A monoclonal antibody against respiratory syncytial virus and a preparation method and application thereof

Abstract

This invention relates to the field of biotechnology, specifically to a monoclonal antibody against respiratory syncytial virus (RSV), its preparation method, and its applications. The RSV monoclonal antibody provided by this invention can recognize specific antigenic epitopes of the pre-F protein of RSV. Results from examples show that the monoclonal antibody has a strong affinity for the RSV F protein and exhibits high neutralizing efficacy against both RSV types A and B, thus preventing RSV infection. The drug for treating RSV provided by this invention contains a monoclonal antibody against RSV and provides good protection against RSV infection. The preparation method of the RSV monoclonal antibody provided by this invention is cost-effective and simple to operate.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a monoclonal antibody against respiratory syncytial virus, its preparation method, and its application. Background Technology

[0002] Respiratory syncytial virus (RSV) is a seasonal, highly contagious RNA virus that is primarily transmitted through droplets and direct contact. After invading the human body, the virus first infects the ciliated cells of the upper respiratory tract, then spreads to the lower respiratory tract, triggering a severe inflammatory response accompanied by high levels of viral replication. In severe cases, it can lead to serious illnesses such as bronchiolitis and pneumonia.

[0003] RSV has become a significant challenge in global public health: It is the leading pathogen among acute lower respiratory tract infections in children under two years old worldwide, with almost all infants experiencing at least one RSV infection within two years of birth. As a leading cause of death from lower respiratory tract infections in infants and young children, RSV causes death in 1 out of every 50 children aged 0-5 years globally, with this mortality burden concentrated primarily in developing countries. Furthermore, age is a key factor in the severity of RSV disease; the elderly, immunocompromised individuals, and those with chronic underlying diseases are more prone to disease progression and even death after infection. Similar to COVID-19 infection, RSV infection does not induce permanent immunity, and individuals may experience repeated infections throughout their lives, further increasing the difficulty of disease control. Therefore, the prevention and treatment of RSV infection has become a pressing global public health issue.

[0004] Currently, the main methods for controlling RSV include vaccines and monoclonal antibodies (hereinafter referred to as "monoclonal antibodies"), but both face significant technical bottlenecks: (1) In the field of vaccines, approved vaccines include Arexy (for the elderly) and AbrySVO (a two-component vaccine for subtypes A and B, for the elderly and pregnant women), both of which are developed based on the RSV pre-fusion conformation protein (pre-F). However, no vaccine products for infants and young children have been approved. RSV vaccine development faces multiple challenges: on the one hand, there is a risk of antibody-mediated infection enhancement, and the phenomenon of vaccine-derived enhanced disease (VDE) in RSV-naïve (unexposed RSV) infants and young children has not been effectively resolved; on the other hand, the immune system of infants and young children is immature, and existing vaccines are difficult to induce a durable and effective protective immune response. Therefore, for infants and young children, two alternative prevention and control strategies are currently mainly adopted: one is to vaccinate pregnant women in late pregnancy to provide passive protection for newborns through placental transfer of antibodies; the other is to directly administer prophylactic neutralizing antibodies to infants and young children.

[0005] (2) In the field of monoclonal antibody drugs, the pre-F protein of RSV has been shown to contain 6 neutralizing epitopes, and the post-F protein contains 4 neutralizing epitopes. Neutralizing antibodies against these epitopes have become the core means of RSV prevention in infants and young children. Currently, two RSV prophylactic monoclonal antibodies have been marketed globally, including palivizumab and nirsevimab (trade name: Beyfortus®, also known as MEDI8897).

[0006] The former targets epitope II of the pre-F protein, but its neutralizing efficacy is low. In clinical applications, it requires high doses and frequent administration, which leads to high costs for preventive treatment of high-risk infants and young children, limiting its widespread use. The latter targets the dominant epitope (SiteØ) of the pre-F protein. Although it is a single-dose formulation, the amino acid sequence of this epitope is poorly conserved and has a high mutation rate. Under immune pressure, it is easy for the virus to escape. In vivo experiments have confirmed that it lacks cross-protection against RSV variants.

[0007] In summary, existing RSV monoclonal antibody drugs suffer from technical problems such as insufficient neutralizing efficacy, poor epitope conservation, and lack of cross-protection against variant strains. Therefore, developing an RSV monoclonal antibody with high neutralizing activity, conserved target epitopes, broad-spectrum protection against variant strains, and controllable cost is of great significance for addressing the challenge of RSV infection control in infants and high-risk groups, and has outstanding clinical value and application prospects. Summary of the Invention

[0008] Therefore, the technical problem to be solved by this invention is to overcome the defects of existing RSV monoclonal antibody drugs, such as insufficient neutralizing efficacy, poor epitope conservation, and lack of cross-protection against variant strains. This invention provides a monoclonal antibody against respiratory syncytial virus, its preparation method, and its application. The monoclonal antibody of this invention has conserved target epitopes and strong neutralizing activity (IC50). 50 (Low), and has a broad-spectrum protective effect against mutant strains.

[0009] Therefore, the present invention provides the following technical solution: The present invention provides a monoclonal antibody against respiratory syncytial virus (RSV), wherein the amino acid sequences of the complementarity-determining regions (CDR-H1, CDR-H2, and CDR-H3) of the heavy chain variable region of the monoclonal antibody against RSV are shown in SEQ ID No. 3 to 5, respectively. The amino acid sequences of the complementarity-determining regions (CDR-L1, CDR-L2, and CDR-L3) of the light chain variable region of the monoclonal antibody against respiratory syncytial virus are shown in SEQ ID No. 6 to 8, respectively. Preferably, the heavy chain amino acid sequence of the monoclonal antibody against respiratory syncytial virus is shown in SEQ ID No. 1, and the light chain amino acid sequence is shown in SEQ ID No. 2. This invention provides a nucleic acid molecule encoding the monoclonal antibody described in the above-mentioned technical solution.

[0010] This invention provides an expression vector comprising the nucleic acid molecule described in the above technical solution.

[0011] This invention provides a genetically engineered biomaterial, including bacteriophages, bacteria, fungi or animal cells, including the expression vector described in the above technical solution.

[0012] This invention provides the application of the monoclonal antibody against respiratory syncytial virus described in the above-described technical solutions, or the nucleic acid molecule described in the above-described technical solutions, or the expression vector described in the above-described technical solutions, or the genetically engineered biomaterial described in the above-described technical solutions, in at least one of the following: 1) Prepare drugs for the prevention and / or treatment of respiratory syncytial virus (RSV); 2) Products for detecting respiratory syncytial virus. This invention provides a product for detecting respiratory syncytial virus (RSV), the product comprising the monoclonal antibody against RSV described in the above technical solution, or the nucleic acid molecule described in the above technical solution, or the expression vector described in the above technical solution, or the genetically engineered biological material described in the above technical solution.

[0013] This invention provides a medicament for treating respiratory syncytial virus (RSV), comprising the anti-RSV monoclonal antibody or the nucleic acid molecule described in the above-described technical solution. Preferably, the product comprises a kit and / or test strips.

