Monoclonal antibody capable of simultaneously identifying multiple pedigree of porcine reproductive and respiratory syndrome virus GP5 protein

Abstract

The invention belongs to the technical field of biology, and discloses a monoclonal antibody capable of simultaneously recognizing multiple pedigree of porcine reproductive and respiratory syndrome virus GP5 protein. The invention discloses a recombinant protein. The recombinant protein is formed by repeatedly connecting two short peptides in series. The GP5 protein is a basis for dividing a PRRSV pedigree, the GP5 protein is taken as a research object, two regions with relatively strong immunogenicity on amino acid of the GP5 protein are repeatedly connected in series, and the two regions are further connected in series with a tag sequence or a Foldon oligopeptide to obtain a recombinant protein. The recombinant protein is used as an immunogen, and a monoclonal antibody capable of simultaneously identifying GP5 proteins of a plurality of pedigree (including pedigree 1, pedigree 3, pedigree 5 and pedigree 8) of PRRSV is prepared and screened.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a monoclonal antibody capable of simultaneously recognizing multiple lineages of porcine reproductive and respiratory syndrome virus (PRRSV) GP5 protein. Background Technology

[0002] For nearly three decades, porcine reproductive and respiratory syndrome (PRRS) has been one of the most important viral infectious diseases affecting swine herds worldwide. The disease causes abortion, stillbirth, and decreased breeding rates in sows, as well as respiratory problems and secondary infections in piglets and finishing pigs. Economic losses to farms can reach as high as 40%.

[0003] The causative agent of PRRS is porcine reproductive and respiratory syndrome virus (PRRSV). This virus has RNA as its genetic material and two genotypes: European and American. The prevalent strains in my country are mainly American strains, while European strains account for a smaller proportion and are mostly attenuated strains. PRRSV viral particles have an envelope and contain seven structural proteins: GP2, E, GP3, GP4, GP5, M, and N. Based on the GP5 protein, American strains can be divided into at least nine lineages. Among these, the prevalent strains in China are mainly lineage 1, lineage 3, lineage 5, and lineage 8.

[0004] The main problems in the prevention and control of PRRS stem from the following aspects: First, the wild-type PRRSV strains circulating in clinical practice and the vaccine strains used have different lineages; second, gene recombination easily occurs between different PRRSV strains; and third, the cross-protection of vaccines with different lineages is relatively weak, resulting in vaccine ineffectiveness.

[0005] There are two methods for controlling PRRS: establishing PRRSV-free farms and using vaccines appropriately. The latter is currently the method used by most pig farms, especially meat pig farms. However, the latter requires suitable testing methods. Viral nucleic acid testing usually requires samples from animals that are currently showing symptoms, making it difficult to determine whether the pig herd has been previously infected with PRRSV or whether there is a risk of latent infection. Currently, commercially available PRRSV antibody test kits do not have the function of identifying different viral lineages, therefore they cannot determine whether the PRRSV antibodies in the pig herd originate from a vaccine or from an infection of a different lineage; nor can they determine the lineage of PRRSV serum antibodies in newly introduced pig herds. This creates significant uncertainty for veterinarians in how to correctly assess the stability of the pig herd's PRRS and how to select vaccines during subsequent rearing.

[0006] This application is hereby submitted. Summary of the Invention

[0007] This invention aims to at least solve one of the technical problems existing in the prior art. Using the GP5 protein as the research object, a kit has been developed that can identify antibodies from multiple PRRSV lineages, for analyzing the PRRSV antibody types in pig herds, providing a precise analytical tool for assessing the immune history and potential infection risk of pig herds.

[0008] The first aspect of this invention is to provide a recombinant protein.

[0009] The second aspect of the present invention aims to provide the application of the recombinant protein of the first aspect of the present invention in the preparation of products that recognize porcine reproductive and respiratory syndrome virus.

[0010] The third objective of this invention is to provide a method for preparing monoclonal antibodies.

[0011] The fourth aspect of this invention aims to provide a monoclonal antibody or an antigen-binding fragment thereof.

[0012] The fifth aspect of this invention aims to provide biological materials related to the recombinant protein of the first aspect of this invention or the monoclonal antibody or antigen-binding fragment thereof of the fourth aspect of this invention.

[0013] The sixth aspect of this invention is to provide a coupling material.

[0014] The seventh aspect of this invention aims to provide the application of the monoclonal antibody or antigen-binding fragment thereof of the fourth aspect of this invention, the biological material of the fifth aspect of this invention, or the conjugate of the sixth aspect of this invention.

[0015] The object of the eighth aspect of the present invention is to provide a product.

[0016] The ninth aspect of this invention aims to provide a method for detecting porcine reproductive and respiratory syndrome virus (PRRSV) GP5 protein for non-diagnostic purposes.

[0017] To achieve the above objectives, the technical solution adopted by the present invention is as follows: In a first aspect, the present invention provides a recombinant protein comprising two short peptides connected in tandem repeats, the short peptides comprising short peptide A and short peptide B; The amino acid sequence of the short peptide A is: A1) SEQ ID NO:4; or A2) an amino acid sequence that has at least 60% homology with SEQ ID NO:4 and has the same or similar function; or A3) an amino acid sequence that has been modified by substitution, deletion or addition of one or more amino acids as shown in SEQ ID NO:4 and has the same or similar function. The amino acid sequence of the short peptide B is: B1) SEQ ID NO:5; or B2) an amino acid sequence that has at least 60% homology with SEQ ID NO:5 and has the same or similar function; or B3) an amino acid sequence that has been modified by substitution, deletion or addition of one or more amino acids as shown in SEQ ID NO:5 and has the same or similar function.

[0018] In some embodiments of the present invention, the amino acid sequence of the short peptide A is as shown in SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8 or SEQ ID NO:10.

[0019] In some embodiments of the present invention, the amino acid sequence of the short peptide B is as shown in SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9 or SEQ ID NO:11.

[0020] In some embodiments of the present invention, the short peptide A shown in SEQ ID NO:4 and the short peptide B shown in SEQ ID NO:5 are combined and tandemly repeated to obtain a recombinant protein.

[0021] In some embodiments of the present invention, the short peptide A shown in SEQ ID NO:6 and the short peptide B shown in SEQ ID NO:7 are combined and tandemly repeated to obtain a recombinant protein.

[0022] In some embodiments of the present invention, the short peptide A shown in SEQ ID NO:8 and the short peptide B shown in SEQ ID NO:9 are combined and tandemly repeated to obtain a recombinant protein.

[0023] In some embodiments of the present invention, the short peptide A shown in SEQ ID NO:10 and the short peptide B shown in SEQ ID NO:11 are combined and tandemly to obtain a recombinant protein.

[0024] In some embodiments of the present invention, the number of repetitions is 2-5 times, such as 2, 3, 4, or 5 times.

[0025] In some embodiments of the present invention, the recombinant protein further includes a tag sequence or a Foldon short peptide.

[0026] In some embodiments of the present invention, the tag sequence includes at least one of His tag, GST tag, Flag tag or HA tag.

[0027] In some embodiments of the present invention, the tag sequence is a His tag, and the amino acid sequence is shown in SEQ ID NO:2.

[0028] In some embodiments of the present invention, the nucleotide sequence of the Foldon short peptide is shown in SEQ ID NO:3.

[0029] In some embodiments of the present invention, the recombinant protein further includes a linker sequence.

[0030] In some embodiments of the present invention, the connection sequence includes at least one of (ggggs)n, (gggs)n, (ggs)n, (g)n, (GS)n, (eaaak)n, (GGCGGCGGCAGC) or (XP)n, where n is a natural number from 0 to 5.

[0031] In some embodiments of the present invention, the linking sequence is used to link short peptide A, short peptide B, a tag sequence, and / or a Foldon short peptide.

[0032] In some embodiments of the present invention, the amino acid sequence of the recombinant protein is: C1) SEQ ID NO:12; or C2) an amino acid sequence having at least 60% homology with SEQ ID NO:12 and having the same or similar function; or C3) an amino acid sequence having the same or similar function after substitution, deletion or addition of one or more amino acids of the amino acid sequence shown in SEQ ID NO:12.

[0033] In some embodiments of the present invention, the amino acid sequence of the recombinant protein is shown in SEQ ID NO:12-15.

