Antibody aiming at 2-87 amino acid fragments of N protein of porcine reproductive and respiratory syndrome virus or antigen binding fragment thereof and application of antibody or antigen binding fragment

Abstract

The invention discloses an antibody aiming at 2-87 site amino acid fragments of PRRSV (porcine reproductive and respiratory syndrome virus) N protein or an antigen binding fragment thereof, the antibody or the antigen binding fragment thereof has broad-spectrum recognition capability on different genotypes of PRRSV, is high in specificity and titer, can be used for preparing a PRRSV detection reagent or kit, realizes rapid and accurate preliminary screening of PRRSV infection, and can be used for detecting PRRSV infection. The problems of single target spot, insufficient specificity, dependence on import of core reagents and the like in the existing detection technology are solved, a key biological reagent is provided for prevention and control of PRRS, and the kit has important practical application value.

Description

Technical Field

[0001] This invention relates to the field of antibody technology, specifically to an antibody or its antigen-binding fragment targeting amino acid fragments 2-87 of the N protein of porcine reproductive and respiratory syndrome virus (PRRSV) and its applications. Background Technology

[0002] Porcine Reproductive and Respiratory Syndrome (PRRS), commonly known as blue ear disease, is a highly contagious disease caused by porcine reproductive and respiratory syndrome virus (PRRSV). This virus can infect pigs of all ages through multiple routes, including contact and airborne transmission, but it is particularly harmful to pregnant sows and piglets. Clinical manifestations are mainly reproductive disorders in sows (such as abortion, stillbirth, mummified fetuses, and weak piglets) and respiratory symptoms in piglets, significantly increasing piglet mortality. More importantly, PRRSV has significant immunosuppressive properties. It can target and invade alveolar macrophages in pigs, destroying the core defense cells of the body's immune system, leading to a sharp decline in the pig's resistance. This makes them highly susceptible to secondary bacterial and other viral infections, creating a vicious cycle of "immunosuppression-multiple infections." This not only increases the difficulty of disease control but also further expands the scope of economic losses, severely restricting the sustainable development of intensive pig farming. Currently, the prevention and control of PRRS mainly relies on comprehensive measures centered on "vaccine immunization + monitoring and eradication," among which accurate and rapid diagnostic technology is the key to achieving effective monitoring and tracing of the source of the epidemic.

[0003] While existing PRRS diagnostic technologies are diverse, they all have insurmountable limitations and cannot fully meet the actual needs of the pig industry, especially at the grassroots level. Virus isolation, as the "gold standard" for pathogen detection, provides accurate results, but its cumbersome and time-consuming process makes it difficult to meet the rapid screening needs at the grassroots level. In terms of molecular biological detection, techniques such as RT-PCR, real-time quantitative PCR, and loop-mediated isothermal amplification (LAMP), while possessing high sensitivity and specificity, rely on sophisticated instruments and specialized operation, making them unsuitable for on-site application in pig farms. Among serological tests, indirect immunofluorescence assays and virus neutralization assays, while specific, are limited by low throughput, high subjectivity, or the delayed production of neutralizing antibodies (1-2 months post-infection), hindering early diagnosis. In contrast, enzyme-linked immunosorbent assay (ELISA) has become the mainstream technology for PRRSV serological detection due to its high throughput, low cost, and ease of operation. However, current ELISA kits still face challenges such as single target targeting, difficulty in distinguishing between wild-type virus infection and vaccine immunization, and reliance on imported core reagents, restricting their accurate application at the grassroots level.

[0004] PRRSV belongs to the genus *Porphyromonas* of the family *Porphyromonasviridae*. It is a single-stranded positive-sense RNA virus with a genome length of approximately 15 kb. The virus is mainly divided into two genotypes: PRRSV-1 (European type) and PRRSV-2 (American type). Currently, PRRSV-2 is predominantly prevalent in my country, and its strains can be further divided into several lineages, with lineage 1 (NADC30-like strains) being the currently dominant circulating strain. The N protein, the nucleocapsid protein of PRRSV, is encoded by the ORF7 gene, has a molecular weight of approximately 15 kDa, and consists of 123 amino acid residues. Its greatest advantage is its extremely high sequence conservation; the amino acid homology of the N protein among different genotypes can reach over 90%, and its content in viral particles is as high as 40%, indicating high expression abundance, making it an ideal broad-spectrum detection target. However, antibodies induced by the entire N protein cannot distinguish between wild-type virus and vaccine-immunized viruses, and some regions may contain homologous sequences with other viral proteins, leading to insufficient specificity.