[0014] The present invention provides a method for detecting respiratory syncytial virus in a sample, the method comprising mixing the monoclonal antibody described in the above technical solution with the sample to be tested, and detecting the level of respiratory syncytial virus in the sample to be tested.

[0015] This invention provides a method for preparing a monoclonal antibody against respiratory syncytial virus, comprising: 1) Obtain peripheral blood from RSV-positive donors to isolate mononuclear cells. 2) Antigen-specific B lymphocytes are sorted using flow cytometry and RSV pre-F tetramer probes; preferably, the markers sorted by flow cytometry include CD20, CD27 and IgD.

[0016] 3) Amplify the variable region of the antigen-specific B lymphocytes; 4) The variable region is recombined and transformed to obtain a monoclonal antibody against respiratory syncytial virus.

[0017] The technical solution of this invention has the following advantages: 1. The monoclonal antibody against respiratory syncytial virus (RSV) provided by this invention can recognize specific antigenic epitopes of RSV pre-F. Results from examples show that the monoclonal antibody exhibits strong affinity activity for RSV F protein, high neutralizing efficacy, and can prevent RSV infection. The action site of the monoclonal antibody on the RSV pre-F protein provided by this invention is conserved in the sequences of strains from previous years, demonstrating good protective effects against RSV infection. The cost of the anti-RSV monoclonal antibody of this invention is controllable, and it can solve the problem of RSV infection prevention and control in infants and high-risk groups.

[0018] 2. The drug for treating respiratory syncytial virus provided by the present invention contains a monoclonal antibody against respiratory syncytial virus or a nucleic acid encoding the monoclonal antibody, and has a good protective effect against RSV infection.

[0019] 3. The method for preparing a monoclonal antibody for treating respiratory syncytial virus provided by this invention is cost-effective and simple to operate. Attached Figure Description

[0020] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0021] Figure 1 This is the flow cytometry detection result of the RSV pre-F-APC tetramer probe in Example 1 of this invention; Figure 2 This is the flow cytometry detection result of the RSV pre-F-BB515 tetramer probe in Example 2 of the present invention; Figure 3 The results of flow cytometry analysis of RSV pre-F specific B cells; Figure 4 Agarose gel electrophoresis identification results of the amplified heavy chain (VH) and light chain (VL) fragments of B cell BCR; Figure 5 The results of PCR agarose gel electrophoresis identification of colonies of heavy and light chain recombinant plasmids pCMV-IGH-VH-05A8 and pCMV-IGL-VL-05A8; Figure 6 The results are from SDS-PAGE identification of monoclonal antibodies. Figure 7 The results are for the purity identification of monoclonal antibodies based on a protein characterization analyzer. Figure 8 This is a graph showing the binding and dissociation curves of monoclonal antibodies. Figure 9 This is a hydrogen-deuterium exchange mass spectrometry analysis of a monoclonal antibody. Figure 10 IC50 of monoclonal antibody against RSV A2 strain 50 picture; Figure 11 IC50 of monoclonal antibody against RSV B strain 50 picture; Figure 12 The graph shows the changes in body weight of infected mice in each group after administration of monoclonal antibodies. Figure 13 This is a diagram showing the binding site and amino acid polymorphism analysis of monoclonal antibodies on RSV F. Detailed Implementation

[0022] The following embodiments are provided to better understand the present invention and are not limited to the preferred embodiments described. They do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the protection scope of the present invention.

[0023] For experiments not specifically described in the examples, the procedures or conditions should be followed according to the conventional experimental procedures described in the literature in this field. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products.

[0024] Example 1: Construction and Specificity Verification of pre-F Tetramer Probe The pre-F tetramer probe was constructed using the biotin-streptavidin system, and the specific process is as follows: 1. Biotinylation of pre-F protein (1) RSV pre-F protein dissolution: Add RSV pre-F (Essential Biotech, cat: 11049-VNAS) to 100µL ddH2O to dissolve and obtain pre-F protein solution. Aliquot 10µL and store at -80℃.

[0025] (2) EZ-Link™ Sulfo-NHS-LC-Biotin (A39257) was brought to room temperature and dissolved in 180µL DMSO to make the concentration of Sulfo-NHS-LC-Biotin in the solution 10mM, and the biotin solution was dispensed into 5µL containers and dried and stored at -20℃.

[0026] (3) Based on the molar ratio of pre-F protein to biotin (1:20), mix the pre-F protein solution from step (1) with the biotin solution from step (2) and incubate at 4°C for 3 hours.

[0027] (4) After the incubation in step (3) is completed, the solution is transferred to a 10KD ultrafiltration tube, washed and ultrafiltered twice to obtain 40µL of biotinylated pre-F protein solution with a concentration of 0.65mg / mL.

[0028] 2. Tetramer Assembly and Purification (1) Tetramerization reaction: APC-streptavidin (BD Bioscience, 564453) and BB515-streptavidin (BioLegend, 405207) were mixed with the biotinylated pre-F protein prepared in step 1 at a molar ratio of 1:6. After mixing thoroughly, the mixture was incubated at room temperature in the dark for 45 min.

[0029] (2) After step (1) is completed, add free D-Biotin (Beyotime, ST2051) with a final concentration of 5µM to each tube and incubate at 4℃ in the dark for 1h to block excess streptavidin binding sites.

[0030] (3) After the incubation in step (2), the solution was transferred to a 100KD ultrafiltration tube, washed and ultrafiltered twice, and then replenished with DPBS to 25µL. The solution was stored at 4℃ to obtain the pre-F tetramer probe solution (pre-F-APC or pre-F-BB515).

[0031] (4) Identification of probe binding effect: 4µL of anti-mouse antibody coated magnetic beads (BD Bioscience, catalog number 552843), 1.5µL of mouse anti-RSV-F monoclonal antibody 7E2 (prepared using hybridoma technology, see Example 13) and 1.5µL of pre-F tetramer probe were mixed with mouse IgG1 isotype control antibody (Thermo Fisher Scientific, catalog number 02-6100) as negative control (NC). The mixture was incubated at room temperature for 15 min and then at 4°C for 30 min. After washing twice, the mixture was analyzed by flow cytometry.

[0032] The results are as follows Figure 1 and Figure 2 As shown, the pre-F-APC and pre-F-BB515 tetramer probes can effectively bind to specific antibodies on magnetic beads.

[0033] Example 2: Sorting of RSV pre-F specific B cells 1. Isolation of PBMCs from RSV-positive specimens DP2 is a DPBS buffer containing 2% (v / v) fetal bovine serum (FBS, EcoSy, FSD500).

[0034] ① Use blood collection tubes containing EDTA-K2 anticoagulant to collect peripheral blood from RSV-positive laboratory personnel, infectious disease nurses, and patients in the fever clinic. Place the samples vertically and transport them to the laboratory at room temperature. Add DP2 to the peripheral blood at a volume ratio of 1:1 to obtain a diluted peripheral blood sample (final volume of 10-20 ml).

[0035] ② Add 15.5 mL of Ficoll-Paque lymphocyte separation solution to a 50 mL PBMC separation tube, centrifuge at 1200 g and 20 °C for 1 min, so that all the separation solution settles to the bottom layer of the sponge pad in the separation tube.