[0034] A second aspect of the present invention provides the use of the recombinant protein of the first aspect of the present invention in the preparation of products that recognize porcine reproductive and respiratory syndrome virus.

[0035] In some embodiments of the present invention, the product includes antibodies.

[0036] In some embodiments of the present invention, the antibody includes a polyclonal antibody or a monoclonal antibody.

[0037] In some embodiments of the present invention, the product further includes reagents, kits, test strips, and antibody chips.

[0038] A third aspect of the present invention provides a method for preparing a monoclonal antibody, comprising the step of immunizing an animal with a recombinant protein from the first aspect of the present invention as an immunogen.

[0039] In some embodiments of the present invention, the preparation method further includes fusing spleen cells of an immunized animal with myeloma cells and screening to obtain hybridoma cell lines.

[0040] In some embodiments of the present invention, the preparation method further includes culturing the hybridoma cell line to obtain a monoclonal antibody.

[0041] In a fourth aspect, the present invention provides a monoclonal antibody or an antigen-binding fragment thereof, prepared by the preparation method of the third aspect of the present invention.

[0042] In some embodiments of the present invention, the monoclonal antibody or its antigen-binding fragment is 1C11 or 1A7; wherein, the heavy chain variable region of 1C11 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO:26-SEQ ID NO:28; the light chain variable region of 1C11 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO:29-SEQ ID NO:31; the heavy chain variable region of 1A7 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO:32-SEQ ID NO:34; the light chain variable region of 1A7 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO:29-SEQ ID NO:31; the CDR is defined according to the IMGT definition scheme.

[0043] In some embodiments of the present invention, the amino acid sequence of the heavy chain variable region of 1C11 is as shown in SEQ ID NO:20, or an amino acid sequence that is functionally identical or similar to the amino acid sequence shown in SEQ ID NO:20 after substitution, deletion or addition of one or more amino acids. In some embodiments of the present invention, the amino acid sequence of the light chain variable region of 1C11 is as shown in SEQ ID NO:22, or is an amino acid sequence that is functionally identical or similar to the amino acid sequence shown in SEQ ID NO:22 after substitution, deletion or addition of one or more amino acids.

[0044] In some embodiments of the present invention, the amino acid sequence of the heavy chain variable region of 1A7 is as shown in SEQ ID NO:24, or is an amino acid sequence that is functionally identical or similar to the amino acid sequence shown in SEQ ID NO:24 after substitution, deletion or addition of one or more amino acids.

[0045] In some embodiments of the present invention, the amino acid sequence of the light chain variable region of 1A7 is as shown in SEQ ID NO:22, or an amino acid sequence that is functionally identical or similar to the amino acid sequence shown in SEQ ID NO:22 after substitution, deletion or addition of one or more amino acids.

[0046] A fifth aspect of the present invention provides biological materials related to the recombinant protein of the first aspect of the present invention or the monoclonal antibody or antigen-binding fragment thereof of the fourth aspect of the present invention, wherein the biological material is any one of a1)-a12): a1) A nucleic acid molecule encoding a recombinant protein of the first aspect of the present invention or a monoclonal antibody or antigen-binding fragment thereof of the fourth aspect of the present invention; a2) An expression cassette containing the nucleic acid molecule of a1); a3) A recombinant vector containing the nucleic acid molecule of a1); a4) A recombinant vector containing the expression cassette of a2); a5) A recombinant microorganism containing the nucleic acid molecule of a1); a6) A recombinant microorganism containing the expression cassette of a2); a7) A recombinant microorganism containing the recombinant vector of a3); a8) A recombinant microorganism containing the recombinant vector of a4); a9) A transgenic cell line containing the nucleic acid molecule of a1); a10) A transgenic cell line containing the expression cassette of a2); a11) A transgenic cell line containing the recombinant vector of a3); a12) A transgenic cell line containing the recombinant vector of a4).

[0047] In some embodiments of the present invention, the transgenic cell line does not contain propagation material.

[0048] In some embodiments of the present invention, the nucleotide sequence of the nucleic acid molecule encoding the recombinant protein of the first aspect of the present invention is shown in SEQ ID NO:16-19.

[0049] In some embodiments of the present invention, the nucleic acid molecule encoding the monoclonal antibody or antigen-binding fragment thereof of the fourth aspect of the present invention comprises a nucleic acid molecule of a heavy chain encoding the monoclonal antibody or antigen-binding fragment thereof of the fourth aspect of the present invention and a nucleic acid molecule of a light chain encoding the monoclonal antibody or antigen-binding fragment thereof of the fourth aspect of the present invention.

[0050] In some embodiments of the present invention, the nucleotide sequence of the heavy chain of the nucleic acid molecule encoding the monoclonal antibody or its antigen-binding fragment of the fourth aspect of the present invention is shown in SEQ ID NO:21 or SEQ ID NO:25.

[0051] In some embodiments of the present invention, the nucleotide sequence of a nucleic acid molecule of a light chain encoding a monoclonal antibody or an antigen-binding fragment thereof of the fourth aspect of the present invention is shown in SEQ ID NO:23.

[0052] Considering the degeneracy of codons, the gene sequence encoding the above-mentioned antibodies can be modified in its coding region without changing the amino acid sequence to obtain a gene encoding the same antibody amino acid sequence; alternatively, the gene can be artificially synthesized and modified according to the codon preference of the host expressing the antibody to improve the expression efficiency of the antibody.

[0053] In some embodiments of the present invention, the expression cassette refers to DNA capable of expressing the recombinant protein, monoclonal antibody, or its antigen-binding fragment in a host cell. This DNA may include not only a promoter to initiate transcription of the gene containing the recombinant protein, monoclonal antibody, or its antigen-binding fragment, but also a terminator to terminate transcription. Furthermore, the expression cassette may also include an enhancer sequence.

[0054] In some embodiments of the present invention, the vector may be a plasmid, a granule, a bacteriophage, or a viral vector.

[0055] In some embodiments of the present invention, the recombinant cells include prokaryotic cells and eukaryotic cells. The prokaryotic cells include bacteria or algae. The eukaryotic cells include fungi, mammalian cells (such as any one of 293 cells, 293T cells, 293FT cells, CHO cells, COS cells, mouse L cells, LNCaP cells, 633 cells, Vero cells, BHK cells, CV1 cells, HeLa cells, MDCK cells, Hep-2 cells, and Per6 cells) or insect cells.

[0056] A sixth aspect of the present invention provides a conjugate comprising a recombinant protein of the first aspect of the present invention or a monoclonal antibody or antigen-binding fragment thereof of the fourth aspect of the present invention, and a conjugate portion comprising a detectable marker.

[0057] Detectable markers refer to substances that have properties that can be directly observed by the naked eye or detected or detected by instruments, such as luminescence, color development, radioactivity, etc. These properties enable qualitative or quantitative detection of the corresponding target.

[0058] In some embodiments of the present invention, the detectable marker is selected from radioactive isotopes, fluorescent substances, chemiluminescent substances, enzymes that catalyze substrate color development, nanoparticle markers, or any combination thereof. In actual use, those skilled in the art can select appropriate markers according to detection conditions or actual needs. Regardless of the marker used, it falls within the protection scope of the present invention.

[0059] In some embodiments of the present invention, the fluorescent dyes include, but are not limited to, fluorescein dyes and their derivatives (e.g., including but not limited to fluorescein isothiocyanate (FITC), hydroxyfluorescein (FAM), tetrachlorofluorescein (TET), etc., or their analogues), rhodamine dyes and their derivatives (e.g., including but not limited to red rhodamine (RBITC), tetramethylrhodamine (TAMRA), rhodamine B (TRITC), etc., or their analogues), and Cy series dyes and their derivatives (e.g., including but not limited to Cy2, Cy3, Cy3B, Cy3.5, Cy...). 5. At least one of Cy5.5, Cy3, etc. or similar substances), Alexa series dyes and their derivatives (including but not limited to Alexa Fluor 350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 33, 647, 680, 700, 750, etc. or similar substances) and protein dyes and their derivatives (including but not limited to phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), polydiophytoxanthin-chlorophyll protein (preCP), etc.).

[0060] In some embodiments of the present invention, the enzyme that catalyzes the color development of the substrate includes, but is not limited to, at least one of horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and glucose-6-phosphate dehydrogenase.

