A monoclonal antibody targeting PPRV HN and its application

Abstract

The application belongs to the technical field of monoclonal antibodies, and particularly relates to a monoclonal antibody targeting PPRV HN and application thereof. The monoclonal antibody targeting PPRV HN is characterized by comprising a light chain and a heavy chain. The amino acid sequence of the variable region of the light chain comprises QSIVHSSGNTY, KYS and FQGSHVPYT. The amino acid sequence of the variable region of the heavy chain comprises GFNIKDYY, IDPENGYT and AELGRGSY. The monoclonal antibody provides a new effective tool and solution for veterinary detection diagnosis, prevention and treatment of PPRV serotypes.

Description

Technical Field

[0001] This invention belongs to the field of monoclonal antibody technology, specifically relating to a monoclonal antibody targeting PPRV HN and its applications. Background Technology

[0002] Pestos petits ruminants (PPR), commonly known as "sheep plague," is a highly contagious viral disease that is widespread in Africa, the Middle East, Europe, and Asia. The PPR virus is abbreviated as PPRV. PPRV has a single, non-segmented, negative-sense RNA genome. The PPRV genome encodes eight proteins, including six structural proteins: nucleocapsid protein, phosphoprotein, matrix protein, fusion protein, hemagglutinin-neuraminidase protein, and large polymerase protein, as well as two non-structural proteins, C and V. Specifically, nucleocapsid protein is abbreviated as N; phosphoprotein as P; matrix protein as M; fusion protein as F; hemagglutinin-neuraminidase protein as HN; and large polymerase protein as L. F and HN play a crucial role in the early stages of PPRV replication. The PPRV HN protein possesses both hemagglutinin and neuraminidase activities. It mediates viral attachment and infection by recognizing and binding to SLAM and Nectin-4 receptors on the surface of host cells. After attachment, the HN protein triggers a conformational change in the F protein, executing the fusion of the viral envelope with the host cell membrane, thereby completing the injection of the viral genome.

[0003] Monoclonal antibodies, as a cornerstone of modern biomedicine, owe their highly efficient and specific targeting capabilities to their precise molecular structure. The core of a monoclonal antibody is its variable region, which directly determines the antibody's specificity in recognizing and binding antigens. Therefore, the sequences of the heavy and light chains of a monoclonal antibody are crucial to its functional specificity.

[0004] In our previous study, we first analyzed the transmembrane region and dominant antigenic epitope region of the PPRV HN protein using bioinformatics methods. Subsequently, we successfully expressed a truncated form of the PPRV HN protein (223aa~551aa) using a prokaryotic expression system. This truncated HN protein was then expressed and purified using the prokaryotic expression system and used to immunize BALB / c mice. After detecting mice with positive serum antibody reactions, we fused their spleen cells with myeloma cells and screened them using HAT selective medium. Finally, we successfully obtained a hybridoma cell line that stably secretes a PPRV HN monoclonal antibody, named 1D4-4D9. Therefore, we aim to develop a sequence-controlled monoclonal antibody that specifically recognizes the HN site for the prevention and treatment of PPRV virus. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a monoclonal antibody targeting PPRV HN and its application.

[0006] To facilitate understanding of the contents of this specification, the names and abbreviations of the substances of this invention are listed below: Hemagglutinin-neuraminidase protein is abbreviated as HN. Monoclonal antibodies targeting PPRV HN are abbreviated as HN. 1D4-4D9 Heavy chain, abbreviated as V H And light chains, abbreviated as V. L The variable region, abbreviated as CDR. Hybridoma cells for PPRV HN monoclonal antibodies, abbreviated as 1D4-4D9.

[0007] The purpose of this invention is to provide a monoclonal antibody targeting PPRV HN, comprising a light chain and a heavy chain: The amino acid sequence of the variable region of the light chain includes: QSIVHSSGNTY, KVS, and FQGSHVPYT.

[0008] The amino acid sequence of the variable region of the heavy chain includes: GFNIKDYY, IDPENGYT, and AELGRGSY.

[0009] Preferably, the amino acid sequence of the light chain variable region is as shown in SEQ ID NO.1. (See reference...) Figure 4 V in A L CDR1~CDR3 in the middle.

[0010] Preferably, the amino acid sequence of the variable region of the heavy chain is shown in SEQ ID NO.2. (See also...) Figure 4 V in A H CDR1~CDR3 in the middle.

[0011] Preferably, the complete genome sequence of the light chain is shown in SEQ ID NO.3. (See reference...) Figure 4 A in V L The complete genome sequence of the heavy chain is shown in SEQ ID NO.4. (See reference...) Figure 4 V in A H .

[0012] Another object of the present invention is to provide a method for preparing a monoclonal antibody targeting PPRV HN, comprising the following steps: The pET28a-HN(V-HN) plasmid was constructed and transformed into DH5α to obtain DH5α target bacteria. The DH5α target bacteria were induced to express pET28a-HN(V-HN) protein by IPTG, purified, and obtained truncated recombinant HN protein. The truncated recombinant HN protein was used as an antigen to obtain hybridoma cell lines 1D4-4D9 through three immunizations. The hybridoma cells were injected into mice via intraperitoneal injection, and ascites fluid was collected, centrifuged, and the supernatant was collected, purified, and obtained monoclonal antibody.