[0036] ③ Slowly pour the diluted peripheral blood sample into the upper layer of the separation tube sponge pad, and centrifuge at 1200g, 20℃, with rising and falling accelerations of 1 and 0 respectively for 20 minutes.

[0037] ⑤ After centrifugation, pour the supernatant (including the white film layer) into a new 50 mL centrifuge tube.

[0038] ⑥ Use DP2 to add liquid to the centrifuge tube to 40 mL, centrifuge at 800 g, 4 °C, with both rising and falling accelerations of 6, for 10 min.

[0039] ⑦ After centrifugation, discard the supernatant, add 3 mL of ACK erythrocyte lysis buffer (Beyotime, C3702), mix thoroughly by pipetting, and lyse at room temperature for 3 min.

[0040] ⑧ Add 12 mL of DP2 to terminate the erythrocyte lysis reaction, and centrifuge at 500g, 4℃ for 5 min. Discard the supernatant and resuspend the PBMCs in 5 mL of DP2. Take 10 µL of the PBMC suspension and mix it with 10 µL of trypan blue, then pipette 10 µL onto a cell counting plate to calculate the number and viability of PBMC viable cells. Centrifuge the PBMC suspension at 500g, 4℃ for 5 min, discard the supernatant, and freeze the cells or continue to the next experiment.

[0041] 2. B cell enrichment 1) Every 1×10 7 Each PBMC cell was resuspended in 80 µL of cold buffer; the buffer was PBS buffer containing 0.5% (v / v) BSA and a final concentration of 2 mM EDTA. 2) Add 20µL of human CD19 MicroBeads (Medlan, 130-050-301) to the cell suspension obtained in step 1) and mix gently with a pipette; 3) Incubate at 4℃ for 15 min; wash once with 5 mL buffer; centrifuge, discard the supernatant, and resuspend in 500 µL buffer to obtain PBMC cell suspension; 4) Take one LS column (Mitteni, 130-042-401), rinse the column once with 3 mL buffer, and set aside; then add 500 µL of PBMC cell suspension to each LS column and pass it through the column, CD19 + B cells have been adsorbed into the separation column by magnetic beads; 5) After all the PBMC cell suspension in the aliquoted LS column has flowed out, wash each column three times with 3 mL buffer. 6) Remove the magnetic rack from the sorting column, add 5 mL of buffer, and use the syringe plunger to push the magnetic beads down to completely elute the adsorbed B cells. Collect the eluent, which is CD19. + B cells, centrifuged and supernatant discarded; CD19 resuspended. + B cells were counted and transferred to a 96-well plate for staining.

[0042] 3. Cell staining and sorting of specific B cells (1) Centrifuge the 96-well plate obtained in step 2 at 500g and 4℃ for 5 min, discard the supernatant, add 100µL LFACS buffer to the sample wells. The FACS buffer contains Fc receptor blocker (BD Bioscience 422302, 1:100) and Live / Dead (BD Bioscience, L34957, 1:500), and incubate at room temperature in the dark for 30 min.

[0043] (2) After staining in step (1), add 100µL of pre-cooled PBS buffer to the sample well, mix gently, centrifuge at 500g and 4℃ for 5min, and discard the supernatant.

[0044] Add the pre-prepared surface antibody and probe mixture (containing antiCD20-PE-cy7, antiCD27-PE, antiIgD-BV421, pre-F-APC, and pre-F-BB515) to the sample wells, with a total volume of 100 µL / well, and stain at 4°C for 30 min. The composition of the surface antibody / probe mixture is shown in Table 1.

[0045] Table 1 Summary of information on surface antibody / probe mixtures

[0046] (3) After staining with surface antibodies and probes, wash twice with pre-chilled FACS, discard the supernatant, add 300µL of FACS buffer to each well, and gently resuspend the cells by pipetting. Immediately transfer the resuspended cell suspension to the flow cytometer for sorting. Based on CD20 + CD27 + IgD - / + and pre-F + By combining biomarkers, specific B cells were screened, and the BDAria III Single Cell sorting program was started. After sorting, the PCR plate was placed in a centrifuge and centrifuged at 2400 rpm for 1 min. After centrifugation, the plate was immediately sealed. The sealed PCR plate can be directly stored at -80℃.

[0047] The test results showed that the probe was double positive (i.e., pre-F-APC). + pre-F-BB515 + The cells showed clear clustering, and their proportion of memory B cells ranged from 0.037% to 0.11% in healthy individuals (HC1, HC2), patients in fever clinics, infectious disease nurses, and children's tonsil samples 11 and 12 (see [link to relevant documentation]). Figure 3 ). This will express CD19+ CD20 + IgD - CD27 + Specific memory B cells that are positive for the pre-F probe were sorted into 96-well PCR plates at a rate of 1 cell / well.

[0048] Example 3: Single B cell BCR amplification and sequencing 1. Reverse transcription (1) Configure the reverse transcription system. The composition of the reverse transcription system is shown in Table 2.

[0049] Table 2 Composition of the reverse transcription system

[0050] (2) Add 3.5 µL of reverse transcription mixture to each well of the single cell well obtained in Example 2, and centrifuge at 2400 rpm for 1 min.

[0051] (3) Place the sample in a PCR instrument and perform reverse transcription. The program is as follows: 25℃, 10 min; 42℃, 120 min; 85℃, 5 min; 16℃, hold; (4) Centrifuge the reverse transcription product at 2400 rpm for 1 min, wrap it in plastic wrap, and store it at 4℃ overnight or at -20℃ for several days.

[0052] 2. Nested PCR (1) Primer preparation: Centrifuge the primers (dry powder) at high speed for 1 min, dilute with ultrapure water to 100 µM, vortex mix for 30 s, and centrifuge briefly. Then dilute the concentration of each primer to the storage concentration of 5 µM.

[0053] (2) First Wheel PCR system 20µL.

[0054] The reaction system is as follows: 2×PrimeSTAR® Max, 10µL; upstream primer 1µL; downstream primer 1µL; DNA template (reverse transcription product obtained in step 1) 1µL; H2O 7µL; total system volume 20µL.

[0055] The primers are as follows: the upstream primer for the IgH heavy chain is VH-1-F, and the downstream primer is VH-1-R; The upstream primer for the Igκ light chain is VK-1-F, and the downstream primer is VK-1-R; The upstream primer for the Igλ light chain is VL-1-F, and the downstream primer is VL-1-R; the nucleotide sequences of the primers are shown in Table 3.

[0056] The PCR procedure is as follows: Pre-denaturation: 95℃, 30s, cycle number 1; Denaturation: 95℃, 15s; Annealing: 58℃, 15s; Extension: 72℃, 30s; Denaturation, Annealing, and Extension cycles: 35; Complete Extension: 72℃, 5min, cycle number 1; Finally, the PCR product was stored at 10℃.

[0057] (3) Take 2 µL of the first round PCR product and use it directly for the second round of PCR amplification. second Wheel The PCR system is 50µL, and the reaction system is as follows: 2×PrimeSTAR® Max 25µL, upstream primer 2µL, downstream primer 2µL, DNA template (first round PCR product) 2µL, H2O 19µL, total 50µL.