[0005] Current technologies fail to accurately screen for conserved fragments with unique antigenic epitopes in the N protein, hindering the full realization of target advantages. Therefore, developing a PRRSV antibody with precise target targeting, high specificity, high titer, and independent intellectual property rights, along with a detection technology based on this antibody, is crucial to overcoming existing technological bottlenecks and is of great significance for promoting precise PRRS prevention and control and ensuring the healthy development of the pig farming industry. Summary of the Invention

[0006] The present invention aims to provide an antibody or antigen-binding fragment of the N protein of porcine reproductive and respiratory syndrome virus (PRRSV) targeting amino acid fragments 2-87, which can be used to detect the level of PRRSV N protein in a sample. The N protein-based detection method can cover most prevalent strains, has strong adaptability to viral genotype variations, and is suitable for preliminary screening of PRRSV infection.

[0007] To achieve the above objectives, the first aspect of the present invention provides an antibody targeting amino acid fragments 2-87 of the N protein of porcine reproductive and respiratory syndrome virus, wherein the amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO:1 and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:2.

[0008] The optimized antibody is an IgG2a type antibody, and the light chain is a kappa light chain.

[0009] A second aspect of the present invention provides an antigen-binding fragment that specifically binds to the antibody described in the first aspect of the present invention.

[0010] A third aspect of the present invention provides a nucleic acid molecule encoding the antibody described in the first aspect, the nucleic acid molecule comprising a nucleotide sequence encoding a heavy chain variable region as shown in SEQ ID NO:3 and a nucleotide sequence encoding a light chain variable region as shown in SEQ ID NO:4.

[0011] A fourth aspect of the present invention provides a recombinant expression vector comprising the nucleic acid molecule described in the third aspect.

[0012] Furthermore, the vector is a pET-32a(+) expression vector.

[0013] In a fifth aspect, the present invention provides a host cell comprising the recombinant expression vector described in the fourth aspect.

[0014] The sixth aspect of the present invention provides a recombinant protein, which is obtained by inducing expression of the recombinant expression vector of the fourth aspect in the host cell of the fifth aspect.

[0015] In a seventh aspect, the present invention provides the use of a recombinant protein in the immunization preparation of antibodies against porcine reproductive and respiratory syndrome virus N protein.

[0016] In an eighth aspect, the present invention provides the use of the antibody or antigen-binding fragment thereof described in the first aspect in the preparation of a reagent or kit for detecting porcine reproductive and respiratory syndrome virus N protein.

[0017] Working principle and beneficial effects of the present invention: This invention relates to an antibody or its antigen-binding fragment prepared targeting amino acid fragments 2–87 of the N protein of porcine reproductive and respiratory syndrome virus (PRRSV). This antibody or its antigen-binding fragment can specifically recognize the N protein of the virus. Based on the highly conserved nature of the N protein, this antibody or its antigen-binding fragment can be used to detect the presence or content of the N protein of PRRSV in samples. The corresponding detection method can cover most prevalent strains and has strong adaptability to viral genotype variations. At the same time, it provides key biological reagents for establishing a stable and sensitive detection method for PRRSV and is suitable for the preliminary screening of this virus infection. Attached Figure Description

[0018] Figure 1 This is an SDS-PAGE result of the bacterial cells induced to express protein in Example 1; where M is the protein molecular weight standard (Marker), 1 is the bacterial protein without induced expression, and 2 is the bacterial protein after induced expression. Figure 2 This is the result of SDS-PAGE of the sample purified by affinity chromatography in Example 1; where M is the protein molecular weight standard (Marker) and 1 is the sample eluted with 250mM imidazole. Figure 3This is a graph showing the results of serum titer detection in mice after immunization in Example 2; Figure 4 This is a graph showing the results of ascites titer detection in mice after injection of cell line N87-3 in Example 3; Figure 5 This is the result of SDS-PAGE analysis of the purified N87-3 monoclonal antibody in Example 3; where M is the protein molecular weight standard (Marker) and 1-3 are the purified N87-3 monoclonal antibody; Figure 6 This is a graph showing the results of the N87-3 monoclonal antibody specificity detection in Example 3. Detailed Implementation