[0013] Preferably, the application is for preparing a PPRV vaccine.

[0014] Preferably, a vaccine is obtained by combining a monoclonal antibody with an adjuvant.

[0015] Preferably, the adjuvant comprises an aluminum salt adjuvant, an emulsion adjuvant, a TLR agonist, a STING agonist, a saponin adjuvant, a nanoparticle adjuvant, or purified water.

[0016] Preferably, the aluminum salt adjuvant comprises aluminum hydroxide, aluminum phosphate, and potassium aluminum sulfate.

[0017] Preferably, when the adjuvant is water, the concentration is 7.5 mg / mL.

[0018] Compared with the prior art, the present invention has the following beneficial effects: 1. The monoclonal antibody targeting PPRV HN of the present invention comprises a light chain and a heavy chain: wherein the amino acid sequence of the variable region of the light chain includes: QSIVHSSGNTY, KVS, FQGSHVPYT. The amino acid sequence of the variable region of the heavy chain includes: GFNIKDYY, IDPENGYT, AELGRGSY. H Belongs to the IgH subtype, V L It is of the κ type. The amino acid sequences of the light chain CDR1~CDR3 prepared in Example 1 of this invention are QSIVHSSGNTY, KVS, FQGSHVPYT, and the amino acid sequences of the heavy chain CDR1~CDR3 are GFNIKDYY, IDPENGYT, AELGRGSY, and HN, respectively. 1D4-4D9 It has excellent neutralizing and immune response properties against PPRV, and can be used to prepare PPRV for detection, diagnosis, prevention and treatment. Attached Figure Description

[0019] Figure 1This is a screening diagram of hybridoma cell lines targeting the PPRV HN protein in Example 1 of the present invention. A shows the PCR amplification results of the PPRV truncated HN gene. B shows the induced expression and Western blot verification analysis of the pET28a-PPRV HN (223-551aa) prokaryotic plasmid. C shows the reaction characteristics and antibody titer of purified hybridoma ascites analyzed by Western blot.

[0020] Figure 2 This is a HybSeq-HT™ sequencing analysis diagram of the hybridoma cell line antibody gene targeting the PPRV HN protein with neutralizing activity in Example 2 of the present invention. In this diagram, A is a schematic diagram of the HybSeq-HT™ sequencing process for the hybridoma cell line antibody. B represents the hybridoma cell line V... H and antibody V L Percentage of gene sequence.

[0021] Figure 3 V is the V-type plasmid of the hybridoma cell line antibody gene chimeric plasmid in Example 3 of the present invention. H and V L Gene map. Where A is V, the chimeric plasmid of the antibody gene from the hybridoma cell line. H Gene map. B is the V of the hybridoma cell line antibody gene chimeric plasmid. L Gene map.

[0022] Figure 4 This refers to the antigen complementarity-determining region (A) in the antibody gene of the hybridoma cell line in Example 3 of the present invention. Wherein, A is the antigen complementarity-determining region of hybridoma cell line V. H and antibody V L Gene sequence analysis and its CDR sequence analysis. B is the hybridoma cell line V. H and V L Schematic diagram of the amino acid sequence structure of CDR1, CDR2, and CDR3. C is a Western blot analysis of the immunoreactivity of hybridoma cell lines lacking CDR1, CDR2, and CDR3 antigens.

[0023] Figure 5 This is a graph showing the neutralizing antibody activity of the monoclonal antibody from the hybridoma cell line in Example 4 of the present invention. In the graph, A represents the Western blot analysis of exogenously transfected hybridoma cell line V. H and V L It can inhibit PPRV virus replication in Vero-SN cells. B is an exogenous transfection of the hybridoma cell line V. H and V L TCID that can inhibit the replication of Vero-SN extracellular PPRV virus 50 Assay and analysis. C exogenous transfection of hybridoma cell line V H and V LFluorescence observation showing inhibition of PPRV-EGFP replication. D represents exogenously transfected hybridoma cell line V. H Flow cytometry analysis showed that VL inhibited PPRV-EGFP replication in Vero-SN cells. E represents exogenously transfected hybridoma cell line V. H and V L Statistical analysis of flow cytometry analysis showing inhibition of PPRV-EGFP replication in Vero-SN cells.

[0024] Figure 6 In Example 4 of this invention, the exogenously transfected hybridoma cell line V... H and V L Inhibition experiments on the adsorption of PPRV. Where A is the exogenous transfection of V... H +V L The cells were incubated in Vero-SN cells 1 hour after PPRV infection. D represents exogenous transfection with V. H +V L The experiment involved simultaneous incubation of Vero-SN cells with the PPRV strain. G indicates that PPRV infection preceded exogenous transfection with V. H +V L Incubate for 1 hour in Vero-SN cells. B is V H +V L Western blot analysis of proteins incubated 1 hour prior to PPRV infection. E represents Western blot analysis of proteins incubated with VH+VL and PPRV strains. H represents PPRV infection prior to V... H +V L Western blot analysis of proteins after incubation for 1 hour. C is V. H +V L TCID infected 1 hour before PPRV infection 50 Determination and analysis. F is V H +V L TCID incubated with PPRV strain 50 Determination and analysis. I is PPRV prior to V. H +V L TCID incubated for 1 hour 50 Measurement and analysis.