[0058] The primers are as follows: The primers are as follows: the upstream primer for the IgH heavy chain is VH-2-F, and the downstream primer is VH-2-R; The upstream primer for the Igκ light chain is VK-2-F, and the downstream primer is VK-2-R; The upstream primer for the Igλ light chain is VL-2-F, and the downstream primer is VL-2-R; the nucleotide sequences of the primers are shown in Table 4.

[0059] The PCR procedure is as follows: Pre-denaturation: 95℃, 30s, cycle number 1; Denaturation: 95℃, 15s; Annealing: 58℃, 15s; Extension: 72℃, 30s; Denaturation, Annealing, and Extension cycles: 35; Complete Extension: 72℃, 5min, cycle number 1; Finally, the PCR product was stored at 10℃.

[0060] Table 3 First Wheel PCR primers (concentration 5µM / primer)

[0061] Table 4, Second Wheel PCR primers (concentration 5µM / primer)

[0062] (4) Identification: The amplified fragments of VH (heavy chain variable region), VL (λ light chain variable region), and VK (κ light chain variable region) are approximately 350-400 bp in size. Take 5 µL of the PCR product, run it on a 2000 bp marker gel, and irradiate the gel. See [link to gel analysis]. Figure 4 PCR results showed that both the heavy chain 05A8-VH and light chain 05A8-VL genes were specifically amplified.

[0063] Example 4: Construction of vector using pCMV-IGH / IGL-VH / VL 1. After the second round of PCR products obtained in Example 3 were identified as fragments of 300-500bp through gel electrophoresis, they were purified using the FastPure Gel DNA Extraction Mini Kit (Novizan DC301-01) to obtain purified VH and VL DNA fragment solutions. The DNA concentration was measured using a UV spectrophotometer. If the DNA concentration was greater than 20ng / µL and the OD 260 / 280 ratio was between 1.8 and 2.0, it was ready for use.

[0064] 2. pCMV-IG vector double enzyme digestion reaction system Taking a 250µL reaction system as an example, the hG double digestion system (5µg) consists of: 5µg pCMV-IGH plasmid, 25µL 10× digestion buffer, 5µL AfeI site restriction endonuclease, 5µL NheI-HF site restriction endonuclease, and enzyme-free water to make up to 250µL.

[0065] Taking a 250µL volume reaction system as an example, the hL double enzyme digestion system (5µg) consists of: 5µg pCMV-IGL plasmid, 25µL 10× enzyme digestion buffer, 5µL AfeI site restriction endonuclease, 5µL KpnI-HF site restriction endonuclease, and enzyme-free water to make up to 250µL.

[0066] The pCMV-IGH plasmid, based on pCMV modification, has the nucleotide sequence shown in SEQ ID No. 50:

[0067] The pCMV-IGH plasmid, based on pCMV modification, has the nucleotide sequence shown in SEQ ID No. 51:

[0068] 3. Homologous recombination (1) Calculation of the amount of carrier fragment used: According to the instructions, the mass of VH / VL DNA is approximately 400 × 0.04 = 16 ng, and the mass of pCMV-IG vector is approximately 4000 × 0.02 = 80 ng.

[0069] The reaction system is shown in Table 5: Table 5. Homologous recombination system of pCMV-IGH / IGL-VH / VL plasmid

[0070] (2) Place the reaction system in a constant temperature water bath and incubate at 37°C for 30 min. After the reaction is terminated, place it on ice for later use.

[0071] (3) Transformation 1) Thaw competent Escherichia coli DH5α at 37℃.

[0072] 2) Take 4µL of homologous recombination product (about 2ng DNA) and add 50µL of DH5α, then incubate on ice for 20min.

[0073] 3) Transfer to a 42℃ water bath, heat shock for 1 min, then incubate on ice for 2 min. 4) Add 300µL SOC medium, and revive DH5α at 37℃, 220rpm, and 1h using a shaker.

[0074] 4) Take 50µL of DH5α bacterial culture and spread it on the corresponding resistance plate, and incubate at 37℃ for about 18h.

[0075] (4) Colony PCR identification 1) Using a 200µL pipette tip, select two moderately isolated single colonies from each plate. Immerse them in the pre-prepared PCR reaction solution, mix vigorously, and then take a small amount of the liquid and place it into a pre-prepared 200µL LB solution containing antibiotics in an EP tube.

[0076] 2) Colony PCR system: 7.5µL of 2× prime Taq (Takara), 1µL of upstream primer for second round PCR, 1µL of downstream primer for second round PCR, and 6.5µL of ddH2O.

[0077] The PCR amplification program was as follows: pre-denaturation at 95°C for 30 seconds, cycle number 1; denaturation at 95°C for 15 seconds, annealing at 58°C for 15 seconds, extension at 72°C for 30 seconds, with a total of 35 cycles for denaturation, annealing, and extension; final extension at 72°C for 5 minutes, cycle number 1; and finally, the PCR products were stored at 10°C.

[0078] 3) Use a gel electrophoresis test to identify positive bands in the colony PCR products.

[0079] 4) At the same time, place the EP tube in a shaker and incubate at 200 rpm and 37°C for 3 hours.

[0080] 5) Select clones whose colony PCR product fragment size meets the expectations and perform first-generation sequencing.

[0081] The results are as follows Figure 5 As shown, both the 05A8 heavy chain and light chain recombinant plasmids were able to amplify fragments of the expected size, indicating specific fragment insertion.

[0082] 4. Plasmid DNA extraction 1) Select positive clones containing complete ORF and correct light or heavy chain variable region sequences. Transfer the bacterial cultures of VH and VL to 10 mL of LB broth with a concentration of 50 µg / mL ampicillin and incubate overnight until the bacterial culture is sufficiently turbid.

[0083] 2) Extract DNA for subsequent cell transfection.

[0084] The CDR sequences of the light and heavy chains of antibody 05A8 are shown in Table 6 below.

[0085] Table 6. Clonal types and CDR sequences of the light and heavy chains of antibody 05A8.

[0086] Example 5: Antibody Expression and Identification 1. Transfect CHO cells with recombinant plasmid (using a 20mL expression system as an example) 1) Assess CHO cell viability 24 hours before transfection, requiring a CHO cell survival rate ≥90%. Use 3.5 × 10⁻⁶ cells / cells. 6 CHO cell suspension was aliquoted into shake flasks containing fresh Opti-MEM medium at a seeding density of 1 cell / mL, with a total volume of 20 mL. The flasks were then incubated at 37°C in a shaking incubator (5% CO2, 150 rpm) for 24 h.

[0087] 2) Dilute 30µg of expression plasmid (light chain VL and heavy chain VH expression plasmid mass ratio of 1:1) with Opti-MEM medium (Gibco, 31985-070) to obtain a total volume of 0.5mL DNA solution.

[0088] PEI transfection reagent (Polyscience, catalog number: 24765-100): According to the recommended ratio in the reagent instructions, the ratio of plasmid to PEI transfection reagent is 1µg: 3µg. Mix 90µg of PEI transfection reagent with Opti-MEM medium to a final volume of 0.5mL to obtain the transfection reagent dilution.

