Hybridoma cell strain, West Nile virus monoclonal antibody as well as preparation method and application of West Nile virus monoclonal antibody

Abstract

The invention discloses a hybridoma cell strain, a West Nile virus monoclonal antibody as well as a preparation method and application of the West Nile virus monoclonal antibody. The West Nile virus monoclonal antibody is secreted by a hybridoma cell strain WN-mAb-12, the hybridoma cell strain is preserved in the China Center for Type Culture Collection (the address is Wuhan University, Wuhan, China), the preservation number is CCTCC NO: C2024123, and the preservation date is April 23, 2024. The monoclonal antibody secreted by the hybridoma cell strain WN-mAb-12 can generate a neutralization effect on the West Nile virus, and has a good neutralization effect. By analyzing the biological characteristics of the West Nile virus, a technical support is laid for researching the protection mechanism of the West Nile virus neutralizing antibody and researching and developing clinical specific medicines in the future.

Description

Technical Field

[0001] The present invention relates to the technical field of cell engineering, and particularly relates to a hybridoma cell line, a monoclonal antibody produced thereby, a preparation method thereof, and an application of the monoclonal antibody in detecting and / or neutralizing West Nile virus. Background Art

[0002] West Nile virus is a neurotropic arbovirus mainly transmitted by Culex mosquitoes and Aedes mosquitoes. It belongs to the Japanese encephalitis virus serogroup of the genus Flavivirus in the family Flaviviridae and is a single-stranded positive-sense RNA virus with a full-length genome of 11,029 nt. The structural sequence order is the 5' non-coding region, encoding 3 structural proteins and 7 non-structural proteins, and finally the 3' non-coding region. West Nile fever caused by this virus is a zoonotic infectious disease that can infect humans and other animals with arthropods - mosquitoes as the transmission vector. Birds are the main hosts, and humans and horses are accidental hosts. In 1937, the virus was first isolated from a blood sample of a febrile woman in the West Nile region of Uganda, so it was named West Nile virus (WNV). In the mid-1990s, the disease was only sporadic locally, but with the mutation of the virus strain, the number of human and horse infection cases has increased. It was first introduced into the United States in 1999, resulting in nearly 10,000 people being infected and hundreds of people dying, which first attracted global attention and has been listed as one of the major global epidemics by the World Health Organization (WHO) and the World Organization for Animal Health (OIE). After the disease entered the United States in 1999, Culex pipiens was the main vector for transmission in the northern region; while Culex quinquefasciatus was the main vector for transmission in the southern and western regions. In 2003, WNV virus was found in birds in the UK and caused extensive transmission among birds. In 2010, the first clinical case of WNV was discovered in Spain. Similar to most Flaviviridae viruses, most people infected with WNV have no clinical symptoms and can recover on their own, but about 20% of the infected population will develop WNF, and the symptoms include fever, headache, muscle pain or rash, etc. 1 / 150 - 1 / 250 of the patients will develop severe neurotropic diseases (West Nile neurotropic disease, WNND), including encephalitis, meningitis or flaccid poliomyelitis-like paralysis, which can lead to respiratory system damage. The case fatality rate of patients infected with WNV is about 4.2%, but the case fatality rate of WNND patients is about 9.6%. Currently, there is no report of WNV encephalitis cases in China. However, due to its vast territory, there are often migratory birds and traded livestock such as horses at the borders, which are very likely to be favorable conditions for the transmission of WNV virus into China. In addition, there are a large number of mosquito species, and the mosquito species vary in different altitudes and regions. The main transmission hosts of WNV that have been confirmed, Culex pipiens and Culex quinquefasciatus, are widely distributed in China.

[0003] WNV is transmitted to humans and other animals through mosquito bites. Its primary host is birds. Some birds show symptoms or even die after infection, while those without obvious symptoms become effective transmitters. Mosquitoes become infected by sucking the blood of virus-infected birds, and the virus is stored in the mosquito's salivary glands. It is then transmitted through bites, and some mosquitoes remain infected throughout their lives. At the same time, experiments have shown that WNV can be horizontally transmitted among birds through contact. Mammals have a relatively low level of viremia after infection, usually not sufficient to transmit the virus back to mosquitoes, thus ending the virus transmission cycle. Other mammals, such as horses, dogs, camels, and goats, have been confirmed to be able to be infected with WNV. Humans are generally considered the "dead-end" host of WNV. Additionally, in nature, WNV is mainly transmitted through mosquitoes and birds. Although many different species of mosquitoes can carry and transmit WNV, Culex mosquitoes play the most significant role in natural transmission. Besides mosquito-borne transmission, WNV can also be transmitted through blood transfusion, transplantation, breast milk, and intrauterine transmission. In 2002, a case of transmission through organ transplantation was reported, and WNV was detected in 4 patients who received organ transplants from the same donor infected with WNV. Since its first discovery in Uganda in 1937, there has been evidence of WNV transmission and prevalence on all continents except Antarctica globally.