[0019] The following detailed description illustrates the specific implementation method: Example 1: Obtaining recombinant protein of porcine reproductive and respiratory syndrome virus N (2–87 aa) In this embodiment, N(2–87 aa) recombinant protein was prepared. The specific preparation method includes the following steps: 1. Construction of recombinant expression vectors The construction of the recombinant antigen of the N protein of porcine reproductive and respiratory syndrome virus (PRRSV) NADC30-like strain (GenBank accession number: URX64839.1) is as follows: Bioinformatics analysis of the amino acid sequence of the N protein from the aforementioned strain revealed that the N protein is 123 amino acids in length, lacks a signal peptide and transmembrane region, and readily forms dimers or multimers in its native conformation, serving as a major structural component of the viral nucleocapsid. When the complete ORF7 gene is directly introduced into a prokaryotic expression system, its expression product readily forms inclusion bodies. Epitope prediction and hydrophilicity analysis of the N protein confirmed that it primarily consists of linear epitopes, and the overall sequence is highly conserved. To improve prokaryotic expression characteristics and preserve the main epitope regions, this invention selected the coding sequence corresponding to amino acids 2–87 of the N protein as the expression fragment, and deleted the coding regions corresponding to amino acids 1 and 88–123. After codon optimization of the selected sequence, it was synthesized by Anshengda Biotechnology Co., Ltd., and the optimized sequence was cloned into the pET-32a(+) expression vector to construct the pET-32a-N(2–87 aa) recombinant expression plasmid for subsequent expression of the recombinant N(2–87 aa) protein.

[0020] 2. Expression and purification of N (2–87 aa) protein After amplification and culture of the identified positive BL21(DE3) clone containing the pET-32a-N (2–87 aa) recombinant expression vector, plasmid was extracted, transformed using standard methods, and single colonies were picked and cultured in LB medium containing ampicillin resistance until the bacterial concentration reached A... 600 When the concentration was 0.5, expression was induced for 18 h with 0.1 mM IPTG at 200 rpm and 16℃. Cells were collected by centrifugation at 4℃ and 4000 rpm for 20 min, and SDS-PAGE electrophoresis was performed to detect the induced expression. The results are as follows: Figure 1 As shown.

[0021] The target protein (N(2–87 aa) recombinant protein) was purified by nickel column affinity chromatography: Induced bacterial cells were resuspended and washed with 20 mL PBS, then sonicated in ice bath for 3 min with binding buffer at 30 W for 2 s, followed by a 2 s interval before repeating the sonication until the bacterial suspension was relatively clear. The suspension was then centrifuged at 12000 rpm for 20 min at 4°C to collect the supernatant for purification. A 50% NI-NTA column was packed, washed with 4 mL of deionized water, and then equilibrated with 5 mL of binding buffer. The supernatant containing the N(2–87 aa) recombinant protein was added to the column, and the permeate was collected and re-loaded. Unbound proteins were washed away with binding buffer, followed by 20 mL of washing buffer. Proteins were then eluted with 250 mM imidazole elution buffer. The purity of the eluted sample was determined by SDS-PAGE electrophoresis. The results are shown below. Figure 2 As shown, the N(2–87 aa) recombinant protein obtained by affinity chromatography had high purity. The concentration of the purified N(2–87 aa) recombinant protein was determined by BCA method.