[0089] 3) Incubate the expression plasmid DNA solution and the transfection reagent dilution solution at room temperature in the dark for 5 min, then mix them in equal volumes. After mixing, incubate at room temperature in the dark for 10 min to obtain the plasmid-transfection reagent complex.

[0090] 4) The plasmid-transfection reagent complex was slowly added to the activated CHO cell suspension in step 1) using a dropwise addition method for transfection. During the transfection process, gentle shaking (50 rpm) was used to promote uniform distribution. Then the culture conditions were restored (37℃, 8% CO2, 150 rpm).

[0091] 5) Add 1.1 mL of SMS CHO-SUPA feed solution 24 h after transfection.

[0092] Subsequently, 1.1 mL of SMS CHO-SUPA nutritional supplement was added every 48 hours.

[0093] 6) Terminate the culture on the 7th day after transfection, collect the transfection supernatant by gradient centrifugation at 3500 rpm for 15 min, and store at 4℃.

[0094] 2. Initial screening of transfection supernatant (ELISA identification) (1) Dilute RSV A2 F protein concentration to 1 ng / µL with coating buffer (Abcam, Ab210899), add 100 µL to each well of a 96-well ELISA coated plate, and incubate overnight at 4°C.

[0095] (2) Add 200 µL of blocking solution to each well and incubate at 37°C for 2 hours in a constant temperature incubator. The blocking solution is a 5% skim milk solution prepared using PBS.

[0096] (3) After incubation, use a high-throughput coated plate washer to wash the plate. Add 300 µL of washing solution each time, shake and soak for 30 seconds each time, wash twice, and pat dry after washing.

[0097] (4) Add 100µL of the transfection supernatant obtained in step 1 to each well, and then place it in a constant temperature incubator and incubate at 37℃ for 1h.

[0098] (5) After incubation, wash the plate 4 times and pat it dry. Soak it in washing solution for 30 seconds each time. The washing solution is 0.05% PBST.

[0099] (6) Then, the enzyme-labeled secondary antibody was diluted to 0.133 μg / mL using the secondary antibody dilution buffer. The secondary antibody dilution buffer was PBST with 2% skim milk added.

[0100] Add 100 µL of diluted enzyme-labeled secondary antibody to each well and incubate at 37°C for 1 hour. After incubation, wash the plate 6 times and pat dry, immersing it in 0.05% PBST for 30 seconds each time.

[0101] Next, 100 µL of chromogenic reagent was added to each well, and the mixture was gently tumbled for 2 seconds to mix. The plate was then sealed and incubated at 37°C for 6 minutes. 50 µL of stop solution was added to each well, and the mixture was gently tumbled to mix. The absorbance was measured at 450 nm, yielding several monoclonal antibodies. One of these monoclonal antibodies was named 05A8. ELISA identification results for monoclonal antibody 05A8 showed that the OD of the transfection supernatant of monoclonal antibody 05A8 was... 450 The value was 3.9, indicating that the supernatant of monoclonal antibody 05A8 was positive for binding to pre-F protein, and that monoclonal antibody 05A8 specifically targets RSV pre-F antigen.

[0102] Example 6: Purification of 05A8 Antibody (1) Functionalization pretreatment of magnetic beads ① The storage bottle containing Protein A-coupled magnetic beads (Tiandi Renhe, SM003100) was vertically vortexed for 30 seconds to ensure the uniformity of the magnetic bead suspension system and to obtain a magnetic bead suspension.

[0103] ② Take 1.5 mL from the magnetic bead suspension and place it in a 50 mL centrifuge tube.

[0104] ③ Use a magnetic rack for solid-phase separation for 1 minute to remove the supernatant storage liquid.

[0105] ④ Add 5 mL of binding / washing buffer (150 mM NaCl + 20 mM Na2HPO4, pH 7.4) to the centrifuge tube, manually invert and mix 10 times, then discard the liquid after separation with a magnetic rack. Repeat this process twice to achieve initial cleaning.

[0106] ⑤ Add 3 mL of 0.1 M NaOH solution, incubate at room temperature for 15 min, then separate with a magnetic rack and discard the liquid to obtain magnetic beads.

[0107] ⑥ Wash the magnetic beads twice with 5 mL of binding / washing buffer to obtain pretreated Protein A-coupled magnetic beads.

[0108] (2) Antibody adsorption and magnetic bead washing ① Slowly add the transfection supernatant obtained in Example 5 to the pretreated Protein A magnetic beads, seal with sealing film, mix evenly, and then slowly rotate and incubate at room temperature on a rotator for 3 hours.

[0109] ② After incubation, collect the magnetic beads using a magnetic separator and discard the supernatant. Retain the supernatant for analysis of the purification effect.

[0110] ④ Add 5 mL of binding / washing buffer and mix thoroughly. Collect the magnetic beads using a magnetic rack and discard the supernatant. Repeat the washing step 3 times to obtain the washed magnetic beads.

[0111] (3) Antibody elution ① Add 2 mL of elution buffer (0.1 M glycine-HCl, pH 2.8) to the washed magnetic beads, mix thoroughly, and incubate at room temperature for 5 min, mixing the magnetic beads several times during the incubation period.

[0112] ② Collect the eluent by magnetic separation into a new tube, immediately add 200µL of neutralization buffer (1 M Tris-HCl, pH 8.5), repeat steps ① and ② twice, collect the eluent, and obtain the purified antibody.

[0113] (4) Protein A coupled magnetic bead regeneration ①Wash the Protein A-conjugated magnetic beads three times with 5 mL of binding / washing buffer (0.15 M NaCl + 20 mM Na2HPO4, pH 7.0) to remove residual antibodies.

[0114] ②Wash with 3 mL of 0.1 M NaOH solution for 15 min twice.

[0115] ③ After washing three times with 5 mL of binding / washing buffer, add 2 mL of 20% (v / v) ethanol and store at 4°C.

[0116] (5) The antibody eluent was subjected to SDS-PAGE and Coomassie Brilliant Blue staining to identify the molecular weight and purity of the antibody light and heavy chains. The results showed that the main bands of the antibody light and heavy chains were as expected. Figure 6 ). Example 7: Antibody purity was determined using the Protein Simple protein characterization analyzer. 1. Add 2 µL of 25×IS (internal standard) and 2.5 µL of 14.2 M β-mercaptoethanol to 50 µL of 05A8 antibody solution with a concentration of 0.5 mg / mL. Heat the mixture at 70 °C for 10 min. After cooling to room temperature, centrifuge the sample at 5000 rpm for 5 min, and transfer 50 µL of the supernatant to a 96-well conical plate, placing it in the sample tray of the Maurice® Protein Characterization Analyzer.

[0117] 2. Remove the CE-SDS PLUS cartridge, insert the running capillary electrophoresis buffer vial (containing capillary electrophoresis buffer solution), and place the CE-SDS PLUS cartridge into the slot of the Maurice® Protein Characterization Analyzer for detection.