[0004] Currently, the diagnosis of this disease mainly uses human or animal tissues, cerebrospinal fluid, serum, or whole blood, combined with serological, virological, histopathological, epidemiological, and other methods. However, serological testing has always been one of the most important methods for diagnosing West Nile disease, mainly including: IgM antibody capture enzyme immunoassay (MAC-ELISA), indirect immunofluorescence assay (IFAT), hemagglutination inhibition assay (HI), and plaque reduction neutralization test (PRNT). Several detection methods for West Nile virus nucleic acid have been developed and widely used, mainly including reverse transcription-polymerase chain reaction (PT-PCR), reverse transcription-nested polymerase chain reaction (PT-nPCR), real-time fluorescence quantitative reverse transcription-polymerase chain reaction PCR (Real time RT-PCR), and nucleic acid sequence-based amplification (NASBA).

[0005] However, in the pursuit of convenience, speed, and efficiency, the above detection methods inevitably have some drawbacks that require further exploration for continuous optimization, such as cumbersome and complex experimental operations and high requirements for instruments and equipment. Currently, the main detection method for West Nile virus is the qRT-PCR method, which is not very suitable for rapid self-screening in West Nile virus epidemic areas, and the research and development of a rapid detection method for neutralizing antibodies against West Nile virus have not been completed yet. The monoclonal antibodies obtained in this study have high titer, strong specificity, and significant neutralizing effects, providing a laboratory basis for the establishment of a detection method for West Nile virus neutralizing antibodies. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to overcome the above-mentioned deficiencies of the prior art and provide a hybridoma cell line WN-mAb-12 of monoclonal antibodies against West Nile virus and its monoclonal antibodies. The monoclonal antibodies secreted by the hybridoma cell line WN-mAb-12 of the present invention can be used for biological diagnosis of West Nile virus, including ELISA (enzyme-linked immunosorbent assay), indirect immunofluorescence, and neutralization assay.

[0007] The first aspect of the present invention is to provide a hybridoma cell line WN-mAb-12 (Hybridoma cell line WN-mAb-12) of monoclonal antibodies against West Nile virus. This hybridoma cell line has been deposited with the China Center for Type Culture Collection (address: Wuhan University, Wuhan, China), with the deposit number CCTCC NO: C2024123 and the deposit date of April 23, 2024.

[0008] The second aspect of the present invention is to provide a monoclonal antibody against West Nile virus, which is secreted by the hybridoma cell line WN-mAb-12 with the deposit number CCTCC NO: C2024123. The monoclonal antibody against West Nile virus of the present invention is obtained by immunizing Balb / c mice with West Nile virus amplified in suckling mouse brains and diluted in PBS as an antigen, and using cell fusion technology to obtain the hybridoma cell line WN-mAb-12 that produces this antibody. The antibody secreted by the hybridoma cell is of the IgG2a type.

[0009] The third aspect of the present invention is to provide a preparation method of the monoclonal antibody against West Nile virus as described in the second aspect of the present invention, including the following steps:

[0010] (1) Immunize Balb / c mice with West Nile virus to obtain immunized Balb / c mice;

[0011] (2) The spleen cells of immunized Balb / c mice were fused with mouse myeloma cells SP2 / 0. The fused cells were selectively cultured in HAT selection medium. Positive clones were screened by indirect immunofluorescence method, and the hybridoma cells in the positive wells were cloned by limiting dilution method until the positive rate of the cloned cells was 100%, obtaining a hybridoma cell line;

[0012] (3) The hybridoma cell line obtained in step (2) was transferred into the abdominal cavity of Balb / c mice for culture, ascites were collected, and purified West Nile virus monoclonal antibody was obtained by affinity chromatography.

[0013] The fourth aspect of the present invention is to provide the application of the West Nile virus monoclonal antibody as described in the second aspect of the present invention or the monoclonal antibody prepared by the preparation method as described in the third aspect of the present invention in the preparation of reagents for detecting West Nile virus or in the preparation of reagents for diagnosing and / or predicting the prognosis of West Nile fever.

[0014] The fifth aspect of the present invention is to provide a reagent for detecting West Nile virus, and the reagent includes the West Nile virus monoclonal antibody as described in the second aspect of the present invention or the monoclonal antibody prepared by the preparation method as described in the third aspect of the present invention.

[0015] The sixth aspect of the present invention is to provide a reagent for diagnosing and / or predicting the prognosis of West Nile fever, and the reagent includes the West Nile virus monoclonal antibody as described in the second aspect of the present invention or the monoclonal antibody prepared by the preparation method as described in the third aspect of the present invention.

[0016] The seventh aspect of the present invention is to provide the application of the West Nile virus monoclonal antibody as described in the second aspect of the present invention or the monoclonal antibody prepared by the preparation method as described in the third aspect of the present invention in the preparation of a preparation for treating West Nile fever.

[0017] Preferably, the preparation includes a freeze-dried preparation or a liquid preparation.

[0018] Preferably, the preparation includes an injectable preparation or an oral preparation.

[0019] The eighth aspect of the present invention is to provide a biological preparation for treating West Nile fever, and the preparation includes the West Nile virus monoclonal antibody as described in the second aspect of the present invention or the monoclonal antibody prepared by the preparation method as described in the third aspect of the present invention.