[0022] Example 2: Establishment of N (2–87 aa) recombinant protein monoclonal antibody cell line This embodiment prepared an N(2–87 aa) recombinant protein monoclonal antibody cell line. The specific preparation method includes the following steps: 1. Mouse immunization The N(2–87 aa) recombinant protein obtained in Example 1 was used as an immunogen to immunize Balb / c mice. For the first immunization (day 1 of the first immunization), 100 μg of immunogen was emulsified with Freund's complete adjuvant at a 1:1 volume ratio, administered subcutaneously at five sites on the back and abdomen of each mouse. A second immunization was performed on day 15, using Freund's incomplete adjuvant emulsified with the immunogen at a 1:1 volume ratio, at a dose of 50 μg per mouse, following the same method. A third immunization was performed on day 29, following the same method as the second immunization. On day 36, a small amount of tail blood was collected for ELISA testing. If the antibody titer was greater than 1:10000, antigen pulse immunization and spleen cell fusion could then be performed. If the antibody titer was less than 1:10000, a fourth immunization was performed on day 43, following the same method. Three days before cell fusion, an intraperitoneal pulse immunization was performed, with 100 μg of immunogen injected directly into the peritoneum without adjuvant.

[0023] 2. Mouse serum titer detection (1) Antigen coating: The N(2–87 aa) recombinant protein obtained in Example 1 was adjusted to 1 μg / mL in the coating buffer. 100 μL was added to each well of the ELISA plate and incubated overnight at 4°C.

[0024] (2) Washing: On the second day, discard the liquid in the well, pat dry, and wash twice with PBST in a plate washer.

[0025] (3) Sealing: Add 200 μL of sealing solution to each well and incubate at 37℃ for 1 h.

[0026] (4) Preparation of serum (primary antibody): Blood was collected from the tail of mice, and serum from unimmunized mice was used as a negative control.

[0027] (5) Add primary antibody: Use blocking solution to serially dilute the serum samples to be tested. Use non-immunized mouse serum diluted 1:2500 as a negative control and blocking solution as a blank control. Incubate at 37℃ for 1 h, discard the liquid in the well, pat dry, wash twice with PBST, and pat dry.

[0028] (6) Add enzyme-labeled secondary antibody: Dilute the enzyme-labeled secondary antibody (HRP-goat anti-mouse) 5000 times with blocking buffer, 100 μL / well, incubate at 37℃ for 1 h, discard the liquid in the well, and pat dry.

[0029] (7) Color development and measurement: Add 50 μL of TMB chromogenic solution to each well, incubate at 37℃ in the dark for 15 min, then add 100 μL of 2 M sulfuric acid to terminate the reaction. Measure the A value at 450 nm using a microplate reader. Calculation: The highest antiserum dilution factor when the ratio of the A value of the test well to the A value of the negative control well (P / N) is ≥2.1 is taken as the serum titer. Mice with a dilution greater than 1:100,000 are prepared for the next fusion step. The results are as follows: Figure 3As shown, the reaction of recombinant N(2–87 aa) protein with immune serum was identified by indirect ELISA, with a mouse serum titer of 1:64000.

[0030] 3. Cell fusion Aseptically, spleen cells from immunized mice were mixed with myeloma cells from SP2 / 0 mice at a ratio of approximately 5:1 in a 50 mL centrifuge tube. After washing twice with culture medium, the supernatant was discarded. Over 50 seconds, 0.9 mL of preheated PEG-1500 was slowly added to disperse the cells as evenly as possible. The mixture was allowed to stand for 1 min. 20 mL of preheated serum-free DMEM culture medium at 37°C was slowly added dropwise, 2 mL over the first two minutes and 18 mL over the next two minutes, all within 4 minutes. The mixture was allowed to stand for 3 minutes, then centrifuged at 800 rpm for 5 minutes and the supernatant was discarded. Preheated FBS and HAT culture medium were added, and the mixture was gently pipetted to mix. The mixture was then transferred to 96-well plates at a density of 200 μL per well and incubated.

[0031] 4. Screening of positive hybridoma cells Ten days after cell fusion, when the fused cells filled more than 50% of the wells, hybridoma cells were screened using an indirect ELISA method.

[0032] 5. Subcloning of positive hybridoma cells Subcloning was performed on the positive wells using a limiting dilution method. The number and location of cell clusters in the positive wells were observed under an inverted microscope. Cell clusters were then aspirated using a 200 μL pipette tip in a clean bench, and the cell count was diluted to 1-2 cells per 100 μL. Prepared feeder cells were then added to 100 μL of the diluted cells in 96-well plates, labeled, and incubated at 37°C for 9 days using a 5% CO2 incubator. After three subcloning cycles until one cell was found per well, the titer of the cell supernatant was measured using an indirect ELISA method. The positive rate reached 100%. The culture was expanded, and the cell line was preserved and numbered N87-3.