[0118] 3. The results showed that the internal standard eluted at approximately 1 minute, followed by the antibody light chain and heavy chain peaks. The sum of the surface areas of the antibody light chain and heavy chain peaks represents the antibody purity. The purity of 05A8 reached over 98%. Figure 7 ).

[0119] 05A8 antibody heavy chain amino acid sequence: MGWSLILLFLVAVATRVLSAVHSEVQLVESGGGLVQPGRSLRLSCTASGFSFGHYGVSWVRQAPGKGLEWVAVIRSKDNGRTTEYAASVRGRFIISRDDSKDIAYLQMNSLKTEDTAMYY CARGGDCCNNGVCFTVDNWGQGTLVTVSSAASTKGSSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNT KVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPA PIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID No.1).

[0120] 05A8 antibody light chain amino acid sequence: MGWSCIILFLVATATGVHSASVTSYELTQPPSVSVSPGQTARITCSGDGLPKQYVYWFQQKPGQAPLLVIYKDTEKPSGIPGRFSGSTSGTTVTLTISGVQAEDEADYYCQSGDISASFL VFGGGTKLTVLSQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS (SEQ IDNo.2).

[0121] Example 8 Antibody Affinity Analysis (1) Place the Octet RH16 Sartorius Protein A sensor in 0.05% PBST solution and activate it for 10 min.

[0122] (2) The RSV pre-F protein (i.e. antigen) was diluted with 0.05% PBST solution to concentrations of 500 nmol, 166.66 nmol, 55.55 nmol, 18.51 nmol, 6.17 nmol, 2.05 nmol and 0.68 nmol according to its molecular weight, and a blank control of 0 nmol was prepared.

[0123] (3) Use 0.05% PBST solution to release 05A8 antibody to 1µg / mL.

[0124] (4) Add the prepared antigen, 05A8 antibody, and PBST to the 96-well blackboard according to the program. The sample volume for each well is 200µL, and the specific arrangement is as follows: ① Baseline calibration well: 0.05% PBST buffer; ② Antibody solidification well: 1μg / mL 05A8 antibody solution; ③ Sample loading well: 7 concentration gradients of RSV pre-F protein solution (in order of increasing concentration) and blank control; ④ Dissociation well: 0.05% PBST buffer; ⑤ Regeneration well: pH 1.5 glycine solution and 0.05% PBST buffer.

[0125] (5) Place the activated Protein A sensor into the detection chamber of the Octet RH16, start the "Baseline Run" program in the software, move the sensor down to the PBST baseline calibration well of the 96-well black plate, and start the baseline run. After the baseline stabilizes, place the sensor into the 200µL antibody curing well for curing until the response value reaches the threshold of about 1.0 nm.

[0126] (6) After the antibody has solidified, place the sensor back into PBST to run the baseline until the curve is stable. Place the antibody-loaded sensor in serially diluted RSV pre-F protein solution for 300s to measure the binding process.

[0127] (7) The sensor was dissociated in PBST for 600 s. Then, the sensor was placed in a glycine solution at pH 1.5 for 5 s, followed by PBST for 5 s, repeated three times for regeneration. The binding and dissociation curves were analyzed to obtain the affinity constant. The results are shown in […]. Figure 8 See Table 7. The results showed that the 05A8 antibody was a high-affinity antibody.

[0128] Table 7. Affinity analysis of 05A8 antibody

[0129] Example 9: Hydrogen-deuterium exchange mass spectrometry detection of antibody-binding epitopes RSV pre-F epitope localization was characterized using a hydrogen-deuterium exchange assay. This experiment consisted of protein deuterium labeling, hydrogen-deuterium exchange mass spectrometry (HDX-MS) analysis, and data analysis.

[0130] 1. Protein deuterium labeling (1) RSV pre-F deuterium labeling experiment Take 5 µL of RSV pre-F protein at a concentration of 1 mg / mL and place it in 25 µL of PBS or heavy water buffer (D2O 90% + 10% 10×PBS) solution, and incubate at room temperature for 0, 10 s, 1 min, 10 min, and 60 min, respectively. Then add 30 µL of quenching buffer (3 M GndHCl, 250 mM TCEP, pH 2.3) at a 1:1 volume ratio with the above protein buffer and incubate at 0 °C for 5 min. After completion, use a microsyringe to transfer 50 µL of the above reaction solution into the HDX sample processing system, including the online enzyme digestion column, trapping column, and peptide analysis column.

[0131] (2) Deuterium labeling experiment of antigen-antibody complex 1) RSV pre-F and monoclonal antibody 05A8 were incubated at a molar ratio of 1:3 for 30 min at room temperature to obtain antigen-antibody complexes.

[0132] 2) Take 5 µL of the antigen-antibody complex (1 mg / mL) and incubate it in 25 µL of heavy water buffer at room temperature for 10 s, 1 min, 10 min, and 60 min, respectively. Then add 30 µL of quenching buffer (3 M GndHCl, 250 mM TCEP, pH 2.3) at a 1:1 volume ratio with the protein buffer above, mix well, and incubate at 0 °C for 5 min. After completion, inject 50 µL of the above reaction solution into the HDX sample manager using a microsyringe.

[0133] (3) Hydrogen-deuterium exchange mass spectrometry analysis 1) Online enzymatic hydrolysis reaction After the sample was injected into the HDX sample processing system, the protein sample was enzymatically digested online in 0.1% formic acid aqueous solution at a rate of 100 µL / min using an immobilized pepsin chromatographic column (2.1 × 30 mm, Waters, Milford, MA).

[0134] 2) Peptide separation Peptides obtained from proteolytic digestion were captured on a C18 trap column (ACQUITY BEH, 1.7 μm, Waters, Milford, MA). They were then eluted with a continuous gradient of 5–40% in 0.1% formic acid acetonitrile solution at a flow rate of 40 µL / min. Separation was achieved by pumping the peptides into a reversed-phase column (ACQUITY UPLCBEH C18, 1.0 × 100 mm, 1.7 μM) via a nanoACQUITY binary solvent manager (Waters, Milford, MA).

[0135] 3) Mass spectrometry analysis HDX-MS (Waters, SYNAPT XS) was performed using electrospray ionization (ESI) mode, with the obtained peptides ionized under MSE mode. A reference mass of 200 pmol / mL leucine enkephalin was used, continuously injected at a flow rate of 10 µL / min.

[0136] 8.3 Data Analysis 1) The undeuterated RSV pre-F protein peptides were identified using ProteinLynx Global Server (PLGS) software (v3.0.3) in HD-MSE mode after the mass spectrometry data (Raw Data) acquired in Masslynx were processed.

[0137] 2) Import the RSV pre-F protein sequence database established in PLGS into DynamX (Waters, Milford, MA, v.3.0) software for peptide deuteration analysis. Set the peptide filtering conditions as follows: (i) Minimum mass spectrometry response: 3000; (ii) Minimum fragment per amino acid: 0.2; (iii) Maximum error: 10 ppm.

[0138] See results Figure 9 It can be seen that the 05A8 antibody shows a significant decrease in deuterium exchange rate at epitopes II, III, IV and φ, suggesting that these sites are antigen-antibody binding sites.

