A PRRSV N protein monoclonal antibody, its preparation method and application

Abstract

The application discloses a PRRSV N protein monoclonal antibody and a preparation method and application thereof, and belongs to the technical field of biological materials, and solves the problem that the stability and specificity of the screening result of the PRRSV monoclonal antibody in the prior art are not ideal. The method comprises the following steps: constructing a gene of PRRSV N protein into a plasmid, then performing expression and purification, and obtaining a recombinant N protein; mixing the recombinant N protein, a Modifer reagent, a Quencher reagent and a DR-B reagent, centrifuging to obtain supernatant, resuspending the supernatant in an SB reagent, and separating biotinylated N protein through membrane separation; screening positive B cells combined with the biotinylated N protein by using a Hypercell high-throughput single-cell screening platform, and obtaining a monoclonal antibody. The single B cell screening technology is used to prepare the monoclonal antibody for the PRRSV N protein, and lays a foundation for diagnosis of the PRRSV and related research on the function of the N protein.

Description

Technical Field

[0001] This invention relates to the field of biomaterials technology, and more specifically to a PRRSV N protein monoclonal antibody, its preparation method, and its application. Background Technology

[0002] Porcine reproductive and respiratory syndrome (PRRS) primarily infects pregnant sows, clinically manifesting as lethargy. Abortion rates in infected sows reach 30%, and in severe cases, stillbirths can occur. Finishing pigs primarily exhibit respiratory symptoms after infection, leading to secondary infections. Infected piglets mainly develop respiratory lesions, with a mortality rate reaching 50%. The Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) has undergone numerous gene substitutions, insertions, and deletions during the evolution of Chinese pig populations. According to the latest provisions of the "Animal Epidemic Prevention Law of the People's Republic of China (2021 Revision)," this disease was downgraded from a Class I animal disease to a Class II disease in 2023. PRRSV exhibits high variability, and its immune escape mechanism is extremely complex, posing significant challenges to its control and related vaccine development. Therefore, there is an urgent need to develop highly efficient and stable diagnostic reagents.

[0003] PRRSV belongs to the family Arteritisviridae and the genus β-Arteritis, with a 15 kb single-stranded positive-sense linear RNA genome. The viral nucleocapsid protein (N protein), encoded by the ORF7 gene, is the most evolutionarily conserved component of the structural proteins, containing a core antigenic epitope cluster in its C-terminal α-helical domain. The N protein exhibits high conservation and contains the most antigenic determinants, demonstrating good immunogenicity in pigs and mice. In the early stages of PRRSV infection, intracellular N protein concentration is high, serving as a serological marker for early infection. Therefore, the N protein is an important target for the immunological diagnosis of PRRSV and plays a significant role in research on the pathogenesis of PRRSV. The N protein interferes with TRIM25-mediated RIG-I ubiquitination, and PRRSV N suppresses the host's innate immune response.

[0004] Monoclonal antibodies (mAbs) possess high specificity and can accurately recognize epitopes of specific antigens, facilitating the development of epitope-based subunit vaccines and related serological detection methods. Traditional hybridoma cell technology is limited by low throughput and long cycle times, while phage display technology, although expanding the screening capacity, suffers from high false-positive rates due to antibody heavy / light chain mismatches. Monoclonal antibodies are produced by the body differentiating a large number of B cells specifically targeting different antigens before stimulation. Monoclonal antibodies are produced from a single B lymphocyte clone and are highly homogeneous. Currently, monoclonal antibody preparation techniques include cell fusion technology, but its disadvantages include a small and homogeneous number of antibodies screened and a long screening cycle. Phage display technology has the disadvantages of being labor-intensive, requiring library construction, having limited library capacity, and a complex screening process. Summary of the Invention

[0005] This invention provides a PRRSV N protein monoclonal antibody, its preparation method, and its application, in order to solve the problem that the stability and specificity of PRRSV monoclonal antibody screening results are not ideal in the prior art.

[0006] In a first aspect, the present invention provides a PRRSV N protein monoclonal antibody, wherein the monoclonal antibody is a 253, 295, 365, 570, or 820 monoclonal antibody; wherein the heavy chain variable region nucleotide sequence of the 253 monoclonal antibody is shown in SEQ ID NO.1~SEQ ID NO.3, and the light chain variable region nucleotide sequence of the 253 monoclonal antibody is shown in SEQ ID NO.4~SEQ ID NO.6; the heavy chain variable region nucleotide sequence of the 295 monoclonal antibody is shown in SEQ ID NO.7~SEQ ID NO.9, and the light chain variable region nucleotide sequence of the 295 monoclonal antibody is shown in SEQ ID NO.10~SEQ ID NO.12; the heavy chain variable region nucleotide sequence of the 365 monoclonal antibody is shown in SEQ ID NO.13~SEQ ID NO.15, and the light chain variable region nucleotide sequence of the 365 monoclonal antibody is shown in SEQ ID NO.16~SEQ ID NO.18; and the heavy chain variable region nucleotide sequence of the 570 monoclonal antibody is shown in SEQ ID NO.19~SEQ ID NO.12. As shown in NO.21, the nucleotide sequence of the light chain variable region of the 570 monoclonal antibody is shown in SEQ ID NO.22~SEQ ID NO.24; the nucleotide sequence of the heavy chain variable region of the 820 monoclonal antibody is shown in SEQ ID NO.25~SEQ ID NO.27; and the nucleotide sequence of the light chain variable region of the 820 monoclonal antibody is shown in SEQ ID NO.28~SEQ ID NO.30.

[0007] The amino acid sequences corresponding to the proteins translated from SEQ ID NO.1 to SEQ ID NO.30 are shown in SEQ ID NO.31 to SEQ ID NO.60.

[0008] The PRRSV N protein monoclonal antibody as described above, wherein the nucleotide sequence of the heavy chain of the 253 monoclonal antibody is shown in SEQ ID NO. 61, and the nucleotide sequence of the light chain of the 253 monoclonal antibody is shown in SEQ ID NO. 62;

[0009] The nucleotide sequence of the heavy chain of the 295 monoclonal antibody is shown in SEQ ID NO. 63, and the nucleotide sequence of the light chain of the 295 monoclonal antibody is shown in SEQ ID NO. 64.

[0010] The nucleotide sequence of the heavy chain of the 365 monoclonal antibody is shown in SEQ ID NO.65, and the nucleotide sequence of the light chain of the 365 monoclonal antibody is shown in SEQ ID NO.66.