[0033] Example 3 Preparation and Identification of Monoclonal Antibodies for N (2–87 aa) Protein In this embodiment, a monoclonal antibody against the N(2-87 aa) protein was prepared and its subclass was identified. The specific steps are as follows: 1. Preparation and titer determination of ascites fluid containing N(2-87 aa) protein monoclonal antibody 12-16 week old female BALB / c mice were intraperitoneally injected with 0.5 mL of sterile liquid paraffin. Ten days later, each mouse was injected with 0.5 mL of cell suspension of cell line N87-3 preserved in Example 2 (5 × 10⁻⁶ cells / mL). 5(Each mouse was injected with the cell suspension, and the day of injection was counted as day 1). On day 7, after significant abdominal distension, ascites fluid was collected, centrifuged at 3000 rpm for 20 min, adipose tissue was removed, and the supernatant was collected and stored at -20℃ for later use. The titer of the ascites fluid was determined by indirect ELISA, and the results are as follows: Figure 4 As shown: Ascites antibody titer 1:1280000.

[0034] 2. Purification of monoclonal antibodies Ascites fluid was collected, and N87-3 monoclonal antibody was purified using the caprylic acid-ammonium sulfate precipitation method, as follows: Ascites fluid was collected and centrifuged at 12000 rpm for 5 min at 4°C. The supernatant was collected, and 2 volumes of 0.06 M acetate buffer (pH 4.0) were added to adjust the pH to 4.5. 33 μL of caprylic acid was added per mL of ascites fluid, and the mixture was stirred at room temperature for 30 min. The mixture was then incubated at 4°C for 1 h to allow for complete precipitation of contaminating proteins. The mixture was centrifuged at 1000 g for 30 min at 4°C. 0.277 g of ammonium sulfate powder was added per mL of the supernatant. The beaker was placed on a magnetic stirrer and stirred for 1 h. The mixture was then centrifuged at 10000 g for 20 min at 4°C. The supernatant was discarded, and the precipitate was dissolved in PBS. SDS-PAGE was used to identify the purity of the monoclonal antibody. The results are shown below. Figure 5 As shown: High-purity N87-3 monoclonal antibody was obtained.

[0035] 3. Identification of Monoclonal Antibody Types and Subclasses The mouse subtype identification kit was used for experimental procedures. The N87-3 monoclonal antibody was identified as IgG2a and kappa light chain.

[0036] Meanwhile, GENEWIZ (Suzhou Genewiz Biotechnology Co., Ltd.) was commissioned to sequence the monoclonal antibody produced by the cell line N87-3 preserved in Example 2 (i.e., the N87-3 monoclonal antibody purified in step 2 of Example 3). The results are as follows: The nucleotide sequence of the heavy chain variable region of the N87-3 monoclonal antibody is: SEQ ID NO:3 5’-GATGTGCAGCTTCAGGAGTCGGGACCTGGCCTGGTGAAACCTTCTCAGTCTCTGTCCCTCACCTGCACTGTCACTGGCTACTCAATCACCAGTGAATATGTCTGGAACTGGATCCGGCAGTTTCCAGAAAACACACTGGAGTGGATGGGCTACATAAGTTATAGTGGTAGTGTTACCTACAACCCATCTCTCAAAAGTCGAATATCTCTCACTCGAGACACATCCAAGAACCAGTTCTTCCTGCACTTGAATTCTGTGACTACTGAGGACACAGCCACATATTACTGTGCAAGATACGGCAGCTCGGGCTACGTAGACTGGGGCCAAGGGACTCGGGTCACTGTCTCTGCA-3’ , The corresponding amino acid sequence is: SEQ ID NO:1 DVQLQESGPGLVKPSQSLSLTCTVTGYSITSEYVWNWIRQFPENTLEWMGYISYSGSVTYNPSLKSRISLTRDTSKNQFFLHLNSVTTEDTATYYCARYGSSGYVDWGQGTRVTVSA ; The nucleotide sequence of the light chain variable region is: SEQ ID NO:4 5’-CAAATTGTTCTCACCCAGTCTCCAGCAATCATGTCTGCATCTCCAGGGGAGAAGGTCACCATCTCCTGCAGTGCCAGCTCAAGTGTAACTTACATGTACTGGTACCAGCAGAAGCCAGGATCCTCCCCCAAACCCTGGATTTATCGCACATCCAACCTGGCTTCTGGAGTCCCTGCTCGCTTCAGTGGCAGTGGGTCTGGGACCTCTTACTCTCTCACAATCAGCAGCATGGAGTCTGAAGATGCTGCCACTTATTACTGCCAGCAGTATCATAGTTACCCACGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA-3’ , The corresponding amino acid sequence is: SEQ ID NO:2 QIVLTQSPAIMSASPGEKVTISCSASSSVTYMYWYQQKPGSSPKPWIYRTSNLASGVPARFSGSGSGTSYSLTISSMESEDAATYYCQQYHSYPRTFGGGTKLEIK.