[0139] Example 10: Detection of the neutralizing effect of anti-RSV antibodies 1) Before infection, Hep-2 cells were cultured at a density of 1.5 × 10⁶ cells per well. 4 One cell was seeded into a 96-well plate (100 µL per well) and cultured for 20 h.

[0140] 2) Using serum-free DMEM medium (Gibco, C11995500CP) as the diluent, antibody 05A8, control antibody parizol, and nisvir were serially diluted. The initial concentration of antibody, control antibody parizol, and nisvir in each well was 20 μg / mL. Subsequent 3-fold serial dilutions were performed for a total of 11 wells, with replicates. Cell control (containing only culture medium, no virus, no antibody) and virus control (no antibody) were also set up.

[0141] 3) Virus dilution: RSV-A2 and RSV-B were diluted to 1.5 × 10⁻⁶ with serum-free DMEM. 3 The virus dilution was obtained by using FFU / mL (i.e., 75 FFU / 50 µL).

[0142] 4) Add an equal volume of virus diluent to the diluted antibody wells and virus control wells, mix with the antibody diluent at a volume ratio of 1:1, add the same volume of serum-free DMEM to the blank control group, mix gently and incubate at room temperature for 2 hours.

[0143] 5) Remove the 96-well culture plate containing a monolayer of Hep-2 cells cultured for 20 hours, aspirate the original complete culture medium from the wells, and add 100µL of serum-free DMEM to wash the cells once. Transfer 100µL / well of the serum-free DMEM and antibody-virus mixture incubated in step 4) to the corresponding positions in the 96-well culture plate.

[0144] 6) After adsorption at 37℃ for 2 hours, discard the supernatant and gently add 100µL of DMEM medium containing a final concentration of 2% (v / v) FBS and a final concentration of 0.9% (w / v) sodium carboxymethyl cellulose. Incubate in a CO2 incubator for 48 hours.

[0145] 7) After 48 hours, fix with 4% PFA for 20 minutes, permeabilize with Triton for 20 minutes, then stain with RSV antibody (7E2) and goat anti-mouse IgG-HRP secondary antibody (Yiqiao Shenzhou, AB_2917997). Use an ELISA speckle analyzer to photograph and count the spots. Based on the number of spots, calculate the inhibition rate corresponding to the serially diluted antibody concentrations and the antibody IC50. 50 The results are shown below. Figure 10 , Figure 11 See Table 8.

[0146] Experimental results showed that antibody 05A8 had significantly stronger inhibitory activity against RSV A2 strain (ATCC, VR-1540) and RSV B (18537) strain (ATCC, VR-1580) than parizol antibody, and its neutralizing activity against RSV B strain was stronger than that of nisevivir.

[0147] Table 8 Summary of antibody neutralizing activities

[0148] Example 11 Animal Protection Verification (1) Inactivated virus control group (inactivated RSV-A2): Mice were injected with the same dose of inactivated virus without antibody administration.

[0149] Negative control group (PBS): Mice were injected with the same volume of PBS but did not receive antibody treatment.

[0150] Experimental group (05A8): 24 hours before RSV-A2 virus challenge, mice were administered 100µL of PBS solution containing 05A8 antibody via intramuscular injection, with the solutions containing 3mg / Kg and 15mg / Kg of antibody, respectively. Positive control (Paliz antibody) antibody group: 24 hours before RSV-A2 virus challenge, mice were administered 100µL of PBS solution containing Paliz antibody via intramuscular injection, containing 3mg / Kg and 15mg / Kg antibody, respectively. (2) Each group contained 7 BALB / c mice. After anesthetizing with isoflurane, mice in the negative control group, experimental group, and positive control group were infected with RSV-A2 virus via nasal drops. Each mouse was challenged with 4 × 10⁶ viruses. 6 TICD 50 / 100µL. The inactivated virus control group was treated with an equal volume and amount of inactivated RSV-A2 virus solution for nasal drops.

[0151] (3) Record the weight of mice up to the fifth day after infection with RSV virus fluid.

[0152] The results are as follows Figure 12 As shown, the 05A8 antibody provides significant weight protection against RSV A2 strain infection in mice.

[0153] Example 12: Site analysis and corresponding amino acid polymorphism analysis (1) The amino acid sites on which antibody 05A8 binds to F protein were determined by cryo-electron microscopy.

[0154] (2) Download the RSV F protein sequences from NCBI over the years.

[0155] (3) The F protein polymorphism was analyzed by Mega software. The key binding amino acid sites of 05A8 were superimposed on the polymorphism analysis map to analyze the conservation of amino acids at the key sites.

[0156] The results show: a: Cryo-electron microscopy results show that the key amino acid residues on the F protein to which the 05A8 antibody binds are E60, K168, A170, L172, S173, T174, K176, A177, V178, L188, K196, D200, K201, and D263.

[0157] b: Polymorphism analysis of the 05A8 binding site revealed that the amino acid polymorphism of the amino acid residues bound to 05A8 was less than 5% (see...). Figure 13 The surface of the monoclonal antibody targeting epitope of the present invention is conserved.

[0158] Example 13 Experimental procedures for the preparation of 7E2 monoclonal antibody 1. Immunized animals (1) Antigen preparation: Dilute RSV pre-F protein (same as in Example 1) with PBS to a concentration of 1 mg / mL. Take 50 μL of this protein solution (containing 50 μg RSV pre-F protein) and emulsify it with an equal volume (50 μL) of Freund's complete adjuvant (Sigma, F5881) in a sterile centrifuge tube until a stable water-in-oil emulsion is formed (it does not spread when dropped into water).

[0159] (2) Immunization regimen: Select 6-8 week old female BALB / c mice. The first immunization was performed by intraperitoneal injection, with each mouse injected with 100 μL of the above emulsified antigen (containing 50 μg RSV pre-F protein).

[0160] Booster immunization: Booster immunizations were administered at weeks 2 and 4 following the initial immunization. For booster immunization, 50 μg of RSV pre-F protein was emulsified with an equal volume of Freund's incomplete adjuvant (Sigma, F5506), and 100 μL was injected intraperitoneally into each mouse.

[0161] (3) Titer detection: Seven days after the last booster immunization, about 100 μL of blood was collected from the tail vein of the mice and the serum was separated. The serum antibody titer was detected using an ELISA plate coated with RSV pre-F protein, and mice with high titers (e.g., 1:10000 or higher) were selected for fusion.

[0162] (4) Pre-fusion enhancement: Three days before fusion, the selected mice were given an intraperitoneal injection without adjuvant, and each mouse was injected with 50 μg RSV pre-F protein (dissolved in 50 μL PBS).

[0163] 2. Preparation of myeloma cells (1) Cell resuscitation and culture: Frozen SP2 / 0 myeloma cells were resuscitated and cultured in DMEM medium containing 15% FBS and 1% penicillin-streptomycin (Gibco, 15140122) at 37°C in a 5% CO2 incubator.

[0164] (2) Cell state: Ensure cells are in the logarithmic growth phase and viability >95% before fusion, and collect approximately 1×10⁻⁶ cells. 8 Each cell was washed twice with serum-free DMEM and resuspended in 10 mL of serum-free DMEM for later use.