[0020] The present invention obtained the above hybridoma cell line WN-mAb-12 and the West Nile virus monoclonal antibody it produced through the following specific methods:

[0021] (1) Virus amplification

[0022] The West Nile virus was diluted in DMEM medium and injected into the brains of 3-day-old Balb / c suckling mice for amplification, with an injection dose of 200 μL per mouse. After injection, the survival of the suckling mice was observed daily. When the suckling mice showed symptoms such as refusal to nurse, lying on their sides, and isolation from the group, the mouse brains were taken under sterile conditions, ground and diluted in 1×PBS, and then centrifuged to obtain the supernatant to get the virus suspension.

[0023] (2) Immunize mice

[0024] The prepared virus suspension was used to immunize male Balb / c mice aged 6 - 8 weeks.

[0025] First immunization: The virus suspension was mixed with an equal volume of Freund's adjuvant and subcutaneously injected into Balb / c mice at multiple points at a dose of 400 μL per mouse.

[0026] Second immunization: Two weeks later, the virus suspension was mixed with an equal volume of Freund's adjuvant and subcutaneously injected into Balb / c mice at multiple points at a dose of 400 μL per mouse.

[0027] Third immunization: Another two weeks later, the virus suspension was mixed with an equal volume of Freund's adjuvant and subcutaneously injected into Balb / c mice at multiple points at a dose of 400 μL per mouse. Ten days after the third immunization, blood was collected from the inner canthus of the mice's eyes, and the mouse serum was obtained by centrifugation. A West Nile virus antigen plate was prepared, and the serum titer was detected by indirect immunofluorescence assay. Mice with a titer greater than 1:10 4 were selected, and their spleen cells were fused with myeloma cells.

[0028] Among them, the Freund's adjuvant was a commercial product obtained from the market.

[0029] (3) Determination of immunoserum titer

[0030] The immunoserum titer was determined by indirect immunofluorescence assay.

[0031] Select in T75cm 2Vero cells (African green monkey kidney cells) in good growth condition in a cell culture flask were added with 35 mL of 2% FBS DMEM medium and 5 mL of virus suspension, and cultured in a CO2 incubator while observing the cytopathic effect. When the cells were partially cytopathic and in a semi-detached state, they were digested and pipetted to disperse, then 200 mL of 2% FBS DMEM medium was supplemented and pipetted into a 96-well plate, 200 μL per well, and cultured in a CO2 incubator for 1 - 2 days. The cell supernatant was discarded, and the cells were washed twice with 1×PBS (if the cytopathic effect is severe and washing may cause serious cell detachment, washing can be omitted), 150 μL per well; 150 μL of methanol pre-cooled at -20°C was added per well, and fixed at -20°C for 20 min, then the supernatant was discarded; 150 μL of 0.5% Triton 100× (immunostaining permeabilization solution, solvent is 1×PBS) was added per well, and permeabilized at room temperature for 15 min, then the supernatant was discarded; sample diluent (10 g of 10% BSA, 0.1 g of 0.02% thimerosal) was added for blocking, 150 μL per well, and blocked in a 37°C water bath for more than 3 h, then the supernatant was discarded, and a West Nile virus antigen plate was prepared.

[0032] Take the antigen plate prepared above, dilute the isolated mouse serum with the sample diluent, and perform serial dilution starting from an initial concentration of 1:500. The dilution gradients are 1:500, 1:1000, 1:2000, 1:4000, 1:8000, 1:16000, 1:32000, and 1:64000 in sequence. At the same time, set up a positive control well (WNV positive serum: sample diluent = 1:1000) and a negative control well (mouse serum after blank treatment: sample diluent = 1:1000), 100 μL per well, and incubate in a 37°C water bath for 2 h; wash the plate 4 times with PBS containing 0.05% (V / V) Tween-20 (polysorbate-20), and pat dry; after diluting FITC-goat anti-mouse IgG: enzyme diluent = 1:300, add 50 μL per well, and incubate in a 37°C water bath for 1 h; wash the plate 4 times with PBS (containing 0.05% (V / V) Tween-20), rinse with running water for 5 min and then pat dry; dry in a 37°C incubator for 0.5 - 1 h, then observe the fluorescence intensity under a fluorescence microscope and determine the result. The dilution gradient corresponding to the well immediately above the one with fluorescence intensity between "+++" and "++" is determined as the titer of the serum.