[0061] In some embodiments of the present invention, the radioactive isotopes include, but are not limited to, those mentioned above. 212 Bi、 131 I, 111 In、 90 Y、 186 Re、 211 At、 125 I, 188 Re、 153 Sm、 213 Bi、 32 P, 94 mTc, 99 mTc, 203 Pb, 67 Ga、 68 Ga、 47 Sc、 43 Sc、 110 mIn, 97 Ru、 62 Cu、 64 Cu、 67 Cu、 68 Cu、 86 Y、 88 Y、 121 Sn、 161 Tb,166 Ho、 105 Rh、 177 Lu、 172 Lu and 18 At least one of F.

[0062] In some embodiments of the present invention, the chemiluminescent reagents include, but are not limited to, luminol and its derivatives, luciferin, fluorescein and its derivatives, ruthenium bipyridine and its derivatives, acridine ester and its derivatives, dioxane and its derivatives, rofenine and its derivatives, and peroxazone and its derivatives.

[0063] In some embodiments of the present invention, the nanoparticle-based labeling agents include, but are not limited to, nanoparticles and colloids; nanoparticles include, but are not limited to, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles. Colloids include, but are not limited to, colloidal metals, dispersed dyes, dye-labeled microspheres, and latexes. Colloidal metals include, but are not limited to, colloidal gold, colloidal silver, and colloidal selenium.

[0064] The seventh aspect of the present invention provides the use of the monoclonal antibody or antigen-binding fragment thereof of the fourth aspect of the present invention, the biological material of the fifth aspect of the present invention, or the conjugate of the sixth aspect of the present invention in any one of (1)-(3): (1) preparing a product for detecting porcine reproductive and respiratory syndrome virus; (2) detecting porcine reproductive and respiratory syndrome virus GP5 protein for non-disease diagnostic purposes; (3) preparing a product for detecting porcine reproductive and respiratory syndrome virus GP5 protein.

[0065] In some embodiments of the present invention, the product comprises at least one of reagents, reagent kits, test strips, and antibody chips.

[0066] A seventh aspect of the present invention provides a product comprising a recombinant protein of the first aspect of the present invention, a monoclonal antibody or an antigen-binding fragment thereof of the fourth aspect of the present invention, a biological material of the fifth aspect of the present invention, or a conjugate of the sixth aspect of the present invention.

[0067] In some embodiments of the present invention, the product includes reagents, kits, test strips, and antibody chips.

[0068] In some embodiments of the present invention, the kit further includes at least one of a solid-phase carrier, a coating solution, a coating buffer, a washing solution, a sample diluent, an enzyme-labeled secondary antibody, a chromogenic solution, a stop solution, a negative control, and a positive control.

[0069] In some embodiments of the present invention, the solid support is an enzyme-linked immunosorbent assay (ELISA) plate, such as a NUNC high-binding-strength polystyrene plate.

[0070] In some embodiments of the present invention, the enzyme-labeled secondary antibody can be a commonly used enzyme-labeled secondary antibody, such as horseradish peroxidase-labeled goat immunized mouse IgG, or it can be a conjugate of the sixth aspect of the present invention (the conjugate portion is an enzyme).

[0071] In some embodiments of the present invention, the coating buffer comprises 0.05-0.15 mol / L pH8-9 Tris-HCl buffer.

[0072] In some embodiments of the present invention, the blocking solution comprises 4%-7% skim milk. In some embodiments of the present invention, the washing solution comprises PBST with a pH of 7-7.5. In some embodiments of the present invention, the sample diluent comprises PBST containing 4%-7% BSA (bovine serum albumin).

[0073] In some embodiments of the present invention, the colorimetric solution includes at least one selected from TMB, DAB (Diaminobenzidine), 4-chloro-1-naphthol, 3-amino-9-ethylcarbazole (AEC), and NBT colorimetric solution. In some embodiments of the present invention, the stop solution includes 0.5-1.5M H2SO4.

[0074] In some embodiments of the present invention, the product can be used to detect porcine reproductive and respiratory syndrome virus (PRRSV) GP5 protein and to detect PRRSV.

[0075] A ninth aspect of the present invention provides a method for detecting porcine reproductive and respiratory syndrome virus (PRRSV) GP5 protein for non-diagnostic purposes, comprising the steps of detecting African swine fever virus (ASFV) GP5 protein using a recombinant protein of the first aspect of the present invention, a monoclonal antibody or antigen-binding fragment thereof of the fourth aspect of the present invention, a conjugate of the fifth aspect of the present invention, and / or a product of the eighth aspect of the present invention.

[0076] In some embodiments of the present invention, the method includes the step of using the ELISA method.

[0077] The beneficial effects of this invention are: The GP5 protein is fundamental for classifying PRRSV lineages. This invention uses the GP5 protein as the research object, repeating and tandem two regions with strong immunogenicity on the amino acid sequence of the GP5 protein, and further tandem with a tag sequence or a Foldon peptide to obtain a recombinant protein. Using this recombinant protein as an immunogen, a monoclonal antibody capable of simultaneously recognizing the GP5 protein of multiple PRRSV lineages (including lineage 1, lineage 3, lineage 5, and lineage 8) was prepared and screened.

[0078] This invention provides a monoclonal antibody for detecting PRRSV in swine herds, which can be used to develop diagnostic kits for PRRSV antigens and antibodies in pigs, for analyzing the PRRSV antibody types in swine herds. This can serve as an essential detection method in the PRRSV virus eradication process in intensive pig farms. It provides a precise analytical tool for assessing the immune history and potential infection risk of swine herds. Furthermore, this invention fills a gap in the international field of PRRSV antigen-antibody diagnostics. Attached Figure Description

[0079] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein: Figure 1 The image shows the amino acid sequence alignment results of GP5 proteins from four lineages. The gray area represents the extracellular region. Peptide sequence A is located between amino acids 53 and 62, and peptide sequence B is located between amino acids 187 and 200.

[0080] Figure 2 This is a schematic diagram of the composition of four GP5 recombinant proteins. White cylinders represent sequence B, and gray cylinders represent sequence A. Sequences B and A are linked by a linker. His tags or foldon peptides are represented by dark black cylinders.

[0081] Figure 3 Recombinant proteins GP5-1 (A), GP5-3 (B), GP5-5 (C), and GP5-8 (D) were expressed and purified for E. coli. SDS-PAGE and Western Blot showed that the four proteins were similar in size, all approximately 10-12 kDa.

[0082] Figure 4 ELISA results for the recognition of different lineages of GP5 recombinant proteins by monoclonal antibodies 1C11 and 1A7.

[0083] Figure 5 To analyze the CDR regions of the heavy and light chains of monoclonal antibodies 1C11 and 1A7 using the IMGT method, the highlighted amino acid sequences represent the three CDR regions.

[0084] Figure 6 These are mutants of recombinant proteins GP5-1, GP5-5, and GP5-8. For each recombinant protein, a mutation site is selected in polypeptide sequence A and polypeptide sequence B, and two or three consecutive amino acids at this site are mutated to alanine.

[0085] Figure 7To analyze the recognition sites of monoclonal antibodies on recombinant proteins using indirect ELISA, mutants of the three recombinant proteins GP5-1, GP5-5, and GP5-8 were used to detect monoclonal antibodies 1C11 (A) and 1A7 (B). After ELISA analysis, the recognition sites of monoclonal antibody 1C11 on the recombinant proteins can be preliminarily analyzed as shown by the arrow (C). The amino acid sites in the dashed box may interact with 1A7.

[0086] Figure 8 To competitively detect the blocking effect of mouse serum containing GP5 polyclonal antibody against monoclonal antibody using ELISA: (A) Recombinant proteins GP5-1 and GP5-8 were coated onto the plates, and mouse serum containing antibodies of different lineages was used to block the binding between monoclonal antibody 1C11 and the recombinant proteins; (B) The blocking ability of mouse serum from different lineages against 1A7 was detected. "This indicates significant differences." Detailed Implementation

[0087] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0088] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0089] As used herein, the term "antibody" refers to an immunoglobulin molecule typically composed of two pairs of polypeptide chains, each pair consisting of a "light" (L) chain and a "heavy" (H) chain. Antibody light chains can be classified as κ and λ light chains. Heavy chains can be classified as μ, δ, γ, α, or ε, and antibody isotypes are defined as IgM, IgD, IgG, IgA, and IgE, respectively. Within both light and heavy chains, variable and constant regions are linked by "J" regions of approximately 12 or more amino acids, and the heavy chain also contains "D" regions of approximately 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains (CH1, CH2, and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain, CL. The constant regions of antibodies mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The VH and VL regions can be further subdivided into highly degenerated regions (called complementarity-determining regions (CDRs)) interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4, from the amino terminus to the carboxyl terminus. The variable regions (VH and VL) of each heavy / light chain pair form the antibody binding sites. The allocation of amino acids to various regions or domains follows the definitions in Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883. The term "antibody" is not limited to any particular method of antibody production. For example, it includes recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. Antibodies can be different isotypes of antibodies, such as IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM antibodies.