[0011] The nucleotide sequence of the heavy chain of the 570 monoclonal antibody is shown in SEQ ID NO.67, and the nucleotide sequence of the light chain of the 570 monoclonal antibody is shown in SEQ ID NO.68.

[0012] The nucleotide sequence of the heavy chain of the 820 monoclonal antibody is shown in SEQ ID NO. 69, and the nucleotide sequence of the light chain of the 820 monoclonal antibody is shown in SEQ ID NO. 70.

[0013] Secondly, the present invention provides a method for preparing the PRRSV N protein monoclonal antibody, comprising the following steps: constructing the PRRSV N protein gene into a plasmid, expressing and purifying it to obtain recombinant N protein; mixing the recombinant N protein, Modifer reagent, Quencher reagent and DR-B reagent in a volume ratio of 10:1:1:50, centrifuging and resuspending the supernatant in SB reagent, separating the biotinylated N protein through a membrane; and using the Hypercell high-throughput single-cell screening platform to screen positive B cells that bind to the biotinylated N protein to obtain the monoclonal antibody.

[0014] The preparation method described above, wherein the gene of PRRSV N protein is constructed into a plasmid, includes the following steps: adding a sumo tag to the 5′ end of the PRRSV N gene sequence and adding a TAA stop codon to the 3′ end to construct the recombinant plasmid pET-28a(+)-sumo-N.

[0015] The preparation method described above, wherein the expression conditions are as follows: the constructed recombinant plasmid is transformed into E. coli BL21 competent cells and cultured until OD600. 600 When the concentration of α=0.7, IPTG with a final concentration of 0.8 mmol / L was added at 25℃ and cultured for 20 h.

[0016] The preparation method described above, wherein the purification method is as follows: purification is performed using a nickel column, washing of impurities with 150 mmol / L imidazole, and elution of the target protein with 400 mmol / L imidazole.

[0017] The preparation method described above, wherein the screening of positive B cells that bind to biotinylated N protein using the Hypercell high-throughput single-cell screening platform includes the following steps: enriching CD138 cells with Releasable Rapid Spheres magnetic beads, screening positive cells using the Hypercell high-throughput single-cell screening platform, performing 10x single-cell sequencing, and using the AbFinder platform to perform a comprehensive scoring of the sequences based on subtype, heavy chain peak intensity, and heavy chain molecular tag, thereby screening out sequences that are mouse subtype IgG1 and have a comprehensive score greater than 6.

[0018] Thirdly, the present invention provides the application of the PRRSV N protein monoclonal antibody described above or the PRRSV N protein monoclonal antibody prepared by the preparation method described above in the preparation of diagnostic reagents for porcine reproductive and respiratory syndrome virus.

[0019] As described above, the reagent is used to diagnose porcine reproductive and respiratory syndrome.

[0020] The N protein is a crucial structural protein for assembling PRRSV viral particles. Existing techniques have shown that PRRSV N protein expressed as inclusion bodies using an *E. coli* prokaryotic expression system. Recombinant proteins expressed using this system are often inactive inclusion bodies, and their spatial conformation is easily disrupted during renaturation, leading to reduced or even lost activity. Adding a fusion tag during plasmid construction promotes soluble expression of the recombinant protein. This invention, by optimizing the codons and adding a sumo tag and a TAA stop codon to the 5′ end of the N gene sequence, successfully constructed the recombinant plasmid pET-28a(+)-sumo-N. Expression was performed using SUMO fusion, and the protein was expressed in the supernatant.

[0021] This invention focuses on the N protein of PRRSV, and successfully constructed an engineered bacterium with pET-28a(+)-sumo-N recombinant protein. Soluble target protein was obtained through induced expression. The recombinant antibody gene sequence was obtained through ELISA verification, single B cell screening, and sequencing. Eukaryotic cell transfection expression was performed, and five monoclonal antibodies were screened out. This provides good technical support for further exploring the structural characteristics and function of PRRSV N protein, as well as for the pathogenesis and treatment of PRRSV, and lays a solid foundation for establishing a rapid PRRSV N protein detection kit.

[0022] Single-cell sorting technology is a key strategy for overcoming the bottlenecks of traditional technologies. Single-cell screening technology has advantages such as high specificity, high yield, and good uniformity. This invention uses single-cell screening technology to prepare monoclonal antibodies against the PRRSV N protein, laying the foundation for the diagnosis of PRRSV and related research on the function of the N protein.

[0023] The sequences screened by single B-cell screening technology have naturally paired light and heavy chains, and the prepared monoclonal antibodies exhibit good stability and high specificity. Existing technologies, such as fluorescence-activated flow cytometry (FACS), for sorting B cells suffer from low separation efficiency, limited cell types, limited sorting speed, and susceptibility to contamination. This invention utilizes the Hypercell high-throughput single-cell screening platform, capable of screening 200,000 cells at a time with visualized screening, effectively avoiding cell contamination. The Hypercell platform comprehensively scores the screened sequences based on heavy chain maxima, heavy chain molecular tags, and high-frequency mutations in heavy chain amino acids, further ensuring the integrity of the variable regions of the antibody's light and heavy chains, as well as the specificity and stability of the monoclonal antibody. The monoclonal antibody against PRRSV N protein prepared by this invention has a titer as high as 1:409600, which is higher than that of similar antibodies prepared in existing technologies. Furthermore, the 570 monoclonal antibody prepared by this invention has an affinity KD of 1.00 × 10⁻⁶. -12 M has higher affinity compared to existing monoclonal antibodies. Attached Figure Description

[0024] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0025] Figure 1The diagram shows the construction and identification results of the recombinant plasmid pET-28a(+)-sumo-N provided in this embodiment of the invention. In the diagram, A is the pET28a-sumo-N plasmid map and B is the identification of the recombinant plasmid pET28a-sumo-N (M: DL 2000 Marker; I: pET28a-sumo-N PCR product).

[0026] Figure 2 The recombinant protein expression and purification results provided in the embodiments of the present invention are as follows: A is the N protein expression analysis (M: 14.4-116 kDa protein marker; 1: pET-28a(+)-sumo-N containing bacterial cells before induction; 2: pET-28a(+)-sumo-N containing bacterial cells after induction; 3: pET-28a(+)-sumo-N containing bacterial cells lysed supernatant; 4: pET-28a(+)-sumo-N containing bacterial cells lysed precipitate); B is the N protein purification (M: 14.4-116 kDa protein marker; 1: pET-28a(+)-sumo-N containing bacterial cells lysed supernatant; 2: efflux; 3-6: 150 mmol / L imidazole washing; 7: 400 mmol / L imidazole elution); C is the Western blot identification of the N protein (M: 10-180 kDa protein marker). KDa protein marker; 1: pET-28a-sumo empty vector bacterial protein; 2: N protein).