[0033] (4) Preparation of hybridoma cells

[0034] Take the serum with a titer greater than 1:10 4For mice, 3 days before fusion, the virus suspension was mixed with an equal volume of Freund's adjuvant and then subcutaneously injected into Balb / c mice at multiple points at a dose of 400 μL per mouse for booster immunization. 3 days later, the spleens of the mice were aseptically removed, made into spleen cell suspensions, and mixed with the mouse myeloma cell line SP2 / 0 in the logarithmic growth phase at a ratio of 1:3. After centrifugation at 1000×g for 5 min at room temperature, the supernatant was discarded. The bottom of the centrifuge tube was gently flicked with a finger to loosen the precipitate, 10 mL of fusion buffer was added to resuspend the cells, and then centrifuged at 1600 rpm for 5 min. The supernatant was discarded; again, 10 mL of fusion buffer was added to resuspend the cells, and centrifuged at 1600 rpm for 5 min. The supernatant was discarded; 1 mL of fusion buffer was added to resuspend the cells to obtain a cell mixture for standby; after disinfecting the electrode chamber with 75% alcohol for 30 min, it was gently rinsed 3 times with 2 mL of fusion buffer, placed in a biosafety cabinet to air dry and irradiated with ultraviolet light for 30 min; 100 μL of the prepared cell mixture was aspirated and added into the electrode chamber, and the resistance value was adjusted to between 5000 and 10000 Ω; after adjusting the resistance value, 800 μL of the cell mixture was aspirated and added into the electrode chamber, the resistance value was measured and recorded, and the resistance value should be between 500 and 1500 Ω. Then press the "Start" key to execute the electrofusion program and record the data; after electrofusion, the cell mixture was aspirated from the electrode chamber and added into a preheated 15 mL centrifuge tube containing 1.6 mL of serum-free DMEM medium, and left to stand at 37 °C for 10 min (the serum-free DMEM medium needs to be prepared in advance and preheated at 37 °C), centrifuged at 1600 rpm for 5 min, the supernatant was discarded, and the cells were gently resuspended with HAT culture medium (hypoxanthine (H), aminopterin (A) and thymidine (T) (HAT, Sigma)), and the cells were dispensed into a 96-well plate, 200 μL per well. After culturing for three days, the cell fusion situation was observed, and half of the HAT culture medium was replaced. This was done continuously for several days until clones formed. Seven days after fusion, the cells were cultured with HT culture medium (hypoxanthine (H) and thymidine (T) (HT, Sigma)).

[0035] (5) Screening for hybridoma cells secreting monoclonal antibodies against West Nile virus

[0036] The cell culture supernatant was screened by indirect immunofluorescence assay. Positive clone hybridoma cells with stronger fluorescence signals were selected for subcloning, and continuously cloned 3 - 4 times by the limiting dilution method until a 100% cell positive rate was achieved. Finally, a hybridoma cell line stably secreting monoclonal antibodies against West Nile virus was obtained and labeled as WN-mAb-12. The cells with a 100% positive rate after cloning were amplified and cultured and then cryopreserved in liquid nitrogen.

[0037] (6) Preparation of ascites

[0038] The WN-mAb-12 hybridoma cell line was at 1×10 5Inject the liquid paraffin-pretreated Balb / c male mice aged 8-10 weeks with only the cell mass into the abdominal cavity. After raising and observing for 10-14 days, when the abdomen of the mice is significantly enlarged, extract ascites. Centrifuge the collected ascites at 10000 rpm for 10 min, separate the supernatant, and obtain the West Nile virus monoclonal antibody secreted by the hybridoma cell line WN-mAb-12 in the cell supernatant, and store it at -80 °C for standby.

[0039] The ascites was further purified by affinity chromatography, and the purity of the purified monoclonal antibody was identified by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis). The Ig subtype of the monoclonal antibody was identified according to the instructions of the Pierce Rapid ELISA Mouse mAb Isotyping Kit.

[0040] Compared with the prior art, the present invention has the following beneficial effects:

[0041] The present invention obtains the West Nile virus monoclonal antibody hybridoma cell line WN-mAb-12 and its monoclonal antibody through the above method. The purified monoclonal antibody of the present invention can prove its neutralizing effect on the West Nile virus when used in ELISA, indirect immunofluorescence method, and qRT-PCR. The monoclonal antibody secreted by this hybridoma cell line lays a foundation for further research on the diagnostic reagent of the West Nile virus and provides a powerful tool for verifying its clinical diagnosis and treatment target of West Nile disease.

[0042] The immunogen of the monoclonal antibody of the present invention is the West Nile virus, not specific to a certain protein of the West Nile virus, which improves the preparation efficiency of the monoclonal antibody and reduces the application cost at the same time. The monoclonal antibody of the present invention can be used not only in ELISA and Western to detect the West Nile virus, but also in cell immunofluorescence and qRT-PCR to detect its neutralizing effect on the West Nile virus.

[0043] At present, there is no effective specific antibody against the West Nile virus at home and abroad, while the monoclonal antibody of the present invention has high specificity and sensitivity for detecting the West Nile virus and has a good neutralizing effect. The application of this antibody will provide support for the functional research of the West Nile virus and the verification work of its clinical specimens as markers of West Nile disease. Brief Description of the Drawings

[0044] Figure 1 It is the indirect immunofluorescence identification result of the monoclonal antibody WN-mAb-12 of the present invention.

[0045] Figure 2 It is the ELISA subtype identification result of the monoclonal antibody WN-mAb-12 of the present invention.

[0046] Figure 3It is the IFA detection result of the neutralizing effect of the monoclonal antibody WN-mAb-12 of the present invention.

[0047] Figure 4 It is the evaluation of the neutralizing titer of the monoclonal antibody WN-mAb-12 in the ascites of the present invention. Among them, a, b, c, d, and e are the incubation results of the cell supernatant of the anti-West Nile virus monoclonal antibody WN-mAb-12 prepared from the hybridoma cell CCTCC NO: C2024123 diluted 100, 200, 800, 3200 times, and the negative control respectively.

