Monoclonal antibody 6G2 for resisting DENV NS1 protein as well as preparation method and application of monoclonal antibody 6G2

Abstract

The invention provides a monoclonal antibody 6G2 resisting DENV NS1 protein and application of the monoclonal antibody 6G2. The CDR amino acid sequence of a heavy chain of the monoclonal antibody 6G2 resisting the DENV NS1 protein is shown as SEQ ID No.1, and the CDR amino acid sequence of a light chain of the monoclonal antibody 6G2 resisting the DENV NS1 protein is shown as SEQ ID No.3. The invention also provides application of the monoclonal antibody 6G2 to preparation of drugs for treating dengue virus infection, and the monoclonal antibody 6G2 for resisting DENV NS1 protein has the performances of competitively binding DENV1-4NS1 protein, inhibiting vascular leakage symptoms and protecting DENV infected fatal injury in vivo, and has potential important value for treating severe dengue fever. The monoclonal antibody 6G2 developed by the invention has important significance and application potential for prevention and treatment of dengue virus infection, including severe dengue fever.

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 6G2 against NS1 protein capable of inhibiting vascular leakage symptoms caused by dengue virus infection. Background Art

[0002] Dengue fever is an acute, mosquito-borne infectious disease caused by the dengue virus. Infection with the dengue virus can lead to latent infection, dengue fever, and dengue hemorrhagic fever. Typical clinical manifestations of dengue fever include sudden onset, high fever, headache, severe muscle and joint pain, and, in some cases, rash, bleeding tendency, swollen lymph nodes, decreased white blood cell count, and thrombocytopenia. Dengue fever is primarily prevalent in tropical and subtropical regions and is transmitted by the Aedes mosquito.

[0003] Early symptoms of severe dengue fever are similar to those of typical dengue fever. The condition worsens suddenly between days 3 and 5, with symptoms of meningitis such as severe headache, nausea, vomiting, impaired consciousness, and neck stiffness. Clinical features of severe dengue fever may include severe bleeding, shock due to severe plasma leakage, or respiratory distress. Gastrointestinal symptoms and a bleeding tendency are more pronounced in severe dengue fever. Severe dengue fever can cause severe organ damage, including severe liver damage, acute respiratory failure, acute heart damage, acute renal insufficiency, and encephalopathy.

[0004] Severe bleeding is one of the main differences between severe dengue fever and common dengue fever. Major causes include liver and kidney dysfunction, the use of antipyretic drugs, gastric and urinary catheter insertion, venipuncture, and intramuscular injection. Severe bleeding often occurs at the peak of severe dengue fever and can manifest as subcutaneous hematomas, as well as bleeding in the digestive tract, respiratory tract, urinary tract, vaginal, and intracranial tract. Subcutaneous hematomas are the most common, often induced by intramuscular injections or deep arterial and venous punctures, and often occur in the trunk and limbs. Severe cases can induce hemorrhagic shock. Gastrointestinal bleeding is often accompanied by a history of peptic ulcers, and bleeding can occur in both the upper and lower digestive tracts. Intracranial hemorrhage is often spontaneous, and patients have a poor prognosis.

[0005] Key pathogenic mechanisms of severe dengue fever include antibody-dependent enhancement (ADE), which specifically enhances viral infection, and severe vascular leakage, which is associated with the fatal symptoms and damage of severe dengue fever. However, because the E and prM proteins that make up the viral particles induce both neutralization and ADE, traditional vaccines designed based on the whole DENV, such as attenuated, inactivated, or chimeric vaccines, can mimic natural DENV infection. While providing limited protection against DENV infection, they may induce ADE, increasing the risk of severe dengue fever. This has led to bottlenecks in the development of traditional antiviral drugs and vaccines for dengue virus (DENV).