[0027] Figure 3 The results of antibody titers in mouse tail blood serum provided in the embodiments of the present invention.

[0028] Figure 4 The results of single B cell screening provided in this embodiment of the invention are as follows: A is the detection of the binding effect of serum with biotinylated N protein (1:100); B is the detection of the binding effect of serum with biotinylated N protein (1:1000); C is the negative control; D, E, and F are the detection of the binding effect of CD138-enriched spleen cells with biotinylated N protein: observation of cell aggregation under different fields of view.

[0029] Figure 5 The results of screening and purification of recombinant expressed antibodies provided in the embodiments of the present invention are shown in the figure. A is the cell supernatant titer; B is the purification of monoclonal antibodies (M: 14.4-116 kDa protein marker; 1: 820; 2: 570; 3: 365; 4: 295; 5: 253).

[0030] Figure 6 The titer of the monoclonal antibody against N protein provided in the embodiments of the present invention.

[0031] Figure 7The results of Western blot identification of five monoclonal antibodies against the N protein provided in this embodiment of the invention are shown below. A is the primary antibody using monoclonal antibody 253, B is the primary antibody using monoclonal antibody 295, C is the primary antibody using monoclonal antibody 360, D is the primary antibody using monoclonal antibody 570, and E is the primary antibody using monoclonal antibody 820; M: 10-180 kDa protein marker; 1: PRRSV N protein; 2: pET-28a-sumo empty vector bacterial protein.

[0032] Figure 8 The results show the affinity of the monoclonal antibody against the N protein provided in the embodiments of the present invention.

[0033] Figure 9 The figure shows the nucleotide sequence of the variable region of the heavy chain of the 253 monoclonal antibody. The yellow highlighted part is the complementarity-determining region (CDR) of the heavy chain, and the unhighlighted part is the frame region (FR).

[0034] Figure 10 The figure shows the nucleotide sequence of the variable region of the light chain of the 253 monoclonal antibody. The yellow highlighted part is the complementarity-determining region (CDR) of the light chain, and the unhighlighted part is the frame region (FR).

[0035] Figure 11 The figure shows the nucleotide sequence of the heavy chain variable region of the 295 monoclonal antibody. The yellow highlighted part is the complementarity-determining region (CDR) of the heavy chain, and the unhighlighted part is the frame region (FR).

[0036] Figure 12 The figure shows the nucleotide sequence of the variable region of the light chain of the 295 monoclonal antibody. The yellow highlighted part is the complementarity-determining region (CDR) of the light chain, and the unhighlighted part is the frame region (FR).

[0037] Figure 13 The figure shows the nucleotide sequence of the variable region of the heavy chain of the 365 monoclonal antibody. The yellow highlighted part is the complementarity-determining region (CDR) of the heavy chain, and the unhighlighted part is the frame region (FR).

[0038] Figure 14 The figure shows the nucleotide sequence of the variable region of the light chain of the 365 monoclonal antibody. The yellow highlighted part is the complementarity-determining region (CDR) of the light chain, and the unhighlighted part is the frame region (FR).

[0039] Figure 15 The figure shows the nucleotide sequence of the variable region of the heavy chain of the 570 monoclonal antibody. The yellow highlighted part is the complementarity-determining region (CDR) of the heavy chain, and the unhighlighted part is the frame region (FR).

[0040] Figure 16The figure shows the nucleotide sequence of the variable region of the light chain of the 570 monoclonal antibody. The yellow highlighted part is the complementarity-determining region (CDR) of the light chain, and the unhighlighted part is the frame region (FR).

[0041] Figure 17 The figure shows the nucleotide sequence of the variable region of the heavy chain of the 820 monoclonal antibody. The yellow highlighted part is the complementarity-determining region (CDR) of the heavy chain, and the unhighlighted part is the frame region (FR).

[0042] Figure 18 The figure shows the nucleotide sequence of the variable region of the light chain of the 820 monoclonal antibody. The yellow highlighted part is the complementarity-determining region (CDR) of the light chain, and the unhighlighted part is the frame region (FR). Detailed Implementation

[0043] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions in the embodiments of this invention will be clearly and completely described below in conjunction with the embodiments of this invention. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention. Where specific techniques or conditions are not specified in the embodiments, they shall be performed in accordance with the techniques or conditions described in the literature in this art or in accordance with the product instructions. Reagents or instruments used without specified manufacturers are all conventional products that can be obtained commercially.

[0044] Those skilled in the art should understand that, in the following description of the embodiments of the present invention, the sequence of numbers does not imply the order of execution. Some or all steps may be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present invention.

[0045] Those skilled in the art will understand that the numerical ranges in the embodiments of the present invention should be understood to specifically disclose each intermediate value between the upper and lower limits of the range. Each smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this application. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0046] Unless otherwise stated, the technical / scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains. While only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0047] To address the issue of insufficient stability and specificity in the screening results of PRRSV monoclonal antibodies in existing technologies, this embodiment provides a PRRSV N protein monoclonal antibody, its preparation method, and its application.

[0048] The present invention provides a PRRSV N protein monoclonal antibody in a first aspect, wherein the monoclonal antibody is a 253, 295, 365, 570, or 820 monoclonal antibody; wherein the heavy chain variable region nucleotide sequence (CDR1-CDR3) of the 253 monoclonal antibody is shown in SEQ ID NO.1~SEQ ID NO.3, and the light chain variable region nucleotide sequence (CDR1-CDR3) of the 253 monoclonal antibody is shown in SEQ ID NO.4~SEQ ID NO.6; the heavy chain variable region nucleotide sequence (CDR1-CDR3) of the 295 monoclonal antibody is shown in SEQ ID NO.7~SEQ ID NO.9, and the light chain variable region nucleotide sequence (CDR1-CDR3) of the 295 monoclonal antibody is shown in SEQ ID NO.10~SEQ ID NO.12; and the heavy chain variable region nucleotide sequence (CDR1-CDR3) of the 365 monoclonal antibody is shown in SEQ ID NO.13~SEQ ID NO. As shown in NO.15, the light chain variable region nucleotide sequences (CDR1-CDR3) of the 365 monoclonal antibody are shown in SEQ ID NO.16~SEQ ID NO.18; the heavy chain variable region nucleotide sequences (CDR1-CDR3) of the 570 monoclonal antibody are shown in SEQ ID NO.19~SEQ ID NO.21; the light chain variable region nucleotide sequences (CDR1-CDR3) of the 570 monoclonal antibody are shown in SEQ ID NO.22~SEQ ID NO.24; the heavy chain variable region nucleotide sequences (CDR1-CDR3) of the 820 monoclonal antibody are shown in SEQ ID NO.25~SEQ ID NO.27; and the light chain variable region nucleotide sequences (CDR1-CDR3) of the 820 monoclonal antibody are shown in SEQ ID NO.28~SEQ ID NO.30.