[0048] Figure 5 It is the qRT-PCR detection result of the neutralizing effect of the monoclonal antibody WN-mAb-12 of the present invention.

[0049] Figure 6 It is the preparation flow chart of the hybridoma cell line WN-mAb-12. Specific Embodiments

[0050] To make the present invention easier to understand, the present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention.

[0051] The West Nile virus used in the following examples was passaged and preserved by the Disease Prevention and Control Center of the Southern Theater Command of the Chinese People's Liberation Army.

[0052] Example 1 Preparation and Identification of the Monoclonal Antibody of the Present Invention

[0053] The preparation of the monoclonal antibody WN-mAb-12 includes the following steps:

[0054] (1) Virus Amplification

[0055] The West Nile virus was diluted in DMEM medium and then injected into the brains of 3-day-old Barb / c suckling mice for amplification. The injection dose was 200 μL / mouse. After injection, the survival of the suckling mice was observed daily. When the suckling mice showed symptoms such as refusal to nurse, lying on the side, and leaving the group, the mouse brains were taken under sterile conditions, ground and diluted in 1×PBS, and then centrifuged to obtain the supernatant to obtain the virus suspension.

[0056] (2) Immunization of Mice

[0057] The prepared virus suspension was used to immunize male Balb / c mice aged 6-8 weeks.

[0058] First immunization: The virus suspension was mixed with an equal volume of Freund's adjuvant and then subcutaneously injected into Balb / c mice at a dose of 400 μL / mouse at multiple points.

[0059] Second immunization: After 2 weeks, mix the virus suspension with an equal volume of Freund's adjuvant and then inject Balb / c mice subcutaneously at multiple sites at a dose of 400 μL per mouse.

[0060] Third immunization: After another 2 weeks, mix the virus suspension with an equal volume of Freund's adjuvant and then inject Balb / c mice subcutaneously at multiple sites at a dose of 400 μL per mouse. Ten days after the third immunization, collect blood from the inner canthus of the mouse eyes, and centrifuge to separate the mouse serum. Prepare a West Nile virus antigen plate and detect the serum titer by indirect immunofluorescence assay. Select mice with a titer greater than 1:10 4 and fuse their spleen cells with myeloma cells.

[0061] Among them, the Freund's adjuvant is a commercial product obtained from the market.

[0062] (3) Determination of immune serum titer

[0063] The immune serum titer was determined by indirect immunofluorescence assay (IFA method).

[0064] Select vero cells with good growth status in a T75 cm 2 cell culture flask, add 35 mL of 2% FBS DMEM medium and 5 mL of virus suspension, culture in a CO2 incubator and observe the cytopathic effect. When the cells are partially diseased and in a semi-detached state, digest and pipette to disperse them, then supplement 200 mL of 2% FBS DMEM medium and pipette it into a 96-well plate, 200 μL / well, and culture in a CO2 incubator for 1 - 2 days. Discard the cell supernatant, wash twice with 1×PBS (if the cytopathic effect is severe and washing may cause serious cell detachment, washing can be omitted), 150 μL / well; add methanol pre-cooled at -20 °C, 150 μL / well, fix at -20 °C for 20 min, discard the supernatant; add 0.5% Triton 100× (immunostaining permeabilization solution, solvent is 1×PBS), 150 μL / well, permeabilize at room temperature for 15 min, discard the supernatant; add sample diluent (10% BSA 10 g, 0.02% thimerosal 0.1 g) for blocking, 150 μL / well, block in a 37 °C water bath for more than 3 h, discard the supernatant, and prepare a West Nile virus antigen plate.

[0065] Take the antigen plate prepared above, dilute the separated mouse serum with sample diluent, perform serial dilution starting from an initial concentration of 1:500, and the dilution gradients are 1:500, 1:1000, 1:2000, 1:4000, 1:8000, 1:16000, 1:32000, and 1:64000 in sequence. At the same time, set up a positive control well (WNV positive serum: sample diluent = 1:1000) and a negative control well (mouse serum after blank treatment: sample diluent = 1:1000), 100 μL / well, incubate in a 37°C water bath for 2 h; wash the plate 4 times with PBS (containing 0.05% (V / V) Tween-20) and pat dry; after diluting FITC-goat anti-mouse IgG: enzyme diluent = 1:300, add 50 μL / well and incubate in a 37°C water bath for 1 h; wash the plate 4 times with PBS (containing 0.05% (V / V) Tween-20), rinse with running water for 5 min and pat dry; after drying in a 37°C incubator for 0.5 - 1 h, observe the fluorescence intensity under a fluorescence microscope and determine the result. The dilution gradient corresponding to the well immediately above the one with fluorescence intensity between "+++" and "++" is determined as the titer of the serum.

