all dilution culture wells are positive. Select cells from a single clone well for continuous passage for more than 20 generations to obtain a hybridoma cell line 6D11 that stably and efficiently expresses the anti-DNEV NS1 protein monoclonal antibody 6D11.

[0062] The hybridoma cell 6D11 was sequenced, and it was found that the amino acids and nucleotides of the heavy chain CDR and light chain CDR of the secreted antibody 6D11 were as shown in SEQ ID No.1-SEQ ID No.4.

[0063] Example 3: Preparation of ascites and purification and identification of monoclonal antibodies

[0064] One week after female BALB / c mice aged 6-8 weeks were intraperitoneally injected with 0.5 ml of Freund's incomplete adjuvant, hybridoma cells 6D11 were cultured at 5×10 6 / immunize mice by intraperitoneal injection, observe the ascites production of mice every day, and extract ascites when the mice show obvious abdominal distension and tightness after about 7-10 days. Protein A column method is used to purify monoclonal antibodies in ascites. The process of antibody purification is as follows: ascites treatment, balance, sample loading, washing and elution.

[0065] Subsequently, SDS-PAGE electrophoresis was used to identify the purity of the antibody, and Coomassie Brilliant Blue-stained monoclonal antibody reducing and non-reducing SDS-PAGE gels were analyzed: In reducing SDS-PAGE, antibody 6D11 was observed to have a single band at 55 kDa (heavy chain) and 25 kDa (light chain). Under non-reducing SDS-PAGE conditions, antibody 6D11 was observed to have a single band at approximately 150 kDa (intact antibody). The results are shown in Figure 2 .

[0066] Example 4: Biological identification of monoclonal antibody 6D11

[0067] 4.1 Antibody subtype identification

[0068] The subtype of the antibody was analyzed using a mouse monoclonal antibody immunoglobulin typing kit (Bio-Rad). The purified antibody was appropriately diluted according to the instructions of the kit and its subtype was detected. The IgG1 subtype well of the ELISA test was positive, and the OD value measured at a wavelength of 450nm was 2.497, indicating that the subtype of the monoclonal antibody 6D11 secreted by the hybridoma cell 6D11 was IgG1.

[0069] 4.2 Antibody binding curve identification

[0070] The binding curve of antibody 6D11 and DENV1-4 NS1 protein was determined by indirect ELISA. The operating steps of the indirect ELISA method can refer to 1.2 in Example 1. 1 μg / ml DENV1-4 NS1 protein was coated separately, the primary antibody had a starting concentration of 100 μg / ml, the antibody 6D11 was diluted 10 times, and the secondary antibody was HRP-labeled goat anti-mouse IgG diluted 5000 times. The absorbance at 450nm was measured, the binding curve of antibody 6D11 and DENV NS1 was fitted and its KD value was calculated. The results showed that antibody 6D11 had strong cross-reactivity with four serotypes of DENV NS1 proteins, among which the binding ability with DENV1 and DENV4 NS1 was at the nM level, and the KD values ​​of the other two serotypes of DENV NS1 proteins reached the pM level. The results are shown in Figure 3 .

[0071] 4.3 Identification of antibodies that competitively inhibit NS1 protein binding to target cells

[0072] Four serotypes of DENV NS1 (20 μg / mL each) protein and antibody 6D11 (40 μg / mL) were mixed in a 10% FBS PBS solution and incubated at 37°C for 30 minutes. Cells (HBMEC human brain microvascular endothelial cells, human umbilical vein endothelial cells (HUVEC)) were resuspended in an ice-cold PBS solution containing 10% FBS and 1% sodium azide to about 1×10 6 Cells / mL. Add 100 μL of cell suspension to each centrifuge tube and incubate on ice for 15 minutes. After the cells and antibody-NS1 protein complex are evenly mixed, incubate at 4°C for 30 minutes. Centrifuge at 1000 rpm for 5 minutes, resuspend in pre-cooled PBS, and wash the cells 3 times. Subsequently, use a mouse His-tag monoclonal antibody directly labeled with FITC to bind to the NS1 protein bound to the target cells and incubate at 4°C in the dark for 30 minutes. Centrifuge at 1000 rpm for 5 minutes, resuspend in pre-cooled PBS, and wash the cells 3 times. Finally, resuspend the cells in an ice-cold PBS solution containing 3% BSA and 1% sodium azide. Immediately transfer the cell suspension to a flow tube for detection. The percentage of target cells bound to NS1 protein in the total number of cells was analyzed. Compared with the control group without antibody, the decrease in the percentage represents the competitive binding of NS1 protein by antibody 6D11. The results are consistent with the ELISA experiment in 4.2. Antibody 6D11 can broadly inhibit the binding of four serotypes of DENV NS1 protein to target cells, but the inhibitory effect of DENV1 and DENV4 NS1 protein is weaker than that of DENV2 and DENV3 NS1 protein. Figure 4 .