[0025] Figure 7 V in Embodiment 5 of the present invention H and V L Inhibition experiment on PPRV adsorption infection. Where A represents V at 37℃. H +V L The culture supernatant was used 1 hour prior to PPRV infection to measure the PPRV viral genome content in Vero-SN cells. B represents the PPRV viral genome content at 37°C. H +V LThe PPRV viral genome content in Vero-SN cells was determined by incubating the culture supernatant with the PPRV strain. C represents the pre-infection rate of PPRV at 37°C compared to V. H +V L The PPRV virus genome content in Vero-SN cells was determined by incubating the culture supernatant for 1 hour. D represents the content of PPRV virus genome in Vero-SN cells at 4°C. H +V L The culture supernatant was used 1 hour prior to PPRV infection to measure the PPRV viral genome content in Vero-SN cells. E represents the viral genome content at 4°C. H +V L The PPRV viral genome content in Vero-SN cells was determined by incubating the culture supernatant with the PPRV strain. F represents the pre-infection rate of PPRV at 4°C compared to V. H +V L Culture supernatant was incubated for 1 hour to determine the PPRV virus genome content in Vero-SN cells. Detailed Implementation

[0026] To enable those skilled in the art to better understand and implement the technical solutions of the present invention, the following detailed description, in conjunction with preferred embodiments and accompanying drawings, provides a clear and complete account of the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0027] It should be noted that all technical terms used in this invention are for the purpose of describing specific embodiments only and are not intended to limit the scope of protection of this invention. Unless otherwise specified, all raw materials, reagents, instruments and equipment used in the following embodiments of this invention can be purchased from the market or prepared by existing methods.

[0028] Example 1 A method for preparing a monoclonal antibody targeting PPRV HN includes the following steps: 1. Construction of recombinant plasmid containing pET28a-PPRV HN (223aa~551aa) truncated protein Bioinformatics analysis was used to predict and analyze the transmembrane region and dominant antigenic epitope region of the PPRV HN protein, amplifying the target fragment to a size of 984 bp, yielding the PPRV HN (223aa~551aa) protein. Using the highly virulent PPRV strain pCAGGS-Velogenic HN(VHN)-HA plasmid as a template, polymerase chain reaction (PCR) amplification was performed. The PCR product was verified by 1% agarose gel electrophoresis, and the target fragment was recovered using a gel extraction kit. The gel-recovered fragment was cloned into the prokaryotic expression vector pET28a, which had been digested with restriction endonucleases, using the Clon Express MultiS one-step cloning kit. The ligation product was transformed into DH5α competent cells, plated on 2YT solid medium containing kanamycin, and incubated overnight at 37°C. Suspected positive single colonies were picked and inoculated into 2YT liquid medium. After culturing for 15 hours, the plasmid was extracted and sent to Sangon Biotech Co., Ltd. for sequencing. The positive recombinant plasmid with correct sequencing was obtained as pET28a-Velogenic HN (V-HN), abbreviated as pET28a-HN (V-HN).

[0029] 2. Purification of pET28a-Velogenic HN (V-HN) recombinant protein induced expression The original bacterial culture containing the recombinant plasmid pET28a-HN (V-HN) was inoculated into 1 mL of 2YT liquid medium containing kanamycin at a volume ratio of 1:100, and cultured with shaking at 37℃ and 200 rpm. The OD of the bacterial culture was then measured. 600 When the expression level reached 1.0, IPTG was added to a final concentration of 1 mmol / L for induction. After induction, the bacterial culture was incubated at 37℃ and 200 rpm with shaking for 12 h. Subsequently, the bacterial pellet was collected by centrifugation at 4℃ and 12000 rpm for 2 min. The pellet was resuspended in 1× phosphate buffer and then sonicated. The lysate was centrifuged at 12000 rpm for 15 min, and the supernatant and pellet were separated and collected. The expression pattern of the recombinant protein was analyzed by SDS-PAGE electrophoresis. After large-scale induction of protein expression, the recombinant protein was purified by nickel-nitrotriacetic acid affinity chromatography. The purified protein was verified by SDS-PAGE electrophoresis, and the concentration of the purified protein was determined for subsequent experiments. The purification results are shown below. Figure 1 As shown.