[0066] (4) Preparation of hybridoma cells

[0067] Take the serum with a titer greater than 1:10 4For mice, three days before fusion, the virus suspension was mixed with an equal volume of Freund's adjuvant and then subcutaneously injected into Balb / c mice at a dose of 400 μL per mouse at multiple points for booster immunization. Three days later, the spleens of the mice were aseptically removed, made into spleen cell suspensions, and mixed with the mouse myeloma cell line SP2 / 0 in the logarithmic growth phase at a ratio of 1:3. The mixture was centrifuged at 1000×g for 5 min at room temperature, the supernatant was discarded, and the bottom of the centrifuge tube was gently flicked with a finger to loosen the precipitate. Then 10 mL of fusion buffer was added to resuspend the cells, and the cells were centrifuged at 1600 rpm for 5 min, and the supernatant was discarded; again, 10 mL of fusion buffer was added to resuspend the cells, and the cells were centrifuged at 1600 rpm for 5 min, and the supernatant was discarded; 1 mL of fusion buffer was added to resuspend the cells to obtain a cell mixture for standby; after disinfecting the electrode chamber with 75% alcohol for 30 min, it was gently rinsed 3 times with 2 mL of fusion buffer, placed in a biosafety cabinet to air dry and irradiated with ultraviolet light for 30 min; 100 μL of the prepared cell mixture was aspirated and added to the electrode chamber, and the resistance value was adjusted to between 5000 and 10000 Ω; after adjusting the resistance value, 800 μL of the cell mixture was aspirated and added to the electrode chamber, the resistance value was measured and recorded, and the resistance value should be between 500 and 1500 Ω. Then the "Start" key was pressed to execute the electrofusion program and data recording; after electrofusion, the cell mixture was aspirated from the electrode chamber and added to a pre-warmed 15 mL centrifuge tube containing 1.6 mL of serum-free DMEM medium, and left to stand at 37 °C for 10 min (the serum-free DMEM medium needs to be prepared in advance and pre-warmed at 37 °C), centrifuged at 1600 rpm for 5 min, the supernatant was discarded, and the cells were gently resuspended with HAT culture medium (hypoxanthine (H), aminopterin (A), and thymidine (T) (HAT, Sigma)), and the cells were dispensed into a 96-well plate, 200 μL per well. After culturing for three days, the cell fusion situation was observed, and half of the HAT culture medium was replaced, and this was done continuously for several days until clones formed. Seven days after fusion, the culture was changed to HT culture medium (hypoxanthine (H) and thymidine (T) (HT, Sigma)).

[0068] (5) Screening of hybridoma cells secreting monoclonal antibodies against West Nile virus

[0069] Indirect immunofluorescence assay was used to screen cell lines. The specific steps were as follows: When the density of hybridoma cells in the 96-well plate reached about 50%, 50 μL of sample diluent (10 g of 10% BSA, 0.1 g of 0.02% thimerosal) and 50 μL of cell supernatant were added to the antigen plate prepared in step (3) of Example 1, and incubated in a 37 °C water bath for 2 h; washed with PBST; After diluting FITC-goat anti-mouse IgG: 0.2% Evans blue: enzyme diluent at a ratio of 1:20:300, add 50 μL per well and incubate at 37 °C for 1 h; wash with PBST and then rinse with running water for 5 min and pat dry; dry in a 37 °C incubator for 30 min to 1 h, observe the fluorescence intensity under a fluorescence microscope and determine the result. The cell lines with visible fluorescence signals were determined as positive cell lines.

[0070] The positive cloned hybridoma cells were subcloned. The specific steps were as follows: Add complete medium to a new 96-well cell culture plate. In addition to adding 200 μL of complete medium to each well in the first column A1-H1, add 100 μL of complete medium to the remaining cell wells; After gently dispersing the hybridoma cells, aspirate an appropriate amount of cell suspension into well A1, and serially dilute it from top to bottom to well H1, and then serially dilute it from the first column to the last column to obtain single hybridoma cells.

[0071] The above limited dilution method was used for continuous cloning 3 to 4 times until a 100% cell positive rate was achieved. Finally, a stable hybridoma cell line secreting anti-West Nile virus monoclonal antibody was obtained and labeled as WN-mAb-12. The cells with a 100% positive rate after cloning were amplified and cultured and then cryopreserved in liquid nitrogen.

[0072] (6) Preparation of monoclonal antibody ascites

[0073] First, inject liquid paraffin into the abdominal cavity of 8- to 10-week-old Balb / c male mice at a volume of 0.5 mL per mouse. After 10 days, digest the WN-mAb-12 hybridoma cell line with trypsin and inject it into the abdominal cavity of Balb / c mice pretreated with liquid paraffin at a cell amount of 1×10 5 / mouse. After breeding and observing for 10-14 days, when the abdomen of the mice was significantly enlarged, ascites was extracted. After the ascites collection was completed, it was centrifuged at 10,000 rpm for 10 min in a 4 °C centrifuge to obtain the cell supernatant of the anti-West Nile virus monoclonal antibody secreted by the hybridoma cell line WN-mAb-12, and stored at -80 °C for later use.

[0074] The preparation process of the hybridoma cell line WN-mAb-12 is as Figure 6 shown, including steps such as immunizing Balb / c mice with West Nile virus, obtaining hybridoma cells, and secreting anti-West Nile virus monoclonal antibody.