[0090] As used herein, the term “antigen-binding fragment” of an antibody refers to a polypeptide containing a fragment of the full-length antibody that retains the ability to specifically bind to the same antigen bound by the full-length antibody, and / or competes with the full-length antibody for specific binding to the antigen; it is also referred to as the “antigen-binding moiety.” See also Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed., Raven Press, NY (1989), which is incorporated herein by reference in its entirety for all purposes. Antigen-binding fragments of antibodies can be generated by recombinant DNA technology or by enzymatic or chemical cleavage of the intact antibody. In some cases, antigen-binding fragments include Fab, Fab', F(ab')2, Fd, Fv, dAb, and complementarity-determining region (CDR) fragments, single-chain antibodies (e.g., scFv), chimeric antibodies, diabody antibodies, and polypeptides containing at least a portion of an antibody sufficient to confer specific antigen-binding ability to the polypeptide.

[0091] In some cases, the antigen-binding fragment of an antibody is a single-chain antibody (scFv), in which the VL and VH domains pair to form a monovalent molecule by enabling them to generate linkers as single polypeptide chains (see, for example, Bird et al., Science 242:423 426 (1988) and Huston et al., Proc. Natl. Acad. Sci. USA 85:5879 5883 (1988)). Such scFv molecules may have a general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of a repeating GGGGS amino acid sequence or a variant thereof. For example, a linker having the amino acid sequence (GGGGS)4 can be used, but variants thereof can also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90: 6444-6448). Other connectors that can be used in this invention are described by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol. 31: 94-106, Hu et al. (1996), Cancer Res. 56:3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56 and Roovers et al. (2001), Cancer Immunol.

[0092] In some cases, the antigen-binding fragment of an antibody is a biantibody, i.e., a bivalent antibody, in which the VH and VL domains are expressed on a single polypeptide chain, but the linker is too short to allow pairing between the two domains on the same chain, thus forcing the domain to pair with the complementary domain of another chain and creating two antigen-binding sites (see, for example, Holliger P. et al., Proc. Natl. Acad. Sci. USA 90:6444 6448 (1993), and Poljak RJ et al., Structure 2:1121 1123 (1994)).

[0093] Antigen-binding fragments (e.g., the antibody fragments described above) of a given antibody (e.g., the monoclonal antibody 1H11 provided in this invention) can be obtained using conventional techniques known to those skilled in the art (e.g., recombinant DNA technology or enzymatic or chemical fragmentation methods), and the antigen-binding fragments of the antibody can be specifically screened in the same manner as those used for intact antibodies.

[0094] In this article, unless the context clearly indicates otherwise, when referring to the term "antibody," it includes not only the complete antibody but also the antigen-binding fragment of the antibody.

[0095] As used herein, the term "monoclonal antibody" refers to an antibody or a fragment of an antibody derived from a group of highly homologous antibody molecules—that is, a group of identical antibody molecules except for the possibility of spontaneous natural mutations. Monoclonal antibodies exhibit high specificity for a single epitope on an antigen. Polyclonal antibodies, as opposed to monoclonal antibodies, typically contain at least two or more different antibodies that typically recognize different epitopes on an antigen. Monoclonal antibodies are usually obtained using hybridoma techniques first reported by Kohler et al. (Nature, 256:495, 1975), but can also be obtained using recombinant DNA techniques (see Journal of Virological Methods, 2009, 158(1-2):171-179).

[0096] As used herein, the term "Escherichia coli expression system" refers to an expression system consisting of Escherichia coli (strain) and a vector, wherein the Escherichia coli (strain) is derived from commercially available strains, such as, but not limited to: GI698, ER2566, BL21(DE3), B834(DE3), BLR(DE3).

[0097] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which polynucleotides can be inserted. When a vector enables the expression of a protein encoded by the inserted polynucleotide, it is called an expression vector. Vectors can be introduced into host cells through transformation, transduction, or transfection, allowing the genetic material elements they carry to be expressed in the host cells. Vectors are well-known to those skilled in the art and include, but are not limited to: plasmids; phage particles; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage; and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retrotranscriptoviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40). A vector may contain multiple elements controlling expression, including but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, a vector may contain a replication initiation site.

[0098] As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including but not limited to prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK293 cells, or human cells.

[0099] In this invention, amino acids are typically represented using single-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala.

[0100] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0101] Example 1: Screening and design of amino acid sequences for recombinant proteins PRRSV strains from the Americas originating in China between 2020 and 2022 were collected from the GeneBank database. Then, the amino acid sequences of the GP5 protein from the most prevalent strains were selected, and the positions of their antigenic determinants were analyzed. Strains from lines 1, 3, 5, and 8 were then subjected to amino acid sequence alignment. Finally, the results of antigenic determinant analysis and amino acid sequence alignment were combined to determine the peptide for immunizing mice.

[0102] The lineages of American PRRSV detected in China between 2020 and 2022 were analyzed from GeneBank. The results are shown in Table 1. The strains with clear lineages in China belong to four lineages: 1, 3, 5, and 8. Among them, strains of lineage 1 and lineage 8 account for 83.9% of the total number of strains in the four lineages.

[0103] Table 1. Statistical results of PRRSV in China from 2020 to 2022

[0104] One representative strain from lineage 1 and lineage 8 were selected to predict the location of their GP5 protein antigenic determinants. The prediction website was: http: / / tools.iedb.org / bcell / . The representative strain from lineage 1 was NADC30, with GeneBank accession number JN654459.1, and the representative strain from lineage 8 was HY21, with GeneBank accession number OL687155.1. The prediction results are shown in Table 2. The locations of the antigenic determinants of the GP5 protein in lineages 1 and 8 are roughly the same, differing only in the length of a few amino acids in the polypeptides that make up the antigenic determinants.

[0105] Table 2. Antigenic determinants on the GP5 protein of lineage 1 and lineage 8 strains

[0106] Then, two amino acid sequences of GP5 protein were selected from each of lineages 1, 3, 5, and 8 for alignment. The results are as follows: Figure 1 As shown. Based on the comprehensive antigenic determinant prediction results, two peptide segments were selected as the basis for designing recombinant proteins: sequence A (53AA-62AA) located in the extracellular region and sequence B (187AA-200AA) located at the C-terminus of the intracellular region.

[0107] like Figure 2 As shown, polypeptide sequences A and B from GP5 proteins derived from lineages 1, 3, 5, and 8 were repeated three times, linked by GGGS (Linker, SEQ ID NO:1). The repeating polypeptide from lineage 1 was appended with a His tag (HHHHHHHH, SEQ ID NO:2) and named GP5-1. The repeating polypeptides from lineages 3, 5, and 8 were each appended with a Foldon peptide (GYIPEAPRDGQAYVRKDGEWVLLSTFL, SEQ ID NO:3) and named GP5-3, GP5-5, and GP5-8, respectively. Detailed information on the four lineage-derived strains and the specific amino acid sequences of polypeptide sequences A and B are shown in Table 3.

[0108] Table 3. Information on the strains from which the recombinant protein originated and the amino acid sequences of the peptides.

[0109] Example 2 Expression and purification of recombinant protein 1. Expression of recombinant proteins Several recombinant protein genes were artificially synthesized by adding His tags or Foldon short peptides, and then inserted into the expression vector pET21a+, which was then transformed into Escherichia coli BL21(DE3).

[0110] The specific method for protein expression is as follows: E. coli containing the recombinant protein expression plasmid are added to LB liquid medium containing ampicillin resistance, and incubated at 220 rpm and 37°C until the turbidity (OD) of the bacterial culture is measured. 600 When the absorbance was around 0.6, IPTG was added to a final concentration of 1 mM for overnight induction. The cells were collected by centrifugation, subjected to polyacrylamide gel electrophoresis, and stained with Coomassie brilliant blue to observe the expression of the target protein. The cells were then lysed by sonication, centrifuged at 10,000 rpm at 4°C for 30 minutes, and the precipitate was collected.