[0073] 4.4 Identification of Antibody Protective Efficacy in Vivo

[0074] Using the AG129 immunodeficient mouse model, the monoclonal antibody 6D11 (300 μg / mouse) was pre-immunized and exposed to a lethal dose of DENV2 (5×10 5 PFU / mouse). The changes in physical signs, disease course, body weight and survival rate of mice were continuously observed, and the protective effect of antibodies was evaluated by vascular leakage experiment. The plotted survival curve showed that within 15 days after DENV2 infection, all the mice in the control group that was infected with the virus alone died on the 12th day, while 25% of the mice in the antibody 6D11 pre-immunized group were still alive on the 15th day, showing a good effect in protecting mice from the attack of lethal doses of DENV2. At the same time, the results of the vascular leakage test showed that the vascular leakage of the liver, spleen and small intestine of the mice in the antibody 6D11 pre-immunized group was effectively alleviated, especially showing complete inhibition of the occurrence of splenic vascular leakage. Results are shown in Figure 5 .

[0075] It can be seen that the monoclonal antibody 6D11 has a good preventive and therapeutic value for dengue fever, including severe dengue fever.

[0076] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A monoclonal antibody 6D11 against DENV1-4 NS1 protein, characterized in that: The amino acid sequence of the CDR of the heavy chain of the monoclonal antibody 6D11 is shown in SEQ ID No.1, and the amino acid sequence of the CDR of the light chain is shown in SEQ ID No.

2.

2. The monoclonal antibody 6D11 against DENV1-4 NS1 protein according to claim 1, characterized in that: The CDR nucleotide sequence of the heavy chain of the monoclonal antibody 6D11 is shown in SEQ ID No.3, and the CDR nucleotide sequence of the light chain is shown in SEQ ID No.

4.

3. The monoclonal antibody 6D11 against DENV1-4 NS1 protein according to claim 1, characterized in that: The subtype of the monoclonal antibody 6D11 is IgG1.

4. A method for preparing the monoclonal antibody 6D11 against DENV1-4 NS1 protein according to any one of claims 1 to 3, characterized in that: The preparation method comprises the following steps: (1) Animal immunization: A sequential immunization strategy was used to immunize BALB / c mice with serotypes DV-1, DV-2, DV-3, and DV-4, respectively; (2) Preparation of hybrid cells: Cultivating mouse myeloma cells SP2 / 0 and maintaining them in a good growth state; Using polyethylene glycol-mediated cell fusion method, the spleen lymphocytes of the mouse in step (1) are obtained, mixed with the above-mentioned SP2 / 0 cells and centrifuged to mediate cell fusion, and the fused cells are cultured under appropriate conditions to obtain hybrid cells; (3) Screening of hybridoma cells: culturing the above hybridoma cells in HAT selective medium and screening to obtain positive clones; (4) Cloning of hybridoma cells: selecting the culture well with the highest antibody titer and the growth of a single clone cell, performing limiting dilution again, performing limiting dilution for more than 4 times in succession, and continuously passage for more than 20 generations to obtain a hybridoma cell line 6D11 that stably and efficiently expresses the monoclonal antibody 6D11; the amino acids of the heavy chain CDR and light chain CDR of the monoclonal antibody 6D11 are shown in SEQ ID No. 1 and SEQ ID No. 2, respectively; (5) Production of monoclonal antibodies using BALB / c mice: 8-10 week old BALB / c mice were selected, and an appropriate amount of Freund's incomplete adjuvant was intraperitoneally injected one week later. Then, the PBS buffer solution containing the hybridoma cell line 6D11 was inoculated again in the abdomen. When the abdomen of the mouse was obviously swollen and before the mouse was on the verge of death, the ascites was collected and centrifuged to determine the antibody titer; (6) Purify the monoclonal antibody in the mouse ascites.

5. Use of the monoclonal antibody 6D11 against DENV1-4 NS1 protein according to any one of claims 1 to 3 in the preparation of a drug or vaccine for treating dengue virus infection.

6. Use of the monoclonal antibody 6D11 against DENV1-4 NS1 protein according to any one of claims 1 to 3 in the preparation of a drug or vaccine for inhibiting or treating vascular leakage caused by dengue virus infection.

7. The use according to claim 5 or 6, characterized in that: The dengue virus has serotype DV-1, DV-2, DV-3 or DV-4.

8. A pharmaceutical composition for treating dengue virus infection, characterized in that: A method comprising the monoclonal antibody 6D11 against DENV1-4 NS1 protein according to any one of claims 1 to 3, and a pharmaceutically acceptable carrier.

9. A method for neutralizing dengue virus, characterized in that: The method comprises contacting the dengue virus with the monoclonal antibody 6D11 against the DENV1-4 NS1 protein according to any one of claims 1-3.

10. A nucleotide encoding the heavy chain and / or light chain variable region of the monoclonal antibody 6D11 against DENV1-4 NS1 protein according to any one of claims 1 to 3, wherein the nucleotide sequence of the heavy chain variable region of the monoclonal antibody 6D11 against DENV1-4 NS1 protein is shown in SEQ ID No. 3, and the nucleotide sequence of the light chain variable region is shown in SEQ ID No. 4.

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