[0075] Example 2 Identification of the characteristics of monoclonal antibodies

[0076] (1) Indirect immunofluorescence identification of monoclonal antibody (IFA method): Using the prepared West Nile virus antigen plate, add the cell supernatant of the anti-West Nile virus monoclonal antibody secreted by the hybridoma cell line WN-mAb-12 (diluted 1:1), incubate at room temperature for 1 h, use the SP2 / 0 cell supernatant as the negative control, and use the West Nile virus polyclonal antibody as the positive control (diluted 1:500). Wash the plate 4 times with PBS (containing 0.05% (V / V) Tween-20) and pat dry; after diluting FITC-goat anti-mouse IgG: enzyme diluent at 1:300, add 50 μL per well and incubate in a 37 °C water bath for 1 h; wash the plate 4 times with PBS (containing 0.05% (V / V) Tween-20), rinse with running water for 5 min and pat dry; after drying in a 37 °C incubator for 0.5 - 1 h, observe the fluorescence results under a fluorescence microscope, as Figure 1 shown. Figure 1 In a, the cell supernatant of the anti-West Nile virus monoclonal antibody secreted by the hybridoma cell line WN-mAb-12; in b, the positive control; in c, the negative control. It can be seen from the figure that the cell supernatant of the anti-West Nile virus monoclonal antibody secreted by the hybridoma cell line WN-mAb-12 shows green fluorescence after recognizing the West Nile virus, the positive control shows green fluorescence after recognizing the West Nile virus, and the negative control cannot recognize the West Nile virus, so there is no fluorescence. The results prove that the anti-West Nile virus monoclonal antibody secreted by the hybridoma cell line WN-mAb-12 (hereinafter referred to as monoclonal antibody WN-mAb-12) can recognize the West Nile virus.

[0077] (2) Subtype identification of monoclonal antibody: Use the mouse monoclonal antibody subtype identification kit from Sigma to identify the subtype of the hybridoma cell line. The subtype of monoclonal antibody WN-mAb-12 is IgM type, as Figure 2 shown.

[0078] (3) Monoclonal antibody specificity identification: First, prepare antigen plates for WNV (West Nile virus), YFV (Yellow fever virus), TBEV (Tick-borne encephalitis virus), ZIKA (Zika virus), JEV (Japanese encephalitis virus), MVEV (Murray Valley encephalitis virus), LAN (Langat virus), DENV 1 (Dengue virus-1), DENV 2 (Dengue virus-2), DENV 3 (Dengue virus-3), and DENV 4 in the same way as the WNV antigen plate described above; the cell supernatant of the WNV-positive monoclonal cell line is dissolved in an equal volume of sample diluent at a ratio of 1:1, 100 μL / well, and added to the above virus antigen plates for cross-experiments. At the same time, set the positive control group as the polyclonal antibody of each virus, diluted 1:500 in the sample diluent, 100 μL / well, and the negative control group as the SP 2 / 0 cell supernatant, dissolved 1:1 in an equal volume of sample diluent, 100 μL / well; then the operation steps are the same as those of the above IFA method. Result determination: "++++" is extremely strong; "+++" is strong; "++" is weak; "+" is extremely weak, and "-" indicates no specificity. The specificity identification results of the monoclonal antibody WN-mAb-12 are shown in Table 1. The monoclonal antibody WN-mAb-12 is strongly positive, and there is no cross-reaction between the monoclonal antibody WN-mAb-12 and YFV, TBEV, ZIKA, JEV, MVEV, LAN, DENV1, DENV2, DENV3, and DENV4 viruses.

[0079] Table 1 Specificity identification results of monoclonal antibody WN-mAb-12

[0080]

[0081] (4) Evaluation of monoclonal antibody neutralization effect

[0082] Use the IFA method and qRT-PCR method to qualitatively detect the neutralization effect of the monoclonal antibody WN-mAb-12 respectively. First, seed Vero cells in good growth state into 24-well cell culture plates and 96-well cell culture plates, with the cell amounts being 1×10 5 / well and 3×10 4 / well respectively, and culture overnight in a CO2 incubator.

[0083] The cell supernatant of the monoclonal antibody against West Nile virus secreted by the hybridoma cell line WN-mAb-12 was diluted at 1:100, 1:200, 1:800, and 1:3200 respectively, and then mixed and incubated with the virus suspension diluted 1:5000 times. After that, the incubated mixture was applied to a previously prepared 96-well cell plate and incubated for 2 h. Then, the supernatant was discarded and the cells were cultured in a new 2% FBS DMEM medium for 48 - 60 h. The cell supernatant of the monoclonal antibody against West Nile virus secreted by the hybridoma cell line WN-mAb-12 was diluted 1:5 and then mixed and incubated with the virus suspension diluted 1:5000 times. After that, the incubated mixture was applied to a previously prepared 24-well cell plate and incubated for 2 h. Then, the supernatant was discarded and the cells were cultured in a new 2% FBS DMEM medium for 48 - 60 h.

[0084] The neutralization effect of the monoclonal antibody WN-mAb-12 was detected by indirect immunofluorescence assay on a 96-well cell plate. The detection results are as Figure 3 shown, Figure 3 where a in Figure 3 is the incubation result of the cell supernatant of the monoclonal antibody WN-mAb-12 with West Nile virus; b is the blank control. It can be seen from Figure 3 that after incubation of the cell supernatant of the monoclonal antibody WN-mAb-12 with West Nile virus, no green fluorescence is shown, indicating a neutralization effect.