[0111] 2. Purification of recombinant proteins The specific method for purifying the protein is as follows: Add 400 mL of inclusion body washing buffer to the precipitate and resuspend, mixing thoroughly. Sonicate for 10 minutes. Centrifuge the washed protein suspension at 4℃, 11377×g centrifugal force for 15 minutes. Discard the supernatant after centrifugation, retain the precipitate, and dissolve it with inclusion body denaturing buffer. Then, renature the recombinant protein using a dialysis bag. Renaturation is performed in three steps, using three different inclusion body refolding buffers. The procedure for each step is the same: place the dialysis bag containing the recombinant protein solution into a beaker containing 500 mL of refolding buffer, and dialyze at 150 rpm in a 6℃ chromatography cabinet with slow stirring for 4-6 hours. After the third dialysis step, remove the protein solution and centrifuge at 8000 rpm for 20 minutes at 4℃. Transfer the supernatant to a new centrifuge tube, and measure the protein concentration using a micro spectrophotometer.

[0112] The solution formulation for purifying proteins is as follows: Inclusion body washing solution: Dissolve 10 mL Triton X-100 and 5 mL EDTA (0.2 M) in PBS buffer (0.01 mol / L pH 7.2), adjust pH to 7.2, and bring the volume to 1000 mL. Filter through a 0.22 μm filter membrane and store at 2-8 °C.

[0113] Inclusion body denaturing solution: Dissolve Tris 6.06g, NaCl 11.18g, and guanidine hydrochloride 573.18g in ultrapure water, adjust the pH to 8.0, and bring the volume to 1000mL. Filter through a 0.22μm filter membrane and store at 2-8℃.

[0114] Inclusion body refolding solution 1: Dissolve 286.6 g of guanidine hydrochloride in Tris-NaCl (pH 8.0) buffer, bring the volume to 1000 mL, filter through a 0.22 μm filter membrane, and store at 2-8 °C.

[0115] Inclusion body refolding solution 2: Dissolve 143.2 g of guanidine hydrochloride and 34.84 g of arginine in Tris-NaCl (pH 8.0) buffer, bring the volume to 1000 mL, filter through a 0.22 μm filter membrane, and store at 2-8 °C.

[0116] Inclusion body refolding solution 3: Dissolve 34.84 g of arginine and 100 mL of glycerol in Tris-NaCl (pH 8.0) buffer, bring the volume to 1000 mL, filter through a 0.22 μm filter membrane, and store at 2-8 °C.

[0117] 3. Identification of recombinant proteins Polyacrylamide gel electrophoresis (SDS-PAGE) was performed using a 15% gel, stained with Coomassie Brilliant Blue for approximately 30 minutes, and destained with destaining solution until the target band was clearly visible. The specificity of the target protein was identified by Western blotting. The mouse anti-His tag antibody was diluted 1:3000 (purchased from Sangon Biotech (Shanghai) Co., Ltd., catalog number D191001). The mouse anti-Foldon antibody was prepared in-house using the following method: Foldon was linked to KLH (prepared by Jier Biochemical (Shanghai) Co., Ltd.), emulsified with Freund's adjuvant, and mice were immunized with a subcutaneous injection of 30 micrograms in the abdomen. Immunization was repeated three times at 4-week intervals, with the last immunization without adjuvant. The antigen was injected intraperitoneally. Blood was collected 15 days later, serum was separated, aliquoted, and stored at -80°C for later use.

[0118] Recombinant proteins GP5-1, GP5-3, GP5-5, and GP5-8 were expressed and purified in *E. coli*, and identified by SDS-PAGE and Western Blot. The molecular weights of the four recombinant proteins were similar, approximately 10-12 kDa. Figure 3 ).

[0119] The amino acid sequences of recombinant proteins GP5-1, GP5-3, GP5-5 and GP5-8 are shown in SEQ ID NO:12-15, and the encoded nucleotide sequences are shown in SEQ ID NO:16-19.

[0120] TPVTKISAEQWGRPGGGSTDWLNERFYWGGGSTPVTKISAEQWGRPGGGSTDWLNERFYWGGGSTPVTKISAEQWGRPGGGSTDWLNERFYWHHHHHHHH(SEQ ID NO:12)。

[0121] TPVTKISAERWGHPGGGSTDWLGNKFDWGGGSTPVTKISAERWGHPGGGSTDWLGNKFDWGGGSTPVTKISAERWGHPGGGSTDWLGNKFDWGYIPEAPRDGQAYVRKDGEWVLLSTFL(SEQ ID NO:13)。

[0122] TPITRVSAEQWGRPGGGSTDWLANKFDWGGGSTPITRVSAEQWGRPGGGSTDWLANKFDWGGGSTPITRVSAEQWGRPGGGSTDWLANKFDWGYIPEAPRDGQAYVRKDGEWVLLSTFL(SEQ ID NO:14)。

[0123] TPLTRVSAELWGRLGGGSTDWLAQKFDWGGGSTPLTRVSAELWGRLGGGSTDWLAQKFDWGGGSTPLTRVSAELWGRLGGGSTDWLAQKFDWGYIPEAPRDGQAYVRKDGEWVLLSTFL(SEQ ID NO:15)。

[0124] ACCCCGGTGACCAAAATTAGCGCGGAACAGTGGGGCCGCCCGGGTGGCGGTAGCACCGATTGGTTAAATGAACGCTTTTATTGGGGTGGCGGTAGCACCCCGGTGACCAAAATTAGCGCGGAACAGTGGGGCCGCCCGGGTGGTGGTAGCACTGATTGGTTAAATGAACGCTTTTATTGGGGTGGCGGTAGCACCCCGGTGACCAAAATTAGCGCGGAACAGTGGGGCCGCCCGGGTGGTGGTAGCACCGATTGGTTAAATGAACGCTTTTATTGGCACCACCACCACCACCACCATCAC(SEQ ID NO:16)。

[0125] ACCCCGGTGACCAAAATTAGCGCGGAACGCTGGGGCCATCCGGGCGGTGGTAGCACCGATTGGCTGGGTAATAAATTTGATTGGGGCGGTGGTAGCACCCCGGTGACCAAAATTAGCGCGGAACGCTGGGGCCATCCGGGCGGTGGTAGCACCGATTGGTTAGGTAATAAATTTGATTGGGGCGGTGGTAGCACCCCGGTGACTAAAATTAGCGCGGAACGCTGGGGCCATCCGGGCGGTGGTAGCACCGATTGGTTAGGCAATAAATTTGATTGGGGCTATATCCCGGAAGCGCCGCGCGATGGTCAAGCGTATGTTCGTAAAGATGGCGAATGGGTGCTGCTGAGCACCTTTCTG(SEQ ID NO:17)。

[0126] ACCCCGATTACCCGTGTTAGCGCGGAACAGTGGGGTCGCCCAGGTGGTGGTAGCACCGATTGGTTAGCGAATAAATTTGATTGGGGTGGTGGTAGCACCCCGATTACCCGCGTGAGCGCAGAACAATGGGGCCGTCCAGGCGGTGGTAGCACCGATTGGTTGGCGAATAAATTTGATTGGGGTGGTGGTAGCACCCCGATTACCCGCGTGAGCGCGGAACAATGGGGTCGTCCAGGTGGTGGTAGCACCGATTGGTTAGCGAATAAATTTGATTGGGGCTATATCCCGGAAGCGCCGCGCGATGGTCAAGCGTATGTTCGCAAAGATGGCGAATGGGTGCTGCTGAGCACCTTTCTG(SEQ ID NO:18)。

[0127] ACCCCATTGACCCGTGTTAGCGCGGAACTGTGGGGCCGTTTAGGTGGTGGTAGTACCGATTGGTTAGCGCAGAAATTTGATTGGGGTGGTGGTAGTACCCCGCTGACCCGCGTTAGCGCGGAATTATGGGGTCGTTTGGGTGGTGGCAGCACCGATTGGTTGGCGCAAAAATTTGATTGG GGTGGTGGTAGTACCCCGCTGACCCGCGTGAGCGCGGAATTATGGGGTCGTTTAGGCGGTGGTAGCACCGATTGGTTAGCGCAAAAATTTGATTGGGGTTATATTCCAGAAGCGCCGCGTGATGGCCAAGCGTATGTTCGTAAAGATGGCGAATGGGTGCTGCTGAGCACCTTTCTG (SEQ ID NO:19).