[0165] 3. Spleen cell preparation (1) Aseptic spleen removal after mouse euthanasia: Mice that had been boosted with fusion were euthanized by cervical dislocation and disinfected by soaking in 75% alcohol for 10 minutes. Under aseptic conditions, the abdominal cavity was opened, the spleen was removed and placed in a petri dish containing 10 mL of serum-free DMEM medium.

[0166] (2) Grinding and filtration: The spleen is gently ground with the syringe core and filtered through a 200-mesh cell sieve. The spleen cell suspension is collected into a 50mL centrifuge tube.

[0167] (3) Centrifugation and washing: Centrifuge at 1000 rpm for 5 minutes and discard the supernatant. Wash twice with serum-free DMEM medium, and finally resuspend in 10 mL of serum-free DMEM and count.

[0168] 4. Cell fusion (1) Cell mixing: Mix the prepared spleen cells (approximately 1×10⁻⁶) 8 (number) and myeloma cells (approximately 1 × 10⁻⁶) 7 Mix the contents of the sample in a 50 mL centrifuge tube, add serum-free DMEM to a final volume of 30 mL, centrifuge at 1000 rpm for 5 minutes, and discard the supernatant.

[0169] (2) PEG fusion: Place the centrifuge tube in a 37°C water bath and gently tap the bottom of the tube to loosen the cell pellet. Slowly add 1 mL of 50% PEG (molecular weight 1500, MCE, HY-Y0873E) preheated to 37°C over 1 minute, stirring gently while adding the PEG. Continue to act in the water bath for 1 minute.

[0170] (3) Termination of fusion: Slowly add 10 mL of preheated serum-free DMEM over 2 minutes, then add to 30 mL, centrifuge at 800 rpm for 5 minutes, and discard the supernatant.

[0171] (4) Resuspension and plating: Resuspend the confluent cells in HAT complete medium (proteintech, PR40011) containing 20% ​​FBS, and adjust the cell concentration to approximately 1×10⁻⁶. 6Cells / mL. Add 100 μL (approximately 1 × 10⁶ cells / mL) of cell suspension to each well of a 96-well cell culture plate. 5 (1 cell), and cultured in a 37°C, 5% CO2 incubator.

[0172] 5. Hybridoma screening (1) Change the medium: On the 5th day after fusion, half of the medium was changed with fresh HAT medium. 50 μL of the old medium was removed and 50 μL of new HAT medium was added.

[0173] (2) Screening and detection: 10 days after fusion, when the hybridoma cell colonies have grown to about 1 / 3 of the bottom of the well, 50 μL of culture supernatant was aspirated. The ELISA plate coated with RSV pre-F protein was used for detection (according to the method in Example 5) to screen out the positive wells that could specifically bind RSV pre-F protein.

[0174] Positive well labeling: Marking culture wells that have shown a positive ELISA result.

[0175] 6. Hybridoma cloning (1) Limiting dilution method: Mix the cells in the positive wells by pipetting and perform serial dilutions of 10-fold until the final cell concentration reaches 0.5-1 cells / mL. Add 100 μL of the diluted cell suspension to each well of a 96-well plate and incubate in an incubator.

[0176] (2) Second screening: After the clonal cell colonies are formed, the supernatant is taken again for ELISA detection to screen out monoclonal cell lines that continuously secrete anti-RSV pre-F protein antibodies.

[0177] (3) Repeated cloning: Perform 2-3 limiting dilutions on the positive monoclonal strain to ensure that a stable monoclonal hybridoma cell line is obtained.

[0178] 7. Large-scale antibody preparation (1) In vitro culture: Stable hybridoma cell lines were expanded to T75 culture flasks, and the culture supernatant was collected. Antibodies were purified by protein A-conjugated magnetic beads to obtain high-purity monoclonal antibodies.

[0179] (2) Induction of ascites in vivo: Healthy BALB / c mice were selected, and each mouse was injected intraperitoneally with 0.5 mL of norphyrane (MCE, HY-N7819). Ten days later, each mouse was injected intraperitoneally with 1 x 10 mL of norphyrane. 6 Hybridoma cells were collected. After 12 days, when the mice's abdomens were significantly distended, ascites was collected. After centrifugation to remove cell debris, the ascites was purified using Protein A-conjugated magnetic beads to obtain a large concentration of monoclonal antibody, namely the 7E2 monoclonal antibody.

[0180] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A monoclonal antibody against respiratory syncytial virus, characterized in that, The amino acid sequences of the complementarity-determining regions CDR-H1, CDR-H2, and CDR-H3 of the heavy chain variable region of the monoclonal antibody are shown in SEQ ID No. 3 to 5, respectively; the amino acid sequences of the complementarity-determining regions CDR-L1, CDR-L2, and CDR-L3 of the light chain variable region of the monoclonal antibody are shown in SEQ ID No. 6 to 8, respectively.

2. The monoclonal antibody against respiratory syncytial virus according to claim 1, characterized in that, The heavy chain amino acid sequence of the monoclonal antibody against respiratory syncytial virus is shown in SEQ ID No. 1, and the light chain amino acid sequence is shown in SEQ ID No.

2.

3. A nucleic acid molecule encoding the monoclonal antibody of claim 1 or 2.

4. An expression carrier, characterized in that, Includes the nucleic acid molecule as described in claim 3.

5. A genetically engineered biomaterial, comprising bacteriophages, bacteria, fungi, or animal cells, characterized in that, Includes the expression vector as described in claim 4.

6. The use of the monoclonal antibody against respiratory syncytial virus as described in claim 1 or 2, or the nucleic acid molecule as described in claim 3, or the expression vector as described in claim 4, or the genetically engineered biomaterial as described in claim 5, in at least one of the following: 1) Prepare drugs for the prevention and / or treatment of respiratory syncytial virus (RSV); 2) Products for detecting respiratory syncytial virus.

7. A product for detecting respiratory syncytial virus, characterized in that, The product comprises a monoclonal antibody against respiratory syncytial virus as described in claim 1 or 2, a nucleic acid molecule as described in claim 3, an expression vector as described in claim 4, or a genetically engineered biomaterial as described in claim 5.

8. A drug for treating respiratory syncytial virus, characterized in that, Includes the monoclonal antibody against respiratory syncytial virus as described in claim 1 or 2, or the nucleic acid molecule as described in claim 3.

9. A method for detecting respiratory syncytial virus in a sample, characterized in that, The method includes mixing the monoclonal antibody of claim 1 or 2 with the sample to be tested and detecting the level of respiratory syncytial virus in the sample to be tested.

10. A method for preparing a monoclonal antibody against respiratory syncytial virus, characterized in that, include: 1) Obtain peripheral blood from RSV-positive donors and isolate mononuclear cells; 2) Antigen-specific B lymphocytes were sorted using flow cytometry and RSV pre-F tetramer probes; 3) Amplify the variable region of the antigen-specific B lymphocytes; 4) The variable region is recombined and transformed to obtain a monoclonal antibody against respiratory syncytial virus.

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