[0085] The neutralization effect of the monoclonal antibody was quantitatively detected by qRT-PCR through cell lysis and extraction of intracellular viral RNA on a 24-well plate.

[0086] Using the professional software Primer Express 2.0 (ABI) for designing real-time quantitative PCR primers and probes, specific primers were designed according to the WNV gene sequence as shown in Table 2.

[0087] Table 2 Information on the use of WNV primers and probes

[0088]

[0089] After extracting West Nile virus RNA by the Trizol method, the neutralization effect of the cell supernatant of the monoclonal antibody WN-mAb-12 was detected according to the qRT-PCR reaction system shown in Table 3 and the reaction procedure shown in Table 4. The results are as Figure 5 shown. After incubation of the cell supernatant of the monoclonal antibody WN-mAb-12 with West Nile virus, the amount of WNV virus detected by qRT-PCR was relatively consistent with that of the positive control, while the negative control detected West Nile virus, indicating that the cell supernatant of the monoclonal antibody WN-mAb-12 has a strong neutralization effect on West Nile virus. Compared with Figure 4It is consistent with the IFA test result, that is, the supernatant of monoclonal antibody WN-mAb-12 cells has a strong neutralizing effect on West Nile virus after being diluted 5 times.

[0090] Table 3 qRT-PCR reaction system

[0091]

[0092]

[0093] Table 4 qRT-PCR reaction program

[0094]

[0095] The neutralizing effect of monoclonal antibody WN-mAb-12 ascites was evaluated by the IFA method. The ascites and WNV polyclonal antibody (positive control) were diluted at ratios of 1:100, 1:200, 1:800, and 1:3200 and then applied to the prepared 96-well cell plates. At the same time, blank Balb / c mouse serum was used as a negative control. After incubation with FITC fluorescent antibody, the fluorescence intensity was observed under a fluorescence microscope to judge the neutralization effect. The results are as Figure 4 shown, Figure 4 a, b, c, d, and e in it are the supernatant of monoclonal antibody WN-mAb-12 cells diluted 100, 200, 800, 3200 times, and the negative control respectively. From Figure 4 it can be seen that as the dilution multiple of the supernatant of monoclonal antibody WN-mAb-12 cells increases, the fluorescence gradually weakens, indicating that as the dilution multiple increases, the neutralizing effect of the monoclonal antibody on the virus weakens, suggesting that the neutralization titer of monoclonal antibody WN-mAb-12 mouse ascites reaches above 1:3200.

[0096] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the protection scope of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A hybridoma cell line, characterized in that The hybridoma cell line is WN-mAb-12, which was deposited in the China Center for Type Culture Collection on April 23, 2024, with the deposit number CCTCC NO: C2024123, and the deposit address is Wuhan University, Wuhan, China.

2. A West Nile virus monoclonal antibody, characterized in that: The monoclonal antibody is secreted and produced by the hybridoma cell line WN-mAb-12 according to claim 1.

3. The method for preparing West Nile virus monoclonal antibodies according to claim 2, characterized in that: The following steps are involved: (1) Immunizing Balb / c mice with West Nile virus to obtain immunized Balb / c mice; (2) Spleen cells of immunized Balb / c mice were fused with mouse myeloma cells SP2 / 0. After fusion, the cells were selectively cultured in HAT selection medium, positive clones were screened by indirect immunofluorescence method, and hybridoma cells in positive wells were cloned and cultured by limiting dilution method until the antibody positivity rate of cloned cells reached 100%, thereby obtaining hybridoma cell lines; (3) The hybridoma cell line obtained in step (2) is injected into the peritoneal cavity of Balb / c mice for cultivation, the ascites is collected, and the purified West Nile virus monoclonal antibody is obtained by affinity chromatography.

4. The preparation method according to claim 3, characterized in that: After purification, the neutralization titer of the monoclonal antibody reached over 1:3200.

5. Use of the West Nile virus monoclonal antibody according to claim 2 or the West Nile virus monoclonal antibody prepared by the preparation method according to claim 3 in preparing a reagent for detecting West Nile virus or in preparing a reagent for diagnosing and / or prognosing West Nile disease.

6. A reagent for detecting West Nile virus, characterized in that: The reagent comprises the West Nile virus monoclonal antibody according to claim 2 or the West Nile virus monoclonal antibody prepared by the preparation method according to claim 3.

7. A reagent for diagnosis and / or prognosis of West Nile disease, characterized in that: The reagent comprises the West Nile virus monoclonal antibody according to claim 2 or the West Nile virus monoclonal antibody prepared by the preparation method according to claim 3.

8. Use of the West Nile virus monoclonal antibody according to claim 2 or the West Nile virus monoclonal antibody prepared by the preparation method according to claim 3 in preparing a preparation for treating West Nile fever.

9. The use according to claim 8, characterized in that: The preparation includes a lyophilized preparation or a liquid preparation; the preparation includes an injectable preparation or an oral preparation.

10. A biological preparation for treating West Nile fever, characterized in that: The biological preparation comprises the West Nile virus monoclonal antibody according to claim 2 or the West Nile virus monoclonal antibody prepared by the preparation method according to claim 3.

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