[0128] Example 3: Screening of Monoclonal Antibodies The method for immunizing Balb / c mice, screening for monoclonal antibodies, and preparing hybridoma cell lines is the same as the classic method: recombinant protein is emulsified with Freund's adjuvant, and female Balb / c mice are subcutaneously injected twice, 3 weeks apart, with each mouse receiving 30 μg of recombinant protein. The third immunization is an intraperitoneal injection of unadjuvanted recombinant protein. Five days later, blood is collected from the tail vein to determine the serum titer. If the titer reaches 1:100,000 or higher, spleen cells can be fused with mouse myeloma cells (sp20) using the traditional method. When the antibody in the culture supernatant of the monoclonal cell line specifically recognizes the GP5 recombinant protein, the cell line is further subcloned, expanded, and cryopreserved.

[0129] Indirect ELISA method: First, recombinant proteins from different PRRSV lineages were diluted to 5 μg / mL with coating buffer (0.1 mol / L, pH 8.8 Tris-HCl buffer, i.e., 1000 mL of solution containing 2.1 g Tris1, pH 8.8). The solution was then added to 96-well plates (high-binding polystyrene 96-well plates, brand NUNC, catalog number 468667 MaxiSorp, manufactured by Thermo Fisher Scientific), 100 μL per well, and incubated overnight at 4°C. The next day, the coating buffer was discarded, and the plates were washed once. Then, 300 μL of blocking buffer (5% skim milk, i.e., 100 mL PBS (Na2HPO4•12H2O 3.63 g, KH2PO4 0.24 g, NaCl 8 g, KCl 0.2 g, double-distilled water added to 1000 mL, pH adjusted to 7.2) was added to each well. (g of skim milk powder), incubate at 37°C for 2 hours, wash 3 times (washing buffer is PBST pH 7.2, 1 mL Tween-20 in 1000 mL PBS), pat dry; add monoclonal antibody sample (or appropriately diluted clinical animal serum, the sample dilution method is to add (bovine serum albumin) BSA to 5% concentration in PBST), 100 μL per well, incubate at 37°C for 1 hour, then wash 3 times according to the above washing method; add 1:3000 diluted horseradish peroxidase HRP-labeled goat anti-mouse IgG (Invitrogen, catalog number: 62-6520) (or add HRP-labeled secondary antibody against the corresponding animal IgG), 100 μL per well, incubate at 37°C for 30 minutes, wash 3 times; finally, add TMB chromogenic solution (Sigma, catalog number 34021), 100 μL per well, react at room temperature in the dark for 10 minutes, then add 50 μL of stop solution (1M) to each well. The solution was prepared by slowly adding 54.3 mL of 95% concentrated sulfuric acid dropwise to 900 mL of distilled water while stirring constantly until the solution was diluted to 1000 mL. The OD value was then read. 450 Absorbance value.

[0130] Mouse hybridoma cell lines were screened using recombinant proteins GP5-1, GP5-3, GP5-5, and GP5-8, respectively, with the synthetic peptide Foldon-KLH (synthesized by Jier Biochemical (Shanghai) Co., Ltd.) serving as a control. Results are shown in Table 4. Figure 4 As shown, two monoclonal antibody cell lines were obtained, denoted as 1C11 and 1A7, and the antibodies secreted by them can recognize multiple lineages simultaneously.

[0131] Table 4. Two monoclonal antibodies that recognize GP5 proteins from different lineages.

[0132] Example 4: Identification of Monoclonal Antibody Subtypes The subtypes of monoclonal antibodies produced by the two monoclonal antibody cell lines screened in Example 3 were identified. Specifically, the subtypes of the screened monoclonal antibodies were identified using the "ELISA Kit for Identification of Mouse Monoclonal Antibody Ig Class / Subclass / Subtype" (catalog number C060101-L) manufactured by Wuhan Pure Biotechnology Co., Ltd., in accordance with the instructions for use.

[0133] The subtypes of the heavy and light chains of the two monoclonal antibodies were determined and are shown in Table 5.

[0134] Table 5. Subtypes of heavy and light chains of monoclonal antibodies

[0135] Example 5: Amplification of the gene for the variable region of a monoclonal antibody Hybridoma cell lines 1C11 and 1A7 were fed to their peak growth period. The mRNA encoding the antibody was reverse transcribed from the cells, and the gene encoding the antibody variable region was amplified using nested PCR and sequenced. The hybridoma cell culture medium was RPMI 1640 (Thermo Fisher, catalog number 11875093) containing 15% fetal bovine serum (Gibco, catalog number 10099-141C). The reverse transcription kit, "SuperScript™ IV CellsDirect™ cDNA Synthesis Kit," was purchased from Thermo Fisher (catalog number 11750150). The PCR amplification reagent was "2×High Fidelity PCR Master Mix," purchased from Sangon Biotech (Shanghai) Co., Ltd. (catalog number BN39292-0001).

[0136] After amplifying the genes encoding the heavy and light chains of the variable region of a monoclonal antibody using nested PCR, the sequences were sequenced and translated into amino acid sequences. The antigen-binding sites on the amino acid sequences of the antibody variable region were then analyzed using the IMGT method. Results are as follows: Figure 5 As shown, the CDR regions of monoclonal antibodies 1C11 and 1A7 are different, suggesting that the antigen sites recognized by these two antibodies may also be different.

[0137] The amino acid sequences of the heavy and light chains of monoclonal antibody 1C11 are shown in SEQ ID NO:20 and SEQ ID NO:22, respectively, and the encoded nucleotide sequences are shown in SEQ ID NO:21 and SEQ ID NO:23, respectively.

[0138] Heavy chain of monoclonal antibody 1C11: GPELVKPGASVRISCKAS GYTFTSYYIHWVKQRPGQGLEWIGW IYPGNVNT KYNEKFKGKATLTADKSSSTVYMQLSSLTSEDSAVYFC ARGQLGPDY WGQGTTLT (SEQ ID NO:20), the underlined portion is the three complementary determination regions (CDRs).

[0139] GGACCTGAGCTGGTGAAGCCTGGGGCTTCAGTGAGGATATCCTGCAAGGCTTCTGGCTACACCTTCACAAGCTACTATATACACTGGGTGAAGCAGAGGCCTGGACAGGGACTTGAGTGGATTGGATGGATTTATCCTGGAAATGTTAATACTAAGTACAA TGAGAAGTTCAAGGGCAAGGCCACACTGACTGCAGACAAATCCTCCAGCACAGTCTACATGCAGCTCAGCAGCCTGACCTCTGAGGACTCTGCGGTCTATTTCTGTGCAAGAGGTCAACTGGGTCCTGACTACTGGGGCCAAGGCACCACTCTCACA (SEQ ID NO:21).

[0140] The light chain of monoclonal antibody 1C11: VMTQSPASLAVSLGQRATISYRAS KSVSTSGYSY MHWNQQKPGQPPRLLIY LVS NLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYC QHIRELTR SEGGPSWK (SEQ ID NO:22), the underlined portion represents the three complementarity determination regions (CDRs).

[0141] GTGATGACCCAGTCTCCTGCTTCCTTAGCTGTATCTCTGGGGCAGAGGGCCACCATCTCATACAGGGCCAGCAAAAGTGTCAGTACATCTGGCTATAGTTATATGCACTGGAACCAACAGAAACCAGGACAGCCACCCAGACTCCTCATCTATCTTGTATC CAACCTAGAATCTGGGGTCCCTGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACCCTCAACATCCATCCTGTGGAGGAGGAGGATGCTGCAACCTATTACTGTCAGCACATTAGGGAGCTTACACGTTCGGAGGGGGGACCAAGCTGGAAA (SEQ ID NO:23).

[0142] The amino acid sequences of the heavy and light chains of monoclonal antibody 1A7 are shown in SEQ ID NO:24 and SEQ ID NO:22, respectively, and the encoded nucleotide sequences are shown in SEQ ID NO:25 and SEQ ID NO:23, respectively.