[0006] Immune strategies focused on protecting the body from the lethal damage caused by vascular leakage are an important approach to improving the clinical symptoms of severe dengue. Nonstructural protein 1 (NS1), directly involved in the pathogenic mechanism of vascular leakage, presents a unique antigenic advantage. Targeting the highly conserved NS1 protein can induce broad-spectrum protection against flaviviruses. Antibodies against NS1 protect against the lethal damage associated with severe dengue by blocking vascular leakage caused by NS1-mediated endothelial cell dysfunction, and have not yet been associated with ADE. However, screening for broad-spectrum NS1 antibodies is hampered by the lack of antigenic stimulation from the natural hexamer, low screening efficiency due to the immunological vulnerability of conserved epitopes, and the lack of widespread structural analysis technology. Consequently, the reported NS1 antibodies still lack the ability to simultaneously protect against infection with all four DENV serotypes. Therefore, targeting the NS1 protein to induce, screen, and identify broad-spectrum protective antibodies is of great significance for the development of DENV antiviral treatments or vaccines. Summary of the Invention

[0007] To address the deficiencies of existing technologies, the present invention provides a monoclonal antibody 6G2 that can broadly bind to four serotypes of DENV NS1 and inhibit their vascular leakage effects, a preparation method, and applications thereof.

[0008] The first aspect of the present invention is to provide a monoclonal antibody 6G2 against DENV NS1 protein, the amino acid sequence of its heavy chain CDR is shown in SEQ ID No. 1, and the amino acid sequence of its light chain CDR is shown in SEQ ID No. 3.

[0009] In some embodiments, the subtype of the anti-DENV NS1 protein monoclonal antibody 6G2 is IgG2b.

[0010] The second aspect of the present invention is to provide the use of the monoclonal antibody 6G2 against DENV NS1 protein.

[0011] The anti-DENV NS1 protein monoclonal antibody 6G2 is used in the preparation of a drug or vaccine for preventing and treating dengue virus infection.

[0012] Use of the anti-DENV NS1 protein monoclonal antibody 6G2 in the preparation of a drug or vaccine for inhibiting or treating vascular leakage in severe dengue fever.

[0013] The monoclonal antibodies against DENV NS1 protein described herein are capable of treating at least two, three, or four dengue virus serotypes. Thus, in certain embodiments, the antibody molecules are administered to a patient infected with or at risk of being infected with a dengue virus, when no test is performed to determine the serotype of the dengue virus, e.g., the serotype of the dengue virus may be unknown, in some embodiments of which the dengue virus has a serotype of DV-1, DV-2, DV-3, or DV-4, more preferably DV-2, DV-3, or DV-4.

[0014] The third aspect of the present invention is to provide a pharmaceutical composition for treating dengue virus infection, comprising an active ingredient and a pharmaceutically acceptable carrier, wherein the active ingredient is any one of the above-mentioned monoclonal antibodies 6G2 against DENV NS1 protein.

[0015] In some embodiments, the pharmaceutical composition is a drug preparation or a vaccine.

[0016] The fourth aspect of the present invention provides a method for neutralizing dengue virus, comprising contacting the dengue virus with the monoclonal antibody 6G2 against the DENV NS1 protein.

[0017] The fourth aspect of the present invention is to provide a method for preparing the monoclonal antibody 6G2 against DENV NS1 protein. The preparation method comprises the following steps:

[0018] (1) Animal immunization: A sequential immunization strategy was used to immunize mice with serotypes DV-1, DV-2, DV-3, and DV-4, respectively;

[0019] (2) Culture of mouse myeloma cells: Culture mouse myeloma cells SP2 / 0 and maintain them in a good growth state for cell fusion;

[0020] (3) Cell fusion: using a polyethylene glycol-mediated cell fusion method, obtaining spleen lymphocytes from the mouse in step (1), mixing them with the SP2 / 0 cells in step (2) and centrifuging them to mediate cell fusion, and culturing the fused cells under appropriate conditions to obtain a culture;

[0021] (4) Screening of hybridoma cells: The above culture was cultured in HAT selective medium for screening cells deficient in hypoxanthine phosphoribosyltransferase (HPRT) or thymidine kinase (TK) activity, and positive clones were obtained by screening;

[0022] (5) Cloning of hybridoma cells: selecting the culture well with the highest antibody titer and the growth of a single clone, performing limiting dilution again, performing limiting dilution more than 4 times continuously, and continuously passaged for more than 20 generations to obtain a single specific hybridoma cell line 6G2 that stably and efficiently expresses the anti-DENV NS1 protein monoclonal antibody 6G2; the amino acid sequences of the heavy chain CDR and light chain CDR of the anti-DENV NS1 protein monoclonal antibody 6G2 are shown in SEQ ID No. 1 and SEQ ID No. 3, respectively;

[0023] (6) Preparation of monoclonal antibody ascites: 8-10 week old BALB / c mice were selected and injected intraperitoneally with an appropriate amount of Freund's incomplete adjuvant one week later. Hybridoma 6G2 cells in PBS buffer were then inoculated into the abdomen again. When the abdomen of the mouse was significantly swollen and before the mouse was on the verge of death, the ascites was collected and centrifuged to determine the antibody titer.