[0049] It is understood that monoclonal antibodies consist of a heavy chain and a light chain. The heavy chain comprises a heavy chain variable region and a heavy chain constant region, and the light chain comprises a light chain variable region and a light chain constant region. Both the heavy chain variable region and the light chain variable region consist of a complementarity-determining region (CDR) and a framework region (FR).

[0050] In this invention, the nucleotide sequence of the heavy chain variable region of the 253 monoclonal antibody, as shown in SEQ ID NO.1 to SEQ ID NO.3, refers to the complementarity-determining region (CDR) of the heavy chain variable region of the 253 monoclonal antibody being composed of CDR1 shown in SEQ ID NO.1, CDR2 shown in SEQ ID NO.2, and CDR3 shown in SEQ ID NO.3. Similarly, the nucleotide sequence of the light chain variable region of the 253 monoclonal antibody, as shown in SEQ ID NO.4 to SEQ ID NO.6, refers to the complementarity-determining region of the light chain variable region of the 253 monoclonal antibody being composed of CDR1 shown in SEQ ID NO.4, CDR2 shown in SEQ ID NO.5, and CDR3 shown in SEQ ID NO.6.

[0051] In this invention, the nucleotide sequence of the heavy chain variable region of the 295 monoclonal antibody, as shown in SEQ ID NO.7~SEQ ID NO.9, refers to the complementarity-determining region (CDR) of the heavy chain variable region of the 295 monoclonal antibody being composed of CDR1 shown in SEQ ID NO.7, CDR2 shown in SEQ ID NO.8, and CDR3 shown in SEQ ID NO.9. Similarly, the nucleotide sequence of the light chain variable region of the 295 monoclonal antibody, as shown in SEQ ID NO.10~SEQ ID NO.12, refers to the complementarity-determining region of the light chain variable region of the 295 monoclonal antibody being composed of CDR1 shown in SEQ ID NO.10, CDR2 shown in SEQ ID NO.11, and CDR3 shown in SEQ ID NO.12.

[0052] In this invention, the nucleotide sequence of the heavy chain variable region of the 365 monoclonal antibody, as shown in SEQ ID NO.13~SEQ ID NO.15, refers to the complementarity-determining region (CDR) of the heavy chain variable region of the 365 monoclonal antibody being composed of CDR1 shown in SEQ ID NO.13, CDR2 shown in SEQ ID NO.14, and CDR3 shown in SEQ ID NO.15. The nucleotide sequence of the light chain variable region of the 365 monoclonal antibody, as shown in SEQ ID NO.16~SEQ ID NO.18, refers to the complementarity-determining region of the light chain variable region of the 365 monoclonal antibody being composed of CDR1 shown in SEQ ID NO.16, CDR2 shown in SEQ ID NO.17, and CDR3 shown in SEQ ID NO.18.

[0053] In this invention, the nucleotide sequence of the heavy chain variable region of the 570 monoclonal antibody, as shown in SEQ ID NO.19~SEQ ID NO.21, refers to the complementarity-determining region (CDR) of the heavy chain variable region of the 570 monoclonal antibody being composed of CDR1 shown in SEQ ID NO.19, CDR2 shown in SEQ ID NO.20, and CDR3 shown in SEQ ID NO.21. Similarly, the nucleotide sequence of the light chain variable region of the 570 monoclonal antibody, as shown in SEQ ID NO.22~SEQ ID NO.24, refers to the complementarity-determining region of the light chain variable region of the 570 monoclonal antibody being composed of CDR1 shown in SEQ ID NO.22, CDR2 shown in SEQ ID NO.23, and CDR3 shown in SEQ ID NO.24.

[0054] In this invention, the nucleotide sequence of the heavy chain variable region of the 820 monoclonal antibody, as shown in SEQ ID NO.25~SEQ ID NO.27, refers to the complementarity-determining region (CDR) of the heavy chain variable region of the 820 monoclonal antibody being composed of CDR1 shown in SEQ ID NO.25, CDR2 shown in SEQ ID NO.26, and CDR3 shown in SEQ ID NO.27. Similarly, the nucleotide sequence of the light chain variable region of the 820 monoclonal antibody, as shown in SEQ ID NO.28~SEQ ID NO.30, refers to the complementarity-determining region of the light chain variable region of the 820 monoclonal antibody being composed of CDR1 shown in SEQ ID NO.28, CDR2 shown in SEQ ID NO.29, and CDR3 shown in SEQ ID NO.30.

[0055] In this invention, the amino acid sequences corresponding to the proteins translated from SEQ ID NO.1 to SEQ ID NO.30 are shown in SEQ ID NO.31 to SEQ ID NO.60.

[0056] In some embodiments, the nucleotide sequence of the heavy chain of the 253 monoclonal antibody is shown in SEQ ID NO. 61, and the nucleotide sequence of the light chain of the 253 monoclonal antibody is shown in SEQ ID NO. 62; the nucleotide sequence of the heavy chain of the 295 monoclonal antibody is shown in SEQ ID NO. 63, and the nucleotide sequence of the light chain of the 295 monoclonal antibody is shown in SEQ ID NO. 64; the nucleotide sequence of the heavy chain of the 365 monoclonal antibody is shown in SEQ ID NO. 65, and the nucleotide sequence of the light chain of the 365 monoclonal antibody is shown in SEQ ID NO. 66; the nucleotide sequence of the heavy chain of the 570 monoclonal antibody is shown in SEQ ID NO. 67, and the nucleotide sequence of the light chain of the 570 monoclonal antibody is shown in SEQ ID NO. 68; the nucleotide sequence of the heavy chain of the 820 monoclonal antibody is shown in SEQ ID NO. 69, and the nucleotide sequence of the light chain of the 820 monoclonal antibody is shown in SEQ ID NO. 70.