[0030] 3. Preparation, purification, and titer determination of monoclonal antibodies targeting PPRV HN Four 7-week-old female SPF-grade BALB / c mice were selected and subjected to three rounds of immunization: For the first immunization, purified truncated recombinant HN protein was emulsified at 75 µg / mouse with an equal volume of Freund's complete adjuvant and injected subcutaneously at five points on the back, ≤0.15 mL per point. Two weeks later, for the second immunization, the protein was emulsified with Freund's incomplete adjuvant and injected subcutaneously at the same dose and in the same manner. Two weeks after the second immunization, a third immunization was administered using the same method. Three days before cell fusion, a pulse immunization was performed. To enhance B cell activation, 100 µg / mouse of truncated recombinant HN protein was diluted to 0.3 mL with sterile PBS, thoroughly mixed, and injected aseptically into the intraperitoneum to enhance the immune response. Eight days after the third immunization, blood was collected from the tail vein, and serum antibody titers were detected by indirect ELISA. Mice with titers ≥1:10000 were selected for cell fusion. Immunized mice were euthanized by cervical dislocation, and the spleen was harvested under sterile conditions, ground, and passed through a 200-mesh sieve. SP2 / 0 myeloma cells in logarithmic growth phase with ≥95% viability were mixed with spleen cells at a volume ratio of 5:1 and fusion was mediated using 50% PEG4000. After fusion, the cell suspension was seeded into 96-well plates containing HAT medium and cultured at 37°C in a 5% CO2 incubator. After 7 days, the medium was replaced with HT medium. After 14 days of culture, positive wells were screened using indirect ELISA: using truncated recombinant PPRV HN (223aa~551aa) protein as the antigen, the positivity of antibodies in the supernatant of the wells was detected. Positive wells were subcloned using limiting dilution, with three consecutive subclonings to obtain hybridoma cell lines stably secreting anti-PPRV HN (223aa~551aa) protein. These hybridoma cell lines were designated 1D4-4D9. Seven-week-old mice were selected and intraperitoneally injected with 1 mL of hybridoma cells (1×10⁶ cells). 6 Each mouse was injected with a single antibody. Starting on day 9 post-injection, the abdominal distension of the mice was observed daily. Ascites fluid was collected and centrifuged at 4000 rpm for 8 minutes at 4°C to remove the precipitate. The supernatant was the monoclonal antibody ascites fluid. The ascites antibody was purified using the Nab Protein A / G Spin Columns antibody purification kit. The purified antibody concentration was determined to be 7.5 mg / mL. The purified antibody was aliquoted and stored at -80°C for later use. Figure 1 As shown in C.

[0031] Example 2 V of monoclonal antibodies targeting PPRV HN H and V L Abundance analysis Obtaining the gene sequence of antibodies expressed by hybridomas is a crucial prerequisite for large-scale antibody production and engineering modification. Therefore, this invention commissioned Anshengda Biotechnology Co., Ltd. to perform comprehensive and reliable hybridoma antibody gene sequencing on hybridoma cell lines. The results are as follows: Figure 2As shown. The sequencing process mainly includes: extracting total RNA from cells using the Trizol method, synthesizing cDNA via reverse transcription, and amplifying to obtain antibody V. H With V L The variable region sequence was obtained and cloned into a vector to complete sequencing library construction and sequence analysis, such as... Figure 2 As shown in A. Subsequently, the sequencing results were compared with the IMGT database to extract V. H and V L The complementarity-determining regions (CDRs) in the sequence, including key information from CDR1, CDR2, and CDR3, were analyzed. The 10 most abundant sequences from each sample were selected for abundance statistics, and the sequence with the highest abundance was identified as the target antibody gene sequence.

[0032] Hybseq HTTM hybridoma antibody gene profiling results showed that the V of this cell line H and V L The variable region sequences all exhibited high homogeneity, among which V H The sequence with the highest abundance in the variable region is close to 96%, V L The variable region also showed a similar trend, with its highest abundance value also approaching 95%, such as Figure 2 As shown in B in the figure. Further analysis of V, which has the highest abundance... H Subtype analysis of the VL sequence revealed that the antibody heavy chain belongs to the IgH type, and the light chain is κ. Based on these results, we selected the VL sequence, which had the highest abundance. H With V L The sequence was used for gene synthesis and cloned into the pcDNA3.4 expression vector for subsequent recombinant expression studies of the monoclonal antibody. The results are as follows: Figure 3 As shown.

[0033] The specific experimental steps are as follows: First, total RNA was extracted from hybridoma cells, and the mRNA was reverse transcribed into cDNA using the SuperScript® III First-Strand Synthesis System for RT-PCR kit. Then, the target antibody's V-type PCR protein was amplified using universal specific primers. H and V L Variable region genes. After PCR products were separated by 1% agarose gel electrophoresis, the target band was excised and purified using a gel extraction kit. The purified V... H and V L After the purity of the fragments was verified by electrophoresis, they were cloned into the pMD19-T sequencing vector and sequenced. Finally, the obtained sequences were compared and homology analyzed to confirm their correctness and consistency.