[0024] (7) Purify the monoclonal antibody in mouse ascites to obtain the antibody.

[0025] The fifth aspect of the present invention is to provide a hybridoma cell that secretes the aforementioned monoclonal antibody 6G2 against DENV NS1 protein.

[0026] The sixth aspect of the present invention is to provide a nucleotide encoding the variable region of the heavy chain or light chain of the monoclonal antibody 6G2 against DENV NS1 protein.

[0027] In some embodiments, the CDR nucleotide sequence of the heavy chain of the anti-DENV NS1 protein monoclonal antibody 6G2 is shown as SEQ ID No. 2, and the CDR nucleotide sequence of the light chain is shown as SEQ ID No. 4.

[0028] The monoclonal antibody 6G2 screened and obtained in the present invention exhibits broad binding to the NS1 protein of four DENV serotypes (DV-1, DV-2, DV-3, and DV-4). In in vitro cell models, it competitively inhibits NS1 protein binding to target cells and effectively inhibits the biological functions of NS1 protein. Antibody 6G2 can delay the onset and progression of symptoms in mice in the AG129 mouse model and protect 25% of mice against a lethal dose of DENV2. Monoclonal antibody 6G2 broadly inhibits the binding of DENV NS1 proteins to target cells across all four serotypes, with the greatest inhibition reaching over 95% against DENV1, DENV3, and DENV4 NS1. However, its inhibitory effect against DENV2 NS1 is relatively weak. Therefore, the antibody mutants prepared from the antibody itself or the recombinant expression products obtained through CDR sequence design and modification described in the present invention have potential antiviral applications in the design of dengue vaccine candidates, screening of therapeutic drugs, or the preparation of antiviral drugs for DENV1-4 viral infections.

[0029] The anti-DENV NS1 monoclonal antibody 6G2 described in this invention has the ability to competitively bind to the DENV1-4 NS1 proteins, inhibit vascular leakage symptoms, and protect against the lethal damage of DENV infection in vivo, potentially offering significant value in treating severe dengue fever. The monoclonal antibody 6G2 developed in this invention has significant implications and potential for the prevention and treatment of dengue virus infection, including severe dengue fever. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is the result of SDS-PAGE identification of the purity of monoclonal antibody 6G2.

[0031] Figure 2 This is the result of ELISA identification of the binding curve of monoclonal antibody 6G2 and DENV1-4 NS1 protein.

[0032] Figure 3 This is a flow cytometry result showing the competitive inhibition of NS1 protein binding to target cells by monoclonal antibody 6G2.

[0033] Figure 4 This figure shows the results of validating the ability of monoclonal antibody 6G2 to protect against fatal vascular leakage damage caused by DENV infection in the AG129 mouse model. DETAILED DESCRIPTION

[0034] To facilitate 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 present disclosure more thorough and comprehensive.

[0035] Experimental procedures in the following examples, where specific conditions are not specified, generally followed conventional conditions, such as those in Molecular Cloning: A Laboratory Manual (4th edition, edited by Green and Sambrook, published in 2013), or according to manufacturer recommendations. All commonly used chemical reagents used in the examples were commercially available.

[0036] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as commonly understood by those skilled in the art to which this invention pertains. The terms used in this specification are for the purpose of describing specific embodiments only and are not intended to limit the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0037] 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 site of the antibody molecule. Because the antigen binding site is complementary to the antigen epitope structure, the hypervariable region is also called the complementarity-determining region of the antibody molecule.

[0038] In some embodiments of the present invention, a sequential immunization strategy is used to target four serotypes of DENV NS1 protein to enhance the immune response to conserved epitopes and improve the induction efficiency of broadly cross-reactive antibodies. A monoclonal antibody 6G2 against DENV NS1 protein is obtained. The monoclonal antibody is of IgG2b subtype and can specifically and broadly recognize four serotypes of DENV NS1 protein, DV-1, DV-2, DV-3, or DV-4. The heavy chain CDR amino acid and nucleotide sequence of the anti-DENV NS1 protein monoclonal antibody are shown in SEQ ID No. 1 and SEQ ID No. 2, and the light chain CDR nucleotide and amino acid sequences are shown in SEQ ID No. 3 and SEQ ID No. 4, as follows:

[0039] SEQ ID No. 1: amino acid sequence of heavy chain CDR

[0040] EVKLVESGGALVKPGGSLKLSCAASGFAFSTYDMSWVRQTPEKRLEWVATISSGATYTYYTDSVKGRFTISRDNARNTLYLQMSSLRSEDTAMYYCSRHGPVVATGDYWGQGTTLTVSS

[0041] SEQ ID No. 2: Nucleotide sequence of heavy chain CDR

[0042] GAAGTGAAGCTGGTGGAGTCTGGGGGAGCCTTAGTGAAGCCTGGAGGGTCCCTGAAACTCTCCTGTGCAGCCTCTGGATTCGCTTTCAGTACCTATGACATGTCTTGGGTTCGCCAGACTCCGGAAAAGAGGCTGGAGTGGGTCGCAACCATTAGTAGTGGTGCTACTTACACCTACTATACAGACAGTGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCAGGAATACCCTGTACTTGCAAATGAGCAGTCTGCGTTCTGAGGACACGGCCATGTATTACTGTTCAAGACATGGGCCGGTTGTGGCTACAGGAGACTACTGGGGCCAAGGCACCACTCTCACGGTCTCTTCA

[0043] SEQ ID No.3: Amino acid sequence of the light chain CDR

[0044] DIVMSQSPSSLAASVGETVTLNCKSSQSLLYSSDQKNYLAWYQQKPGQSPKLLIYWASTRDSGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYNYLTFGAGTKLELK

[0045] SEQ ID No.4: Nucleotide sequence of the light chain CDR

[0046] GACATTGTGATGTCACAGTCTCCATCCTCCCTAGCTGCGTCAGTTGGAGAGACGGTTACTTTGAACTGCAAGTCCAGTCAGAGCCTTTTATATAGTAGCGATCAAAAGAACTACTTGGCCTGGTATCAGCAGAAACCAGGACAGTCTCCTAAACTGCTGATTTACTGGGCATCCACTAGGGATTCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTGTGAAGGCTGAGGACCTGGCAGTTTATTACTGTCAGCAATATTATAACTATCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAAC。

[0047] In some embodiments of the present invention, the preparation method of the monoclonal antibody against DENV1-4 NS1 protein includes biosynthesis, cell expression, hybridoma cell secretion, etc., for example, by constructing the corresponding expression gene of the antibody and expressing it through an expression vector and a host cell.

[0048] The preparation method also includes fusing, screening, cloning, and stably passaged BALB / c mouse spleen cells sequentially immunized with DENV1-4 NS1 protein and myeloma cells SP2 / 0 to obtain a hybridoma cell line 6G2, which secretes the monoclonal antibody 6G2.

[0049] The present invention is further described in detail below with reference to specific embodiments.

[0050] Example 1: Preparation of monoclonal antibodies

[0051] 1. Immunization of mice: 6-8 week old BALB / c mice were immunized using a sequential immunization strategy. The mice were immunized in the order of serotypes with an interval of 2 weeks. The specific steps are:

[0052] (1) Eight female BALB / c mice aged 4-6 weeks were randomly divided into two groups: a sequential immunization group of 5 mice and a control group of 3 mice.

[0053] (2) Primary immunization: The sequential immunization group was immunized with DENV1 NS1. Emulsify the DENV1 NS1 protein with equal volumes of Freund's complete adjuvant. The immunization dose was 50 μg / mouse, and the immunization dose was 0.5 mL / mouse. The antigen emulsion was injected intraperitoneally into the mice.

[0054] (3) Booster immunization: Two weeks after the primary immunization, Freund's complete adjuvant was replaced with Freund's incomplete adjuvant. The immunization dose was halved to 25 μg / mouse, and the immunization dose was 0.5 mL / mouse. Emulsified DENV2 NS1, DENV3 NS1, and DENV4 NS1 proteins were injected intraperitoneally into the sequential immunization groups, with an interval of two weeks between each injection. Seven days after the final immunization, the antibody titer of the mouse serum was measured by ELISA.

[0055] 2. Indirect ELISA to measure the titer of immune mouse serum

[0056] Blood was collected from the tail vein and centrifuged to collect serum. Serum was coated with four DENV serotype NS1 proteins and used to identify the antibody titer against each DENV serotype.