[0057] Secondly, the present invention provides a method for preparing the PRRSV N protein monoclonal antibody, comprising the following steps: constructing the PRRSV N protein gene into a plasmid, expressing and purifying it to obtain recombinant N protein; mixing the recombinant N protein, Modifer reagent, Quencher reagent and DR-B reagent in a volume ratio of 10:1:1:50, centrifuging and resuspending the supernatant in SB reagent, separating the biotinylated N protein through a membrane; and using the Hypercell high-throughput single-cell screening platform to screen positive B cells that bind to the biotinylated N protein to obtain the monoclonal antibody.

[0058] In some embodiments, the process of constructing the PRRSV N protein gene into a plasmid includes the steps of: adding a sumo tag to the 5′ end of the PRRSV N gene sequence and adding a TAA stop codon to the 3′ end to construct the recombinant plasmid pET-28a(+)-sumo-N.

[0059] In some embodiments, the expression conditions are as follows: the constructed recombinant plasmid is transformed into E. coli BL21 competent cells and cultured until OD2000. 600 When the concentration of α=0.7, IPTG with a final concentration of 0.8 mmol / L was added at 25℃ and cultured for 20 h.

[0060] In some embodiments, the purification method is as follows: purification using a nickel column, washing with 150 mmol / L imidazole to remove impurities, and eluting the target protein with 400 mmol / L imidazole.

[0061] In some embodiments, the screening of positive B cells that bind to biotinylated N protein using the Hypercell high-throughput single-cell screening platform includes the following steps: enriching CD138 cells with Releasable Rapid Spheres magnetic beads, screening positive cells using the Hypercell high-throughput single-cell screening platform, performing 10x single-cell sequencing, and using the AbFinder platform to perform a comprehensive scoring of the sequences based on subtype, heavy chain peak intensity, and heavy chain molecular tag, thereby screening out sequences that are mouse subtype IgG1 and have a comprehensive score greater than 6.

[0062] Thirdly, the present invention provides the application of the PRRSV N protein monoclonal antibody described above or the PRRSV N protein monoclonal antibody prepared by the preparation method described above in the preparation of diagnostic reagents for porcine reproductive and respiratory syndrome virus.

[0063] In some embodiments, the reagent is used to diagnose porcine reproductive and respiratory syndrome.

[0064] This invention focuses on constructing a prokaryotic expression vector for PRRSV N protein to achieve the expression and purification of the N protein, and preparing monoclonal antibodies against the N protein, while also conducting in-depth research on its characteristics. The results show that the recombinant expression plasmid pET28a-sumo-N was successfully constructed. The N protein was induced to express and purified in *E. coli*, and used to immunize BALB / c mice. Single B cell screening technology was used to screen for specific single B cells against the N protein. The antibody gene sequence was obtained by single-cell sequencing, and a eukaryotic expression plasmid was constructed. Transient transfection of CHO cells to express the monoclonal antibody resulted in the selection of five monoclonal antibodies of the IgG1 subtype (253, 295, 365, 570, and 820). All five monoclonal antibodies specifically reacted with the PRRSV N protein, with antibody titers greater than 10⁵. Among them, antibody 570 exhibited a high affinity dissociation constant. The epitopes of the monoclonal antibodies were determined using biomembrane interference technology, showing that the epitopes of the five monoclonal antibodies were different. In conclusion, this study successfully prepared a specific monoclonal antibody against PRRSV N protein using single B cell technology, laying an experimental foundation for studying the structural characteristics and functional role of PRRSV N protein and constructing an efficient and rapid PRRSV antibody detection method.

[0065] The main reagents used in this invention embodiment are as follows: recombinant plasmid pET-28a(+)-sumo-N was synthesized by Suzhou Junji Gene Technology Co., Ltd.; CHO Chinese hamster ovary cells and pET-28a-sumo empty vector bacterial protein were preserved by the Antibody Collaborative Innovation Center Laboratory of Jilin Medical College; Quick Antibody adjuvant was purchased from Bio-Long Biotechnology Co., Ltd.; Releasable Rapid Spheres magnetic beads, Modifer reagent, Detection Reagent A (DR-A) reagent, Detection Reagent B (DR-B) reagent, Storage Buffer (SB) reagent, Encapsulation Reagent (ER) reagent, and Quencher reagent were purchased from Shanghai Taoxuan Scientific Instruments Co., Ltd.; rProtein G magnetic beads were purchased from Changzhou Tiandi Renhe Biotechnology Co., Ltd.; nickel column was purchased from Siterofan Biotechnology Co., Ltd.; DMEM culture medium, ExpiCHO™ enhancer, and ExpiCHO™ excipient were purchased from Thermo Fisher Scientific Co., Ltd.; IPTG was purchased from Yisheng Biotechnology (Shanghai) Co., Ltd.; 14.4~116 KDa protein markers were purchased from Beyotime Biotechnology Co., Ltd.; 10~180 KDa protein markers were purchased from Beijing Biotechnology Co., Ltd.; and goat anti-mouse IgG was purchased from Icoin (Wuhan) Biotechnology Co., Ltd.

[0066] The main instruments used in this invention embodiment are as follows: AKTA Purifier protein purification system purchased from Sitop Biotechnology Co., Ltd.; Hypercell high-throughput single-cell screening platform purchased from Shanghai Taoxuan Scientific Instruments Co., Ltd.; GatorPrime instrument purchased from Gator Bio; chemiluminescence gel imaging system purchased from BIO-RAD, USA; and PCR instrument purchased from Eppendorf, USA.

[0067] The experimental animals used in this invention were: SPF-grade BALB / c mice, female, 5 weeks old, weighing (15±5) g, purchased from Liaoning Changsheng Biotechnology Co., Ltd. (Production Certificate No. SCXK(Liaoning)2020-0001). The animal experiments were approved by the Medical Ethics Committee of Jilin Medical College (Approval No.: 2023-KJT-041).

[0068] The technical solution of the present invention will be further described below with reference to specific embodiments.

[0069] Example 1

[0070] This embodiment provides an experiment for preparing PRRSV N protein monoclonal antibodies based on single B cell screening technology.

[0071] 1. Construction and identification of prokaryotic expression vectors:

[0072] The PRRSV N gene sequence (AAR19407.1) was obtained from the NCBI database. A TAA stop codon was added to the 3′ end of the PRRSV N gene. The pET28a(+)-sumo vector and the PRRSV N gene were then cloning the recombinant plasmid pET28a(+)-sumo-N using seamless cloning technology. PCR identification was performed using upstream primer T7-F (5'-TAATACGACTCACTATAGGG-3′ (SEQ ID NO. 81)) and downstream primer T7-R (5'-TGCTAGTTATTGCTCAGCGG-3′ (SEQ ID NO. 82)). The primers were synthesized by Jilin Kumei Biotechnology Co., Ltd. The reaction system consisted of 12.5 μL of 2×Taq Mix, 11.3 μL of ddH2O, 0.5 μL each of the upstream and downstream primers, and 0.2 μL of pET28a(+)-sumo-N. The reaction conditions were: 94℃ for 5 min, for 30 cycles, 94℃ for 30 s, 52℃ for 30 s, 72℃ for 1 min, and then extended at 72℃ for 10 min.