[0034] Example 3 V H and V LThe CDR region and the biological function of antibodies The amino acid sequence results of HybSeq-HT™ sequencing of antibody genes in hybridoma cell lines are as follows: Figure 4 As shown, the heavy chain variable region and light chain variable region with the highest abundance (Rank 1) were selected for gene synthesis, and then cloned into the pCDNA3.4 expression vector to construct pcDNA3.4-V. H -1D4-4D9(HN) and pcDNA3.4-V L -1D4-4D9(HN), such as Figure 3 As shown. To further enhance antibody affinity, we performed codon optimization during synthesis and fused the optimized variable region with the constant region of the mouse antibody, thereby constructing a V antibody that retains the characteristics of the variable region. H Expression plasmids and V L Expression plasmid. V H Expression plasmids and V L V in expression plasmid H With V L Gene structure such as Figure 4 As shown. V H With V L Expression plasmids were transfected into HEK-293T cells to achieve secretory expression. Specific experimental steps are as follows: A series of CDR deletion plasmids were designed using SnapGene software, including V... H The plasmids that delete CDR1, CDR2, and CDR3 are V H ΔCDR1, V H ΔCDR2, V H ΔCDR3, V L The plasmids that delete CDR1, CDR2, and CDR3 are V L ΔCDR1, V L ΔCDR2, V L ΔCDR3 was constructed using reverse PCR combined with enzyme digestion and self-ligation to create a mutant plasmid. Sequencing verification ensured the precise deletion site and the absence of frameshift mutations and additional base mutations. Subsequently, functional validation systems were constructed using single or co-transfection strategies. Western blotting experiments were then conducted to analyze the impact of each CDR region on antibody-antigen binding function. A total of 18 experimental systems were set up, including one co-transfection group, co-transfected with V... H V L The plasmid served as a positive control. Two groups were transfected separately, each transfected with V... H plasmid, V L Plasmids were used to verify the necessity of single-chain expression for antibody assembly. Six groups of single CDR deletion transfections were performed. H ΔCDR1, V H ΔCDR2, V H ΔCDR3, VL ΔCDR1, V L ΔCDR2, V L ΔCDR3. Six groups of single CDR deletion co-transfection groups, V H +V L ΔCDR1, V H +V L ΔCDR2, V H +V L ΔCDR3, V H ΔCDR1+V L V H ΔCDR2+V L V H ΔCDR3+V L Three groups of CDR double-deletion co-transfection groups, V H ΔCDR1+V L ΔCDR1, V H ΔCDR2+V L ΔCDR2, V H ΔCDR3+V L ΔCDR3. HEK-293T cells were plated and cultured at 37°C in a 5% CO2 incubator until 75% confluence before transfection. After 21 hours of transfection, cells were frozen at -80°C. Upon thawing, the cells were centrifuged at 3000 rpm for 5 minutes at 4°C, and the supernatant was collected for subsequent Western blot (WB) analysis. WB analysis used lysates of HEK-293T cells transfected with the full-length PPRV HN plasmid pCAGGS-VHN-HA as the sample. Electrophoresis was performed at 80V for 30 minutes and then at 120V for 1 hour. Wet transfer was used, with constant current at 220 mA and ice bath for 2 hours to transfer proteins to an NC membrane. After blocking the NC membrane with 5% skim milk blocking buffer on a shaker at room temperature for 1 hour, the supernatant of each group was used as the primary antibody and incubated overnight at 4°C. The next day, the membrane was washed three times with TBST buffer for 5 minutes each time, and HRP-labeled anti-IgG secondary antibody was added and incubated at room temperature for 1 hour. The membrane was washed three times with TBST again for 5 minutes each time. Finally, ECL chemiluminescence reagent was added, and the specific binding bands were exposed and recorded using a chemiluminescence imaging system.

[0035] Western blot results showed that only when V H With V LOnly when both chains are co-expressed can the specific target band be detected at the expected molecular weight position; expression of either chain alone results in undetectable bands. The results indicate successful recombinant expression of specific antibodies derived from 1D4-4D9 hybridoma cell lines, and the obtained antibodies exhibit good specificity and sensitivity in antigen recognition. The antigen complementarity-determining region (CDR) is the core functional region for antibody specific recognition and binding to antigens, located within the heavy chain variable region and the light chain variable region, and is divided into three conserved subregions: CDR1, CDR2, and CDR3, based on structural and functional characteristics. This invention obtains HN through hybridoma cell gene sequencing. 1D4-4D9 The full-length nucleotide sequence, and its V H and V L The functional areas within the structural domain are visually labeled, such as... Figure 4 As shown in Table 1. To clarify the necessity of CDRs in antibody-antigen binding, a series of CDR deletion plasmids were designed using the specific primers in Table 1, and the CDR deletion region V was deleted. H V L Plasmids were transfected into HEK-293T cells alone. After 24 hours of culture, the supernatant was collected. Western blotting experiments showed no antigen-binding specific bands in either group. Six single CDR deletion co-transfection systems were constructed. H +V L ΔCDR1 / 2 / 3、V H ΔCDR1 / 2 / 3+V L , with V H +V L As a positive control, no specific bands were observed in any of the deletion groups. Further research was conducted to construct six V... H or V L The corresponding CDR double-deletion co-transfection system, V H ΔCDR1 / 2 / 3+V L ΔCDR1 / 2 / 3, Western blotting experiments showed no specific bands in any of the three groups. In summary, HN 1D4-4D9 CDR1, CDR2, and CDR3 are all key target sites for antigen-specific recognition. The loss of any CDR leads to the antibody losing its antigen-binding ability, confirming that CDRs play an irreplaceable core role in antibody-antigen binding.