[0057] 3. Culture and subculture of mouse myeloma cells SP2 / 0: SP2 / 0 myeloma cells were cultured and subcultured using 1640 medium containing 10% bovine serum in a 37°C incubator containing 5% carbon dioxide.

[0058] 4. Cell fusion: BALB / c mouse peritoneal macrophages were used as feeder cells. One day before fusion, BALB / c mouse peritoneal macrophages were inoculated into 96-well culture plates and cultured in 1×HAT selection medium. 5 Blood was collected from the orbital venous plexus of the above mice for serum isolation. Mice were sacrificed by cervical dislocation and soaked in 75% alcohol for 15 minutes. The abdomens of the mice were partially dissected and the spleens were aseptically removed. The spleens were rinsed with pre-chilled serum-free 1640 medium and triturated to obtain a splenocyte suspension.

[0059] Place the above-mentioned sp2 / 0 and spleen cells in centrifuge tubes and dilute to 30 mL with 1640 medium. Centrifuge at 1000 rpm for 5 minutes, discard the supernatant, wash twice with 30 mL of 1640 medium, and finally resuspend in 10 mL of serum-free 1640 medium. Count the cells to determine the concentration and total amount of both cells.

[0060] Mix splenocytes and sp2 / 0 cells in a 5:1 ratio, centrifuge at 1000 rpm for 5 minutes, and discard the supernatant. Gently flick the bottom of the tube to completely resuspend and mix the cell pellet. Initiate cell fusion with 1 mL of preheated 50% PEG solution. After fusion is terminated, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, and add 1× HAT 1640 medium preheated at 37°C. Mix the cell suspension and add it dropwise to a 96-well cell culture plate containing feeder cells using a pipette (100 μl / well). Incubate at 37°C in a 5% CO2 incubator.

[0061] 5. Hybridoma Cell Screening: After 5 days, the 96-well culture plate was cultured with half the volume of 1×HAT medium. After 10 days, the fused hybridoma cells were cultured in the selective medium for approximately two weeks. When the cell colonies reached an appropriate size, the cell culture supernatant was aspirated for enzyme-linked immunosorbent assay (ELISA) to screen for positive clones. Positive hybridoma clones were screened using indirect ELISA.

[0062] 6. Hybridoma Cell Cloning: Hybridoma cell cloning culture was performed using the limiting dilution method. Hybridoma wells that tested positive for antibodies were selected for cell counting. 150-200 cells were prepared using 1640 complete medium containing 1×HT to prepare a cell suspension. 100 μL was inoculated into a 96-well culture plate containing feeder cells. After 10 days, cell growth was observed and the antibody level in the supernatant was assayed. The wells with the highest antibody titer and showing single clonal cell growth were selected for further limiting dilution. This was repeated four or more times until all dilution wells were positive. Cells from a single clonal well were selected for continuous passage for at least 20 generations. This was used to screen for the hybridoma cell line 6G2 that stably and efficiently expressed anti-DNEV NS1 protein monoclonal antibodies.

[0063] 7. Monoclonal Antibody Ascites Preparation: Select healthy 8-10 week old BALB / c mice and intraperitoneally inject each mouse with 0.5 mL of incomplete Freund's adjuvant. After 7-10 days, when the mouse's abdominal cavity is enlarged and ascites develop, intraperitoneally inoculate 5×106 positive hybridoma cells diluted in PBS with 6G2 hybridoma cells in each abdomen. 7-10 days after inoculation, the mouse abdomen will be significantly enlarged. Closely observe the mouse's health and abdominal signs and collect ascites at an appropriate time.

[0064] 8. Purification of monoclonal antibodies: Monoclonal antibody 6G2 was purified from ascites using the Protein A column method.

[0065] 9. Use SDS-PAGE electrophoresis to identify the purity of the antibody. Analyze the reducing and non-reducing SDS-PAGE gels of the monoclonal antibody 6G2 stained with Coomassie Brilliant Blue: In reducing SDS-PAGE, a single band was observed at 55kDa (heavy chain) and 25kDa (light chain) for the monoclonal antibody 6G2. In non-reducing SDS-PAGE conditions, a single band was observed at approximately 150kDa (intact antibody) for the antibody 6G2. The results are shown in Figure 1 .