[0073] 2. Expression and purification of recombinant proteins:

[0074] The obtained recombinant plasmid pET-28a(+)-sumo-N was transformed into Escherichia coli BL21(DE3) competent cells, plated on kanamycin LB solid culture dishes, and single colonies were picked and inoculated into kanamycin liquid LB medium. The cells were then cultured at 37°C until OD200. 600 When the concentration of the protein was 0.7, IPTG was added at 25°C to a final concentration of 0.8 mmol / L. After culturing for 20 h, the bacterial pellet was collected by centrifugation, resuspended in PBS, and sonicated. The supernatant of the bacterial lysis was collected and purified using a nickel column. Impurities were washed with 150 mmol / L imidazole and eluted with 400 mmol / L imidazole to obtain the target protein (recombinant N protein), which was identified by 10% SDS-PAGE electrophoresis and Western blot.

[0075] 3. Animal immunization and tail blood serum titer determination:

[0076] Four immunized mice (labeled as mice 1, 2, 3, and 4) served as the experimental group, and one mouse (labeled as a negative control) served as the negative control. In the experimental group, recombinant N protein and Quick Antibody adjuvant were mixed at a 1:1 volume ratio and injected intramuscularly into the leg of each mouse at a dose of 40 μg / mouse. Each immunization was administered 15 days apart, for a total of three injections. Blood was collected from the tail vein of each mouse under aseptic conditions, and the immune serum titer was measured using ELISA. In the negative control group, physiological saline was injected intramuscularly into the leg of each mouse at a dose of 40 μg / mouse. Each immunization was administered 15 days apart, for a total of three injections. Blood was collected from the tail vein of each mouse under aseptic conditions, and the immune serum titer was measured using ELISA. Three days prior to the single B-cell screening technique, a booster immunization was performed by intraperitoneal injection of the corresponding antigen (100 μg / mouse).

[0077] 4. Single B cell screening:

[0078] 60 μg recombinant N protein, 6 μL Modifer reagent, 6 μL Quencher reagent, and 300 μL DR-B reagent were mixed, centrifuged, and the supernatant was resuspended in SB reagent. The biotinylated N protein was separated by filtration through a 40 μm membrane. 22.5 μL ER reagent, 5 μL DR-A reagent, and 5 μL biotinylated N protein were added to each of three EP tubes. 10 μL of 1:100 diluted serum, 1:1000 diluted serum, and PBS (negative control) were added to each of the three EP tubes, respectively, and the mixture was incubated at 37 ℃ for 1 h to obtain three groups of test samples. 3 μL of each test sample was mixed with 7 μL ER reagent and added to a cell counting chamber to observe the binding ability of serum to biotinylated N protein. The spleen of mice (immunized mice) was aseptically removed, ground on a 70-mesh copper mesh, washed with DMEM medium, and mixed to prepare 1.5 × 10⁻⁶ splenic acid granules. 8 cells / mL of spleen cells. To 1.5 × 10 8 Spleen cells were added to 1.5 mL of PBS solution to prepare a spleen cell suspension. CD138 cells in the spleen cell suspension were enriched using Releasable Rapid Spheres magnetic beads, and the binding ability of cells (positive cells) to biotinylated N protein was observed. Positive cells (cells secreting monoclonal antibodies) were screened using the Hypercell high-throughput single-cell screening platform.

[0079] 5. Recombinant antibody expression, screening, and purification:

[0080] 10x single-cell sequencing (10x Genomics) was performed. Sequences from the selected positive cells were analyzed using the AbFinder platform (Shanghai Taoxuan Scientific Instruments Co., Ltd.) based on subtype, heavy chain peak density, and heavy chain molecular tagging. Sequences identifying the mouse subtype as IgG1 and with a comprehensive score greater than 6 were sent to Suzhou Hongxun Biotechnology Co., Ltd. for sequence synthesis, yielding monoclonal antibodies. The heavy chain variable region sequence of the monoclonal antibody was inserted into the pMT24-mCHG1-VH eukaryotic vector (which itself contains a constant region sequence) to obtain the heavy chain plasmid; the light chain variable region sequence of the monoclonal antibody was inserted into the pMT24-mCHG1-VL eukaryotic vector (which itself contains a constant region sequence) to obtain the light chain plasmid. 10 μg of light chain plasmid and 10 μg of heavy chain plasmid were transfected into 6×10⁶ cells / mL. 6 Cells / mL CHO cells were transiently transfected for 20 h, then incubated with 200 μL of LExpiCHO™ enhancer and 8 mL of ExpiCHO™ adjuvant for 7 days. Cell supernatant was collected by centrifugation and screened using indirect ELISA. Recombinant antibodies were purified using rProtein G magnetic beads and identified by 12% SDS-PAGE electrophoresis.

[0081] 6. Monoclonal antibody titer determination:

[0082] The titer of monoclonal antibodies was determined using an indirect ELISA method. The antibody was coated with 2 μg / mL N protein and blocked with 5% skim milk. The primary antibody was purified monoclonal antibody serially diluted from 1:100, and the secondary antibody was goat anti-mouse IgG diluted from 1:3500. OD 450 Reading time. P is the OD value of the positive well, N is the OD value of the negative well, and the potency is determined when P / N>2.1.

[0083] 7. Monoclonal antibody specificity assay:

[0084] Western blot was used to identify the specificity of five monoclonal antibodies. PRRSV N protein and pET-28a-sumo empty vector bacterial protein (laboratory-preserved) were transferred to PVDF membranes by SDS-PAGE electrophoresis and semi-dry method. The membranes were blocked with 5% skim milk. The primary antibody was purified monoclonal antibody diluted 1:5500, and the secondary antibody was goat anti-mouse IgG diluted 1:4500.

[0085] 8. Monoclonal antibody affinity assay:

[0086] The affinity of the N protein for the interaction with the monoclonal antibody was detected using the principle of biomembrane interference (BLI). 20 μg / mL of monoclonal antibody was immobilized on a Gator Bio instrument, then bound to PRRSV N protein (500 nM) solution, followed by dissociation in PBST to obtain the interaction kinetic curves between the monoclonal antibody and the PRRSV N protein.