[0036] Table 1 Constructs V H With V L CDR plasmid PCR primer sequence Example 4 Exogenous expression of V H +V L Neutralizing antibody activity To verify whether functional antibodies expressed directly from cells without purification possess virus-neutralizing activity, V... HWith V L The plasmid was co-transfected into Vero-SN cells at gradients of 1 µg and 2 µg, and EGFP-PPRV virus was inoculated 21 h after transfection.

[0037] The specific experimental steps are as follows: (1) Co-transfection of V H +V L V H +V L The plasmid was co-transfected into Vero-SN cells with 85% confluence in 12-well plates at gradients of 1 µg and 2 µg. 21 h post-transfection, 12 µL of EGFP-PPRV virus was inoculated. A virus infection group without plasmid transfection served as a positive control, and a virus infection group transfected with the pCDNA3.4 empty vector served as a negative control. After 72 h of culture, the supernatant was collected, and TCID was measured. 50 Cells were lysed to prepare samples, and the expression of viral HN protein was detected by Western blot.

[0038] (2) Co-transfection of V H +V L Collect cell culture supernatant: Incubate the virus with the supernatant, and add V... H +V L The plasmid was co-transfected into HEK-293T cells at 75% confluence in 6-well plates, and pCDNA3.4 empty vector was transfected simultaneously as a negative control. Cell culture supernatant was collected 21 h post-transfection and stored at -80℃ for later use. Before use, the cells were thawed, centrifuged at 3000 rpm for 5 min at 4℃, and the supernatant was used as the antibody source. Vero-SN cells were seeded in 12-well plates and cultured to 95% confluence. Twelve 5 mL EP tubes were divided into experimental and replicate groups, with two replicates per group. In the experimental group, 1 mL, 2 mL, 3 mL, and 4 mL of supernatant were added to the four tubes, respectively. The supernatant from 1 mL to 3 mL was replenished to 4 mL with serum-free DMEM. The negative control group received the supernatant transfected with the empty vector, while the positive control group received no supernatant. 12 µL of PPRV vaccine strain at MOI=1 was added to each tube, and the cells were incubated at 37℃ for 1 h. The incubation solution was then transferred to the cell wells and incubated at 37℃ for 1 h. Discard the old culture medium, wash the cells three times with PBS buffer, add 1 mL of DMEM complete medium containing 10% fetal bovine serum to each well, and incubate at 37°C and 5% CO2 for 72 h. Collect the cell supernatant to determine TCID. 50 Cells were lysed to collect total protein, and the expression level of viral HN protein was detected by Western blotting.

[0039] (3) V H +V LCo-transfection supernatant was incubated 1 hour before virus infection: Vero-SN cells were seeded in 12-well plates and cultured to 95% confluence. Experimental groups, a negative control group with empty vector transfection supernatant, a positive control group with virus infection only, and a replicate group were set up, with two replicates in each group. In the experimental groups, 1 mL, 2 mL, 3 mL, and 4 mL of supernatant were added to each well. 1-3 mL of supernatant was then added, and serum-free DMEM was added to bring the total volume to 4 mL. After incubation at 37°C and 5% CO2 for 1 hour, 12 µL of PPRV vaccine strain with an MOI of 1 was added to each well, and the cells were incubated at 37°C for 1 hour for adsorption. The old culture medium was discarded, and the cells were washed three times with PBS. 1 mL of complete DMEM medium containing 10% fetal bovine serum was added to each well, and the cells were incubated at 37°C and 5% CO2 for 72 hours. The cell supernatant was collected to determine the half-maximal viral load (ICL), and total protein was collected by lysing the cells. Western blot analysis was performed to detect viral HN protein expression.

[0040] (3) V H and V L After co-transfection with supernatant, cells were incubated for 1 hour post-viral infection: Vero-SN cells were seeded in 12-well plates and cultured to 95% confluence. Experimental groups, a negative control group (with empty vector transfection supernatant), and a positive control group (viral infection only) were set up. 12 µL of PPRV vaccine strain with an MOI of 1 was added to each well, and the cells were incubated at 37°C and 5% CO2 for 1 hour, after which the viral load was discarded. For the experimental groups, 1 mL, 2 mL, 3 mL, and 4 mL of supernatant were added. The 1 mL to 3 mL supernatant were then replenished to 4 mL with serum-free DMEM. The negative control group received empty vector transfection supernatant, while the positive control group received no supernatant. After further incubation at 37°C and 5% CO2 for 1 hour, the liquid in the wells was discarded, and the cells were washed three times with PBS buffer and cultured for 72 hours with complete culture medium. After culture, the cell supernatant was collected for viral TCID assay. 50 Cells were lysed to collect total protein samples, and the expression level of viral HN protein was detected by Western blotting.