[0143] Heavy chain of monoclonal antibody 1A7: GAELAKPGASLKMSCKAS GYTFTNYW MHWVKQRPGQGLEWIGY INPHTGYT EYNQKFKNKASLTADKPSSTAYMQLSSLTSEDSAVYFC ARGGSYVGFYY WGQGTL (SEQ ID NO:24), the underlined portion represents the three complementary determination regions (CDRs).

[0144] GGGGCTGAACTGGCAAAACCTGGGGCCTCATTGAAGATGTCCTGCAAGGCTTCTGGCTACACCTTTACTAACTGGATGCACTGGGTAAAACAGAGGCCTGGACAGGGTCTGGAATGGATTGGATACATTAATCCTCACACTGGTTATACTGAGTACAA TCAGAAATTCAAAAACAAGGCCTCATTGACTGCAGACAAACCCTCCAGCACAGCCTACATGCAACTGAGCAGCCTGACATCTGAGGACTCTGCAGTCTATTTCTGTGCAAGAGGGGGGTTCCTACGTGGGATTTTATTACTGGGGCCAAGGGACTCTG (SEQ ID NO:25).

[0145] The light chain of monoclonal antibody 1A7: VMTQSPASLAVSLGQRATISYRAS KSVSTSGYSY MHWNQQKPGQPPRLLIY LVS NLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYC QHIRELTR SEGGPSWK (SEQ ID NO:22), the underlined portion represents the three complementarity determination regions (CDRs).

[0146] GTGATGACCCAGTCTCCTGCTTCCTTAGCTGTATCTCTGGGGCAGAGGGCCACCATCTCATACAGGGCCAGCAAAAGTGTCAGTACATCTGGCTATAGTTATATGCACTGGAACCAACAGAAACCAGGACAGCCACCCAGACTCCTCATCTATCTTGTATC CAACCTAGAATCTGGGGTCCCTGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACCCTCAACATCCATCCTGTGGAGGAGGAGGATGCTGCAACCTATTACTGTCAGCACATTAGGGAGCTTACACGTTCGGAGGGGGGACCAAGCTGGAAA (SEQ ID NO:23).

[0147] Example 6: Identification of the recognition site of monoclonal antibody on recombinant protein GP5 Mutants of GP5 recombinant protein from different lineages (Example 2) were plated, and the binding ability of monoclonal antibodies 1C11 and 1A7 to these recombinant proteins was detected by indirect ELISA (same as Example 3), thereby analyzing the recognition sites of these antibodies on the recombinant proteins.

[0148] First, the recombinant protein is mutated: the method for selecting mutation sites on the recombinant protein is as follows: from polypeptide sequences A and B of GP5-1 and GP5-8, respectively, amino acids with significant differences between different lineages are mutated to alanine; from polypeptide sequences A and B of GP5-5, positions with the same amino acids as GP5-1 and GP5-8 are selected and mutated to alanine. Specific mutation sites are as follows: Figure 6 As shown. The mutated recombinant proteins were obtained using the method of Example 1.

[0149] Then, the mutated recombinant protein and the unmutated recombinant protein were coated onto plates, and the recognition ability of the monoclonal antibody was tested again to identify the binding site of the monoclonal antibody on the GP5 protein. The results are as follows: Figure 7 As shown, monoclonal antibody 1C11 recognizes the antigenic determinant "FYW" or "FDW" in sequence A. Clonal antibody 1A7 binds to both the tested recombinant proteins, suggesting that its binding site is located on other amino acid residues outside the mutation site, such as the amino acid in the dashed box in sequence B.

[0150] Example 7 Purification of IgG from monoclonal antibodies Reagent preparation: 25mM TBS: 3.025g Tris, 4.375g NaCl, dissolved in 400mL distilled water, pH adjusted to 7.4 with HCl, and brought to a final volume of 500mL. Store at 4℃. 50mM Glycine: 0.375g Glycine, dissolved in 80mL distilled water, pH adjusted to 1.9 with HCl, and brought to a final volume of 100mL. Store at 4℃. 1M Tris: 12.1g Tris, dissolved in 80mL distilled water, pH adjusted to 9.0 with HCl, and brought to a final volume of 100mL. Store at 4℃. Protein A+G Agarose: Purchased from Beyotime Biotechnology Co., Ltd., catalog number P2019-10ml.

[0151] Purification steps: First, the culture supernatant of the monoclonal cell line was diluted 1:1 with TBS, filtered through a 0.22 μM filter membrane, and then added to a Protein A+G Agarose column equilibrated with TBS. Unbound serum impurities were then washed with TBS. Next, IgG was eluted by adding 50 mM Glycine dropwise to the column, and the eluted IgG solution was neutralized with Tris at pH 9.0. Finally, the IgG solution was concentrated using a 100 kDa ultrafiltration tube (Millipore Amicon Ultra, catalog number R1HB04151), the concentration was measured, aliquoted, and stored at -80°C.

[0152] Example 8: HRP-labeled monoclonal antibody and testing the working concentrations of antibody and platelet protein. 1. Monoclonal antibody labeling using sodium periodate oxidation method Materials: HRP (RZ 3.0, Reagent grade) was purchased from Beyotime Biotechnology Co., Ltd., catalog number: ST2569-25mg; NaIO4 was purchased from Shanghai Maclean Biochemical Technology Co., Ltd., catalog number: S817518-100g; Ethylene glycol was produced by VETEC GmbH, Germany, catalog number: V900208-500ML; NaBH4 was purchased from Sangon Biotech (Shanghai) Co., Ltd., catalog number: A355262-0001; Jelap dialysis bags were purchased from Hunan Yibo Biotechnology Co., Ltd., catalog number: MD10-5000.

[0153] Steps: First, dialyze the protein sample overnight with PBS at 4°C. Second, prepare the HRP solution as follows: Weigh 1 mg of HRP and dissolve it in 45 μL of ddH2O; weigh 1 mg of NaIO4 and dissolve it in 50 μL of ddH2O; add 50 μL of NaIO4 solution dropwise to the HRP solution while stirring; place the mixed solution at 4°C and let it stand for 30 minutes; take 1 μL of ethylene glycol, add 4 μL of ddH2O and mix well, then add it dropwise to the above mixed solution in the dark while stirring, and let it stand at room temperature for 30 minutes. Fourth, mix the protein sample and HRP solution at a volume ratio of 1:1 and dialyze in PBS in the dark for 6 hours. Fifth, weigh 0.2 mg of NaBH4 and dissolve it in 10 μL of ddH2O. Add 10 μL of NaBH4 solution to the protein sample, wrap it in aluminum foil to protect it from light, and place it on a decolorizing shaker at 4°C for 2 hours. Then dialyze overnight with PBS in the dark. Fifth, centrifuge at 12,000 rpm for 10 minutes, and collect the supernatant, which is the enzyme conjugate. Add 20% glycerol, aliquot, and store at 4°C or -20°C. For subsequent ELISA activity testing, dilute according to volume ratios (e.g., 1:1000, 1:2000, etc.).

[0154] 2. Testing the working concentrations of antibodies and platelet proteins. The optimal ratio of monoclonal antibodies to plate-coating proteins was analyzed using a checkerboard titration method. The specific method was as follows: First, monoclonal antibodies 1C11 and 1A7 were ligated to HRP. Then, recombinant proteins of different concentrations (GP5-1 and GP5-8) were used for plate coating. Next, HRP-labeled monoclonal antibodies of different dilutions were added, followed by the addition of TMB solution (an ELISA substrate). After color development and termination with sulfuric acid, the OD was measured. 450 Absorbance value (refer to Example 3 for ELISA detection process).

[0155] The results are shown in Table 6-9. The table lists suitable OD values. 450 Absorbance values ​​are marked with bold and underline.

[0156] Table 6. Optimal concentration ratio of monoclonal antibody 1C11 to platelet protein GP5-1 determined using checkerboard titration.

[0157] Table 7. Optimal concentration ratio of monoclonal antibody 1C11 to platelet protein GP5-8 determined using checkerboard titration.

[0158] Table 8. Optimal concentration ratio of monoclonal antibody 1A7 to platelet protein GP5-1 determined using checkerboard titration.

[0159] Table 9. Optimal concentration ratio of monoclonal antibody 1A7 to platelet protein GP5-8 determined using checkerboard titration.