[0087] 9. Monoclonal antibody epitope grouping assay:

[0088] Biolayer interferometry (BLI) was used to group the epitopes of monoclonal antibodies binding to the N protein. One antibody (10 μg / mL) was immobilized on a Gator Bio instrument and then bound to the N protein (500 nM). An irrelevant antibody (10 μg / mL) was used as a negative control to block the probe, and then another antibody (10 μg / mL) was bound to determine whether there was competition between the antibodies.

[0089] Example 2

[0090] This embodiment provides the experimental results of Embodiment 1.

[0091] 1. Identification of prokaryotic expression vectors:

[0092] like Figure 1 As shown in Figure A, the PRRSV N gene was directionally inserted into the multiple cloning site of the pET-28a(+)-sumo vector using seamless cloning technology. The recombinant plasmid pET28a(+)-sumo-N was approximately 6013 bp in size. After amplification using T7-F and T7-R primers, the results of PCR amplification of the PRRSV N gene product were detected by 1.5% agarose gel electrophoresis, as shown in Figure A. Figure 1 As shown in Figure B, the amplified target band is clearly visible at 800 bp, consistent with expectations. These experimental results demonstrate the successful construction of the recombinant plasmid pET28a(+)-sumo-N.

[0093] 2. Expression and purification of recombinant proteins:

[0094] Using SnapGene software, the molecular weight of the protein (expressed by the recombinant plasmid pET28a(+)-sumo-N) was predicted. The inserted N gene (PRRSV N gene) is 800 bp in length, encoding 266 amino acids, with a theoretical molecular weight of approximately 29.1 kDa. The theoretical molecular weight of the protein expressed by the recombinant plasmid pET28a(+)-sumo-N is 34 kDa. Figure 2As shown in Figure A, after induction with IPTG at a final concentration of 0.8 mmol / L for 20 h at 25℃, 10% SDS-PAGE analysis showed that the target protein (expressed by the recombinant plasmid pET28a(+)-sumo-N) was present at 34 kDa in the bacterial cell lysis supernatant, which highly matched the theoretical molecular weight and was expressed in a soluble manner. Figure 2 As shown in Figure B, the target protein (recombinant N protein) was obtained after purification using a nickel column. 10% SDS-PAGE gel electrophoresis showed that the purity of the recombinant N protein band reached 90%. Figure 2 As shown in C, Western blot analysis indicates that the recombinant N protein can be specifically recognized by the anti-His tag antibody, consistent with expectations. These experimental results demonstrate the successful purification of the recombinant N protein.

[0095] 3. Determination of mouse tail blood titer:

[0096] The antibody titer of mouse tail blood serum was measured using an indirect ELISA method. The results were analyzed using Origin 2024 software. Figure 3 As shown, the titer of the polyclonal antibody obtained from immunizing mice reached 1:102400.

[0097] 4. Single B cell screening:

[0098] The binding effect of mouse serum to biotinylated N protein was detected. The binding effect of 1:100 diluted mouse serum to biotinylated N protein was as follows: Figure 4 The binding effect of A, 1:1000 diluted mouse serum with biotinylated N protein is as follows: Figure 4 In the B, negative control, as shown Figure 4 C. The binding effect of CD138-enriched spleen cells on biotinylated N protein was detected as follows: Figure 4 In the D, E, and F samples, three cell clumps (cells clustered together) were observed in both serum and cell detection, indicating that both serum and cells specifically and effectively bind to the biotinylated N protein. These experimental results demonstrate the successful screening of positive cells.

[0099] 5. Recombinant antibody expression, screening, and purification:

[0100] The AbFinder platform was used to comprehensively score the sequences of positive cells based on subtype, heavy chain peak intensity, and heavy chain molecular tag. Sequences with a mouse subtype of IgG1 and a comprehensive score greater than 6 were selected, resulting in five plasmids (named 253 monoclonal antibody, 295 monoclonal antibody, 365 monoclonal antibody, 570 monoclonal antibody, and 820 monoclonal antibody). Indirect ELISA was used to screen the five cell supernatants. Figure 5In the A-cell supernatant, the titer was 1:1600. Five plasmids screened using the AbFinder platform were predicted using SnapGene software: the theoretical molecular weight of the heavy chain of all five plasmids in the pMT24-mCHG1-VH eukaryotic vector was approximately 50 kDa, and the theoretical molecular weight of the light chain of all five plasmids in the pMT24-mCHG1-VL eukaryotic vector was approximately 25 kDa. SDS-PAGE electrophoresis results are shown below. Figure 5 As shown in Figure B, the target protein appeared at 50 kDa and 25 kDa, with sizes consistent with expectations and clear heavy and light chain bands. These results indicate the successful screening and purification of five monoclonal antibodies that specifically bind to the PRRSV N protein.

[0101] The nucleotide sequences (3 sequences per plasmid) of the complementarity-determining regions (CDRs) in the heavy chain variable regions of monoclonal antibodies 253, 295, 365, 570, and 820, and the nucleotide sequences (3 sequences per plasmid) of the CDRs in the light chain variable regions of monoclonal antibodies 253, 295, 365, 570, and 820 are shown in SEQ ID NO. 1 to SEQ ID NO. 30, respectively. The amino acid sequences corresponding to the proteins translated from SEQ ID NO. 1 to SEQ ID NO. 30 are shown in SEQ ID NO. 31 to SEQ ID NO. 60, respectively. The nucleotide sequence of the heavy chain variable region of monoclonal antibody 253 is shown in... Figure 9 As shown, the nucleotide sequence of the light chain variable region of the 253 monoclonal antibody is as follows: Figure 10 As shown; the nucleotide sequence of the heavy chain variable region of the 295 monoclonal antibody is as follows. Figure 11 As shown, the nucleotide sequence of the light chain variable region of the 295 monoclonal antibody is as follows: Figure 12 As shown; the nucleotide sequence of the heavy chain variable region of the 365 monoclonal antibody is as follows. Figure 13 As shown, the nucleotide sequence of the light chain variable region of the 365 monoclonal antibody is as follows: Figure 14 As shown; the nucleotide sequence of the heavy chain variable region of the 570 monoclonal antibody is as follows. Figure 15 As shown, the nucleotide sequence of the light chain variable region of the 570 monoclonal antibody is as follows: Figure 16 As shown; the nucleotide sequence of the heavy chain variable region of the 820 monoclonal antibody is as follows. Figure 17 As shown, the nucleotide sequence of the light chain variable region of the 820 monoclonal antibody is as follows: Figure 18As shown. SEQ ID NO.5, SEQ ID NO.11, SEQ ID NO.17, SEQ ID NO.23, and SEQ ID NO.29 are sequences with fewer than 10 well-defined nucleotides: SEQ ID NO.5: TACACATCA, SEQ ID NO.11: CATGCAAAC, SEQ ID NO.17: CTTGCATCC, SEQ ID NO.23: GACACATCC, SEQ ID NO.29: GACACATCC. These sequences were intentionally skipped during sequence listing. SEQ ID NO.35, SEQ ID NO.41, SEQ ID NO.47, SEQ ID NO.53, and SEQ ID NO.59 are sequences with fewer than 4 specifically defined amino acids: SEQ ID NO.35: YTS, SEQ ID NO.41: HAN, SEQ ID NO.47: LAS, SEQ ID NO.53: DTS, SEQ ID NO.59: DTS. These sequences were intentionally skipped during sequence listing.