[0066] 10. Monoclonal Antibody Subtype Identification: Analysis was performed using a Bio-Rad Mouse Monoclonal Antibody Immunoglobulin Identification Kit. The purified monoclonal antibody was appropriately diluted and tested, strictly following the kit instructions. The ELISA assay was positive for the IgG2b subtype, with an OD value of 1.864 at 450 nm, indicating that the monoclonal antibody 6G2 secreted by hybridoma cell 6G2 was of the IgG2b subclass.

[0067] The hybridoma cell 6G2 was sequenced, and the amino acid and nucleotide sequences of the heavy chain CDR and light chain CDR of the secreted antibody 6G2 were shown in SEQ ID No. 1 to SEQ ID No. 4.

[0068] The hybridoma cell line 6G2 was cloned four times and cultured continuously for more than four months, and the antibody secretion was stable.

[0069] Example 2: Binding curve of antibody 6G2 and NS1 protein identified by indirect ELISA

[0070] DENV1-4 NS1 proteins were diluted to 1 μg / mL with PBS and added to a 96-well ELISA plate at 100 μL / well. The plates were coated overnight at 4°C. Excess incubation solution was discarded, the plates were washed three times with PBST, and patted dry. 5% skim milk powder solution was added at 300 μL / well and blocked at 37°C for 2 h. The blocking solution was discarded, the plates were washed three times with PBST, and patted dry. The antibody to be tested was diluted 10-fold starting at 100 μg / mL in eight gradients, and 100 μL / well was added to the ELISA plate. The plates were incubated at 37°C for 60 min. The negative control was PBS-immunized mouse serum, and the blank control was PBS solution. The serum samples were discarded, the plates were washed three times with PBST, and patted dry. 100 μL / well of HRP-labeled goat anti-mouse secondary antibody solution diluted 5000 times in PBS was added and the plates were incubated at 37°C for 60 min. The secondary antibody solution was discarded, the plates were washed five times with PBST, and patted dry. TMB substrate was added for color development (component A: component B = 1:1), 100 μL / well, and color was developed at room temperature for 10 minutes. 50 μL / well of 2M concentrated sulfuric acid was added to stop color development, and the absorbance (A) of each well was measured at a wavelength of 450 nm using a microplate reader. The calculated KD value of antibody 6G2 showed that antibody 6G2 had strong cross-reactivity with all four serotypes of DENV NS1 protein, with the binding ability to DENV2 NS1 being at the nM level, and the KD values ​​of the other three serotypes of DENV NS1 protein all reaching the pM level. Figure 2 .

[0071] Example 3: Flow cytometry verification of antibody 6G2 competitively inhibiting the binding of NS1 protein to target cells

[0072] HBMEC and HUVEC (human brain microvascular endothelial cells, human umbilical vein endothelial cells) were routinely passaged and cultured to the logarithmic growth phase. After trypsinization, the cells were harvested and the supernatant discarded. DENV1-4NS1 protein (20 μg / mL) and antibody 6G2 (40 μg / mL) were mixed in a 10% FBS-PBS solution and incubated at 37°C for 30 minutes. All reagents were pre-chilled at 4°C and all operations were performed on ice. Cells were resuspended in ice-cold PBS containing 10% FBS and 1% sodium azide to approximately 1 × 106 cells / mL. 100 μL of cell suspension was added to each centrifuge tube and incubated on ice for 15 minutes. After the cells and antibody-NS1 protein complex were mixed evenly, they were incubated at 4°C for 30 minutes. The cells were centrifuged at 1000 rpm for 5 minutes, resuspended in pre-chilled PBS, and washed three times. Subsequently, a mouse-derived His-tag monoclonal antibody directly labeled with FITC was used to bind to the NS1 protein bound to the target cells, and the cells were incubated 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 cytometer, protect from light, and detect on the machine as soon as possible. The percentage of target cells bound to the NS1 protein in the total number of cells was analyzed. Compared with the control group without antibody, the decrease in the proportion represents that the NS1 protein was competitively bound by antibody 6G2. The results showed that antibody 6G2 can broadly inhibit the binding of four serotypes of DENV NS1 proteins to target cells. Among them, the inhibitory effect on DENV1, DENV3 and DENV4 NS1 was the best, all reaching more than 95%, but the inhibitory effect on DENV2 NS1 was weak, which is consistent with the ELISA experimental results in Example 2. See the results. Figure 3 .