[0041] The results are as follows Figure 5 and Figure 6 As shown, the results indicated that compared with the negative control transfected with empty vector, the expression level of HN protein in cells was reduced by Western blot analysis, and the inhibitory effect of the 2µg plasmid transfection group was stronger than that of the 1µg group, indicating that V H With V L The expressed antibodies can inhibit viral replication, and the inhibitory effect is positively correlated with the antibody expression level, such as... Figure 5 As shown. The results show that TCID in the viral supernatant 50 The results show that V H +V LThe co-transfection group significantly inhibited extracellular PPRV virus replication in Vero-SN cells in a dose-dependent manner. Based on the fluorescently labeled PPRV strain, compared with the empty pCDNA3.4 and the positive control group, the co-transfection group significantly inhibited the fluorescence intensity within Vero-SN cells in a dose-dependent manner, indicating that the co-transfection group can significantly inhibit the intracellular replication ability of PPRV-EGFP. Figure 5 As shown in C. Also, as... Figure 5 Flow cytometry analysis also showed that, compared with empty pCDNA3.4 and the positive control group, the co-transfection group could significantly inhibit the replication capacity of PPRV-EGFP cells.

[0042] To further systematically investigate the virus-neutralizing activity and dose-dependent characteristics of this antibody, this invention designed three incubation strategies: PPRV vaccine strain and exogenously transfected V... H +V L Incubate together with supernatant, and exogenously transfect V H +V L The supernatant was incubated with the PPRV vaccine strain for 1 hour prior to infection and exogenous transfection with V. H +V L The supernatant was incubated 1 hour after infection with the PPRV vaccine strain. Results were as follows: Figure 6 As shown.

[0043] The results showed that in the group where the virus was incubated with the supernatant, Western blot analysis revealed that, compared to the positive control group, the expression level of PPRV HN protein in the experimental group decreased in a gradient manner with increasing antibody concentration. Therefore, V H and V L Transfection supernatant specifically inhibited viral proliferation in Vero-SN cells. TCID 50 The measurement results are as follows Figure 6 As shown in Figure C, the results indicated that the extracellular viral titer in the experimental group decreased in a gradient with increasing antibody concentration, further confirming its inhibitory effect on extracellular viral proliferation. The supernatant was incubated 1 hour before viral infection. Compared to the positive control group, the expression level of PPRV HN protein in the cells of the experimental group decreased in a gradient with increasing antibody concentration. TCID 50 The measurement results are as follows Figure 6 As shown in F, the results indicate that the extracellular viral titer in the experimental group decreased in a gradient with increasing antibody concentration, suggesting that the antibody still possesses specific inhibitory activity under this incubation strategy. In the group incubated 1 hour after viral infection with supernatant, WB and TCID levels... 50 The test results all indicate that V H +V L The transfection supernatant did not show a specific inhibitory effect on the proliferation of PPRV in Vero-SN cells, nor did it exhibit neutralizing antibody biological activity.

[0044] Example 5 Exogenous expression of V H +V L Inhibit PPRV adsorption infection To confirm exogenous co-transfection of V H +V L During the virus neutralization phase of the culture supernatant, this invention sets up three incubation strategies: PPRV virus co-transfected with exogenous V... H +V L Incubate together with supernatant, and exogenously transfect V H +V L The supernatant was incubated with PPRV virus for 1 hour prior to inoculation and exogenous co-transfection with V. H +V L The supernatant was incubated for 1 hour after PPRV infection. Samples were collected immediately after virus adsorption at 37°C for 1 hour, and the viral genome copy number was detected by quantitative real-time PCR to assess the viral genome replication level.

[0045] The specific experimental steps are as follows: Vero-SN cells were seeded into 12-well plates and cultured until 90% confluence. Six 5mL EP tubes were taken and divided into experimental and replicate groups, with each group repeated three times. The experimental group was treated with 3mL of exogenous transfected Vero-SN cells. H +V LFor the supernatant, the negative control group received the supernatant from empty vector transfection, while the positive control group received no supernatant. Add 12 µL of PPRV vaccine strain (MOI=1) to each tube and incubate at 37°C for 1 h. After pre-cooling the cells to 4°C, wash three times with cold PBS, transfer the incubation solution to cell wells, incubate at 4°C for 1 h, wash three times with cold PBS, add 500 µL of Trizol to each well, and incubate at room temperature for 10 min to lyse the cells. Transfer the lysis buffer to enzyme-free EP tubes, add 200 µL of chloroform, vortex for 15 s, incubate on ice for 5 min, and centrifuge at 4°C, 12000 rpm for 15 min. Aspirate the upper aqueous phase, add an equal volume of isopropanol, incubate on ice for 5 min, and centrifuge at 4°C, 12000 rpm for 20 min. Wash the precipitate twice with 1 mL of 75% ethanol, centrifuge at 4°C, 9500 rpm for 5 min, air dry in a clean bench, and dissolve the RNA in 50 µL of enzyme-free water. Take 2 µg of RNA, add RNase-free ddH2O and 4×g DNA wiperMix, and incubate at 42℃ for 2 min to remove genomic DNA. Add 5×HiScript II qRT SuperMix II, and react at 50℃ for 15 min and 85℃ for 5 s to complete reverse transcription. Perform real-time PCR using cDNA as a template: the upstream primer is PPRN8a-F, with the nucleotide sequence SEQ ID NO.17: CACAGCAGAGGAAGCCAAAC. The downstream primer is PPRN9b-R, with the nucleotide sequence SEQ ID NO.18: TGTTTTGTGCTGGAGGAAGGA. The reaction conditions are: 95℃ pre-denaturation for 30 s, 95℃ denaturation for 10 s, 60℃ annealing for 20 s, 40 cycles; melting curve: 95℃, 10 s; 60℃, 5 s. Each sample is tested in triplicate to analyze the number of viral RNA copies adsorbed by cells. Two strategies were employed: incubating the supernatant 1 hour before viral infection and incubating the supernatant 1 hour after viral infection. Simultaneous quantitative fluorescence analysis was performed.