[0102] The nucleotide sequence of the heavy chain of the 253 monoclonal antibody is shown in SEQ ID NO. 61, and the nucleotide sequence of the light chain of the 253 monoclonal antibody is shown in SEQ ID NO. 62; the nucleotide sequence of the heavy chain of the 295 monoclonal antibody is shown in SEQ ID NO. 63, and the nucleotide sequence of the light chain of the 295 monoclonal antibody is shown in SEQ ID NO. 64; the nucleotide sequence of the heavy chain of the 365 monoclonal antibody is shown in SEQ ID NO. 65, and the nucleotide sequence of the light chain of the 365 monoclonal antibody is shown in SEQ ID NO. 66; the nucleotide sequence of the heavy chain of the 570 monoclonal antibody is shown in SEQ ID NO. 67, and the nucleotide sequence of the light chain of the 570 monoclonal antibody is shown in SEQ ID NO. 68; the nucleotide sequence of the heavy chain of the 820 monoclonal antibody is shown in SEQ ID NO. 69, and the nucleotide sequence of the light chain of the 820 monoclonal antibody is shown in SEQ ID NO. 70.

[0103] The amino acid sequence of the heavy chain of the 253 monoclonal antibody is shown in SEQ ID NO. 71, and the amino acid sequence of the light chain of the 253 monoclonal antibody is shown in SEQ ID NO. 72; the amino acid sequence of the heavy chain of the 295 monoclonal antibody is shown in SEQ ID NO. 73, and the amino acid sequence of the light chain of the 295 monoclonal antibody is shown in SEQ ID NO. 74; the amino acid sequence of the heavy chain of the 365 monoclonal antibody is shown in SEQ ID NO. 75, and the amino acid sequence of the light chain of the 365 monoclonal antibody is shown in SEQ ID NO. 76; the amino acid sequence of the heavy chain of the 570 monoclonal antibody is shown in SEQ ID NO. 77, and the amino acid sequence of the light chain of the 570 monoclonal antibody is shown in SEQ ID NO. 78; the amino acid sequence of the heavy chain of the 820 monoclonal antibody is shown in SEQ ID NO. 79, and the amino acid sequence of the light chain of the 820 monoclonal antibody is shown in SEQ ID NO. 80.

[0104] The nucleotide and amino acid sequences of each monoclonal antibody were obtained by sequencing using a 10X single-cell sequencer.

[0105] 6. Monoclonal antibody titer determination:

[0106] The titers of five monoclonal antibodies were determined using an indirect ELISA method, and the results are as follows: Figure 6 As shown, the titers of monoclonal antibodies 570 and 820 were both 1:409600, while the titers of monoclonal antibodies 253, 295, and 365 were slightly lower, all at 1:204800. These results indicate that all five monoclonal antibodies exhibit good binding activity to the PRRSV N protein.

[0107] 7. Monoclonal antibody specificity assay:

[0108] Western blot results are as follows Figure 7 As shown, all five monoclonal antibodies can recognize PRRSV N protein, with a distinct band at 34 kDa. None of the five monoclonal antibodies recognize the pET-28a-sumo empty vector bacterial protein, indicating that all five monoclonal antibodies can specifically recognize PRRSV N protein.

[0109] 8. Monoclonal antibody affinity assay:

[0110] The results of the BLI method for determining the affinity of monoclonal antibodies are as follows: Figure 8As shown in Table 1, the monoclonal antibodies 253, 295, 365, 820, and 570 have a concentration of 8.72 × 10⁻⁶. -9 M, 1.41×10 -8 M, 1.14×10 -10 M, 1.54×10 -11 M and 1.00×10 -12 The smaller the M.KD value, the better the affinity. 820 and 570 monoclonal antibodies have high affinity, while 253 and 365 monoclonal antibodies have medium affinity. Figure 8 The results in Table 1 show that all five monoclonal antibodies have good affinity for PRRSV N protein.

[0111] Table 1. Monoclonal antibody affinity assay

[0112]

[0113] 9. Monoclonal antibody epitope grouping assay:

[0114] The epitopes of monoclonal antibodies were determined using the BLI method. Five monoclonal antibodies were subjected to a 5×5 reciprocal cross pairing experiment. The results are shown in Table 2. The results indicate that there is no competition among the five monoclonal antibodies, which means that the epitopes of the five monoclonal antibodies are different.

[0115] Table 2. Monoclonal antibody epitope assay

[0116]

[0117] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

[0118] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A PRRSV N protein monoclonal antibody, characterized in that, The monoclonal antibody is a 253 monoclonal antibody; The nucleotide sequences of the CDR region of the heavy chain variable region of the 253 monoclonal antibody are shown in SEQ ID NO.1 to SEQ ID NO.3, and the nucleotide sequences of the CDR region of the light chain variable region of the 253 monoclonal antibody are shown in SEQ ID NO.4 to SEQ ID NO.

6.

2. The PRRSV N protein monoclonal antibody according to claim 1, characterized in that, The nucleotide sequence of the heavy chain of the 253 monoclonal antibody is shown in SEQ ID NO. 61, and the nucleotide sequence of the light chain of the 253 monoclonal antibody is shown in SEQ ID NO.

62.

3. The use of the PRRSV N protein monoclonal antibody according to any one of claims 1 to 2 in the preparation of diagnostic reagents for porcine reproductive and respiratory syndrome virus.

4. The application according to claim 3, characterized in that, The reagent is used to diagnose porcine reproductive and respiratory syndrome.

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