[0073] Example 4: Verification of the Protective Efficacy of NS1 Broad-Spectrum Antibodies in the Immunodeficient Mouse AG129 Model

[0074] Using the AG129 immunodeficient mouse model, the monoclonal antibody 6G2 (300 μg / mouse) was pre-immunized and then exposed to a lethal dose of DENV2 (5×105 PFU / mouse). The changes in the physical signs, course of disease, body weight and survival rate of the mice were continuously observed, and the vascular leakage experiment was used to evaluate the protective effect of the antibody. The results of the plotted survival curve showed that while all the mice in the control group that were only challenged with DENV2 died on the 12th day, the antibody 6G2 was able to protect the mice from the attack of a lethal dose of DENV2, and 25% of the mice were still alive on the 20th day after infection. The results are shown in Figure 4 Middle A.

[0075] At the same time, the results of the vascular leakage experiment based on Evans Blue showed that antibody 6G2 could significantly improve the degree of vascular leakage in the liver, spleen and small intestine of mice, especially the vascular leakage in the spleen was completely alleviated (P>0.05). Figure 4 Middle B.

[0076] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be determined by the appended claims.

Claims

1. A monoclonal antibody 6G2 against DENV NS1 protein, characterized in that: The amino acid sequence of the heavy chain CDR is shown in SEQ ID No.1, and the amino acid sequence of the light chain CDR is shown in SEQ ID No.

3.

2. The anti-DENV NS1 protein monoclonal antibody 6G2 according to claim 1, characterized in that The subtype of the anti-DENV NS1 protein monoclonal antibody 6G2 is IgG2b.

3. Use of the monoclonal antibody 6G2 against DENV NS1 protein according to claim 1 or 2 in the preparation of a drug or vaccine for preventing and treating dengue virus infection.

4. Use of the monoclonal antibody 6G2 against DENV NS1 protein according to claim 1 or 2 in the preparation of a drug or vaccine for inhibiting or treating vascular leakage in severe dengue fever.

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

6. A pharmaceutical composition for treating dengue virus infection, characterized in that it comprises an active ingredient and a pharmaceutically acceptable carrier, wherein the active ingredient is the monoclonal antibody 6G2 against DENV NS1 protein according to claim 1 or 2.

7. The pharmaceutical composition for treating dengue virus infection according to claim 6, characterized in that: The pharmaceutical composition is a drug preparation or a vaccine.

8. A method for neutralizing dengue virus, characterized in that: The method comprises reacting dengue virus with the monoclonal antibody 6G2 against DENV NS1 protein according to claim 1 or 2.

9. A method for preparing monoclonal antibody 6G2 against DENV NS1 protein, characterized in that: The preparation method comprises the following steps: (1) Animal immunization: A sequential immunization strategy was used to immunize mice with serotypes DV-1, DV-2, DV-3, and DV-4, respectively; (2) Cultivation of mouse myeloma cells: Cultivating mouse myeloma cells SP2 / 0 and maintaining them in a good growth state for cell fusion; (3) Cell fusion: using a polyethylene glycol-mediated cell fusion method, obtaining spleen lymphocytes from the mouse in step (1), mixing and centrifuging with the SP2 / 0 cells in step (2) to mediate cell fusion, and culturing the fused cells under appropriate conditions to obtain a culture; (4) Screening of hybridoma cells: culturing the above culture in HAT selective medium and screening to obtain positive clones; (5) 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 6G2 that stably and efficiently expresses the anti-DENV NS1 protein monoclonal antibody 6G2; the amino acid sequences of the heavy chain CDR and light chain CDR of the anti-DENV NS1 protein monoclonal antibody 6G2 are shown in SEQ ID No. 1 and SEQ ID No. 3, respectively; (6) Preparation of monoclonal antibody ascites: 8-10 week old BALB / c mice were selected and injected intraperitoneally with an appropriate amount of Freund's incomplete adjuvant one week later. Then, hybridoma cells 6G2 were inoculated again in the abdomen with PBS buffer. 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. (7) Purify the monoclonal antibody in mouse ascites to obtain the antibody.

10. A nucleotide encoding the variable region of the heavy chain or light chain of the monoclonal antibody 6G2 against DENV NS1 protein according to claim 1, wherein the nucleotide sequence encoding the variable region of the heavy chain of the monoclonal antibody 6G2 against DENV NS1 protein is shown in SEQ ID No. 2; The nucleotide sequence encoding the light chain variable region of the monoclonal antibody 6G2 against DENV NS1 protein is shown in SEQ ID No.4.

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