[0037] 4. Specificity detection of monoclonal antibodies The following proteins were used as antigens: porcine reproductive and respiratory syndrome virus (PRRSV) N (2-87 aa) protein (N), PRRSV GP5 protein (GP5), PRRSV Nsp7 protein (NSP), porcine pseudorabies virus gE (52-260 aa) protein (gE-52-260), porcine pseudorabies virus gB (59-337 aa) protein (gB-59-337), porcine pseudorabies virus gB (507-734 aa) protein (gB-507-734), porcine foot-and-mouth disease virus 3ABC protein (3ABC), African swine fever virus I226R protein (I226R), African swine fever virus CD2v protein (CD2v), African swine fever virus p30 protein (p30), and African swine fever virus p72 protein (p72). All of the above antigens were obtained by inducing prokaryotic expression in the laboratory.

[0038] The ELISA plate was coated under the same conditions, using the N87-3 monoclonal antibody purified in step 2 of Example 3 as the primary antibody and HRP-labeled goat anti-mouse IgG as the secondary antibody. A values ​​were read on the ELISA reader. 450 The values ​​were calculated by taking the average of three replicates for each sample. The specificity of the monoclonal antibody was detected using an indirect ELISA method (refer to Example 2). The results are as follows: Figure 6 As shown, the monoclonal antibody produced by the cell line N87-3 preserved in Example 2 (i.e., the N87-3 monoclonal antibody purified in step 2 of Example 3) has good specificity and can be used for qualitative or quantitative detection of porcine reproductive and respiratory syndrome virus N protein in the sample to be tested.

[0039] It should be noted that those skilled in the art can make various modifications and improvements without departing from the technical solution of this invention. These modifications and improvements should also be considered within the scope of protection of this invention, and will not affect the effectiveness of the invention or the practicality of the patent. The scope of protection claimed in this application shall be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. An antibody targeting amino acid fragments 2-87 of the N protein of porcine reproductive and respiratory syndrome virus, characterized in that, The amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO:1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

2.

2. The antibody according to claim 1, characterized in that, The antibody is an IgG2a type antibody, and the light chain is a kappa light chain.

3. An antigen-binding fragment that specifically binds to the antibody as described in claim 1 or 2.

4. A nucleic acid molecule encoding the antibody of claim 1 or 2, characterized in that, The nucleic acid molecule contains a nucleotide sequence encoding the heavy chain variable region as shown in SEQ ID NO:3 and a nucleotide sequence encoding the light chain variable region as shown in SEQ ID NO:

4.

5. A recombinant expression vector comprising the nucleic acid molecule of claim 4.

6. The carrier according to claim 5, characterized in that, The vector is the pET-32a(+) expression vector.

7. A host cell, characterized in that, The host cell comprises the recombinant expression vector of claim 5.

8. A recombinant protein of amino acid fragments 2-87 of the N protein of porcine reproductive and respiratory syndrome virus, characterized in that, The recombinant protein is obtained by inducing expression of the recombinant expression vector of claim 5 in the host cells of claim 6.

9. The use of the recombinant protein according to claim 8 in the immunization preparation of antibodies against porcine reproductive and respiratory syndrome virus N protein.

10. The use of the antibody or antigen-binding fragment thereof according to any one of claims 1-3 in the preparation of reagents or kits for detecting porcine reproductive and respiratory syndrome virus N protein.

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