[0046] like Figure 7 As shown, qPCR results indicated that the viral genome copy number in the supernatant co-incubation group and the group incubated with supernatant 1 hour prior to virus infection was significantly lower than that in the positive control group. However, the intracellular viral genome copy number in the group incubated with supernatant 1 hour after virus infection showed no significant difference from the positive control group. This result is consistent with previous neutralization activity assays. These data suggest that exogenous transfection with V... H +V L The main neutralization mechanism of the supernatant is to target the early replication process of the virus, rather than to act on the invasion process after the virus is adsorbed.

[0047] To further clarify exogenous transfection of V H +V L The neutralization target of the supernatant was investigated by qPCR at 4°C to detect the effect of the supernatant on the PPRV adsorption process. Figure 7 As shown, compared with the positive control group, the number of viral RNA copies adsorbed in cells was significantly lower in the group incubated with supernatant 1 hour before virus infection and in the group incubated with supernatant and virus co-infected (p<0.001). However, there was no statistically significant difference between the group incubated with supernatant 1 hour after virus infection and the positive control group. These results clearly confirm that exogenous transfection with V... H +V L The supernatant can exert neutralizing activity by inhibiting the adsorption process of PPRV virus to Vero-SN cells.

[0048] When preparing monoclonal antibodies using hybridoma technology, the first step is to obtain and analyze the rearranged, specific heavy and light chain variable region gene sequences. These sequences are not only the molecular basis for understanding antibody binding specificity but also the starting point and core target for genetic engineering modifications such as antibody humanization and affinity maturation. However, to achieve efficient and stable expression in non-lymphocyte cells such as CHO or HEK293 cells, the variable region sequences alone are far from sufficient. They must be combined with constant region sequences to construct a complete immunoglobulin genome sequence containing the promoter, signal peptide sequence, and the complete coding region. This complete sequence ensures that the antibody molecule can complete correct transcription, translation, folding, and secretion within the host cell, ultimately forming a biologically functional complete antibody molecule.

[0049] This invention employs monoclonal antibody hybridoma sequencing technology to extract total RNA from hybridoma cell lines 1D4-4D9. Using this mRNA as a template, cDNA corresponding to the antibody gene is synthesized through reverse transcription. Further, specific PCR amplification is used to obtain gene fragments of the variable regions of the antibody heavy and light chains. Cloning and sequencing analysis successfully resolved the nucleotide sequences of the variable regions of the heavy and light chains of the monoclonal antibody targeting the PPRV HN protein with neutralizing antibody activity.

[0050] It should be noted that when numerical ranges are involved in this invention, it should be understood that both endpoints of each numerical range and any value between the two endpoints can be selected. Since the steps and methods used are the same as in the embodiments, preferred embodiments are described in this invention to avoid redundancy. Although preferred embodiments of this invention have been described, those skilled in the art, once they understand the inventive concept of this invention, can make other changes and modifications to these embodiments, and all such changes and modifications fall within the scope of this invention.

[0051] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. If such modifications and variations fall within the scope of equivalents of this invention, then this invention also intends to include these modifications and variations.

Claims

1. A monoclonal antibody targeting PPRV HN, characterized in that, Composed of light chains and heavy chains: The nucleotide sequence of the light chain is shown in SEQ ID NO.1; the amino acid sequence of its variable region CDR1 is QSIVHSSGNTY; the amino acid sequence of CDR2 is KVS; and the amino acid sequence of CDR3 is FQGSHVPYT. The nucleotide sequence of the heavy chain is shown in SEQ ID NO.2; the amino acid sequence of its variable region CDR1 is GFNIKDYY; the amino acid sequence of CDR2 is IDPENGYT; and the amino acid sequence of CDR3 is AELGRGSY.

2. The application of the monoclonal antibody targeting PPRV HN according to claim 1 in the preparation of antibody products, characterized in that, The antibody product is a vaccine.

3. The application of the monoclonal antibody targeting PPRV HN according to claim 2 in the preparation of antibody products, characterized in that, The vaccine is obtained by combining monoclonal antibodies with adjuvants.

4. The application of the monoclonal antibody targeting PPRV HN according to claim 3 in the preparation of antibody products, characterized in that, The adjuvant includes aluminum salt adjuvants, emulsion adjuvants, TLR agonists, STING agonists, saponin adjuvants, nanoparticle adjuvants, or purified water.

5. The application of the monoclonal antibody targeting PPRV HN according to claim 4 in the preparation of antibody products, characterized in that, The aluminum salt adjuvant comprises aluminum hydroxide, aluminum phosphate, and potassium aluminum sulfate.

6. The application of the monoclonal antibody targeting PPRV HN according to claim 4 in the preparation of antibody products, characterized in that, When the adjuvant is water, the concentration of the monoclonal antibody is 7.5 mg / mL.

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