[0160] Example 9: Competitive ELISA verification of the blocking effect of mouse serum on monoclonal antibodies Balb / c mice were immunized with recombinant proteins GP5-1 and GP5-8 to prepare polyclonal antibodies against GP5 protein, as follows: Recombinant proteins GP5-1 or GP5-8 were emulsified with Freund's adjuvant and administered subcutaneously to female Balb / c mice twice, 3 weeks apart, with each mouse receiving 30 μg of recombinant protein. The third immunization was performed via intraperitoneal injection of the unadjuvanted recombinant protein. Five days later, blood was collected from the tail vein to determine the serum titer. Once the titer reached 1:10,000 or higher, blood was collected, serum was separated, and cryopreserved.

[0161] The blocking effect of mouse serum on monoclonal antibodies was verified using a competitive ELISA. The procedure was as follows: GP5-1 or GP5-8 protein was coated onto the plate at a concentration of 0.5 μg / mL, 100 μL / well, and incubated overnight at 4°C. Then, the plate was blocked with 300 μL / well of 5% skim milk at 37°C for two hours. The plate was washed three times with PBST, and 150 μL / well of mouse serum diluted 1:1000 was added. The plate was incubated at 37°C for 30 minutes, followed by three washes with PBST. 100 μL / well of HRP-labeled GP5 monoclonal antibody was added, and the plate was incubated at 37°C for 3 minutes, followed by three washes with PBST. 100 μL / well of TMB chromogenic buffer was added, and the plate was incubated in the dark for 10 minutes. Finally, 50 μL of 1 M sulfuric acid was added to each well to stop the reaction.

[0162] Competitive ELISA results showed that the binding between the monoclonal antibody 1C11 and the recombinant protein could be blocked by homologous mouse serum, while heterologous serum also showed blocking ability, albeit weaker. Figure 8 (A). The binding between monoclonal antibody 1A7 and recombinant protein can be blocked by serum from lineage 8 mice, while the blocking ability of serum from lineage 1 is weaker. Figure 8 (B). This result indicates that the specific binding between monoclonal antibody 1C11 and recombinant protein is superior to that between monoclonal antibody 1A7 and recombinant protein.

[0163] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments, and various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. A recombinant protein, characterized in that, The recombinant protein comprises two short peptides tandemly repeated, the short peptides being short peptide A and short peptide B; The amino acid sequence of the short peptide A is as follows: A1) SEQ ID NO:4; or A2) An amino acid sequence that has at least 60% homology with SEQ ID NO:4 and is functionally identical or similar; or A3) The amino acid sequence shown in SEQ ID NO:4 is modified by substitution, deletion or addition of one or more amino acids and has the same or similar function. The amino acid sequence of the short peptide B is as follows: B1) SEQ ID NO:5; or B2) An amino acid sequence that has at least 60% homology with SEQ ID NO:5 and is functionally identical or similar; or B3) The amino acid sequence shown in SEQ ID NO:5 is modified by substitution, deletion or addition of one or more amino acids, and has the same or similar function.

2. The recombinant protein according to claim 1, characterized in that, The repetitions are performed 2-5 times. Preferably, the recombinant protein further includes a linker sequence; Preferably, the connection sequence includes at least one of (ggggs)n, (gggs)n, (ggs)n, (g)n, (GS)n, (eaaak)n, (GGCGGCGGCAGC) or (XP)n, where n is a natural number from 0 to 5; Preferably, the recombinant protein further includes a tag sequence or a Foldon short peptide; Preferably, the tag sequence includes at least one of His tag, GST tag, Flag tag, or HA tag; Preferably, the nucleotide sequence of the Foldon short peptide is as shown in SEQ ID NO:3; Preferably, the amino acid sequence of the short peptide A is as shown in SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8 or SEQ ID NO:10; and / or, the amino acid sequence of the short peptide B is as shown in SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9 or SEQ ID NO:

11.

3. The use of the recombinant protein according to claim 1 or 2 in the preparation of products that recognize porcine reproductive and respiratory syndrome virus; Preferably, the product includes antibodies; Preferably, the antibody comprises a polyclonal antibody or a monoclonal antibody.

4. A method for preparing a monoclonal antibody, comprising the step of immunizing an animal with the recombinant protein of claim 1 or 2 as an immunogen; Preferably, the preparation method further includes fusing spleen cells from immunized animals with myeloma cells and screening to obtain hybridoma cell lines.

5. A monoclonal antibody or its antigen-binding fragment, prepared by the preparation method of claim 4; Preferably, the monoclonal antibody or its antigen-binding fragment is 1C11 or 1A7; wherein, The heavy chain variable region of the 1C11 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO:26-SEQ ID NO:28, respectively. The light chain variable region of the 1C11 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO:29-SEQ ID NO:31, respectively. The heavy chain variable region of the 1A7 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO:32-SEQ ID NO:34, respectively. The light chain variable region of the 1A7 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO:29-SEQ ID NO:31, respectively. The CDR is defined using the IMGT definition scheme; Preferably, the amino acid sequence of the heavy chain variable region of 1C11 is as shown in SEQ ID NO:20, or is an amino acid sequence that is functionally identical or similar to the amino acid sequence shown in SEQ ID NO:20 after substitution, deletion or addition of one or more amino acids. Preferably, the amino acid sequence of the light chain variable region of 1C11 is as shown in SEQ ID NO:22, or is an amino acid sequence that is functionally identical or similar to the amino acid sequence shown in SEQ ID NO:22 after substitution, deletion or addition of one or more amino acids. Preferably, the amino acid sequence of the heavy chain variable region of 1A7 is as shown in SEQ ID NO:24, or is an amino acid sequence that is functionally identical or similar to the amino acid sequence shown in SEQ ID NO:24 after substitution, deletion or addition of one or more amino acids. Preferably, the amino acid sequence of the light chain variable region of 1A7 is as shown in SEQ ID NO:22, or is an amino acid sequence that is functionally identical or similar to the amino acid sequence shown in SEQ ID NO:22 after substitution, deletion or addition of one or more amino acids.

6. Biological material relating to the recombinant protein of claim 1 or 2 or the monoclonal antibody of claim 5 or its antigen-binding fragment, wherein the biological material is any one of a1)-a12): a1) A nucleic acid molecule encoding the recombinant protein of claim 1 or 2 or the monoclonal antibody of claim 5 or its antigen-binding fragment; a2) An expression cassette containing the nucleic acid molecule described in a1); a3) A recombinant vector containing the nucleic acid molecules described in a1); a4) A recombinant vector containing the expression cassette described in a2); a5) Recombinant microorganisms containing the nucleic acid molecules described in a1); a6) Recombinant microorganisms containing the expression cassette described in a2); a7) Recombinant microorganisms containing the recombinant vector described in a3); a8) Recombinant microorganisms containing the recombinant vector described in a4); a9) Transgenic cell lines containing the nucleic acid molecules described in a1); a10) Transgenic cell lines containing the expression cassette described in a2); a11) Transgenic cell lines containing the recombinant vector described in a3); a12) Transgenic cell lines containing the recombinant vector described in a4).

7. A conjugate comprising the recombinant protein of claim 1 or 2 or the monoclonal antibody of claim 5 or an antigen-binding fragment thereof, and a conjugation portion comprising at least one of a detectable marker and an enzyme.

8. The use of the monoclonal antibody or its antigen-binding fragment as described in claim 5, the biological material as described in claim 6, or the conjugate as described in claim 7 in any one of (1)-(3): (1) Prepare products for detecting porcine reproductive and respiratory syndrome virus; (2) Detection of porcine reproductive and respiratory syndrome virus GP5 protein for non-disease diagnosis purposes; (3) Prepare products for detecting porcine reproductive and respiratory syndrome virus GP5 protein.

9. A product comprising the recombinant protein of claim 1 or 2, the monoclonal antibody of claim 5 or its antigen-binding fragment, the biomaterial of claim 6 or the conjugate of claim 7; Preferably, the product includes reagents, kits, test strips, and antibody chips.

10. A method for detecting porcine reproductive and respiratory syndrome virus (PRRSV) GP5 protein for non-diagnostic purposes, comprising the steps of detecting African swine fever virus (ASFV) GP5 protein using the recombinant protein of claim 1 or 2, the monoclonal antibody of claim 5 or its antigen-binding fragment, the conjugate of claim 7, and / or the product of claim 9.

Images