# US 2025/0155432 A1 · NANOBODY CLASSICAL SWINE FEVER VIRUS (CSFV)-E0-Nb1 AGAINST CSFV E0 PROTEIN, GENE ENCODING THE SAME AND USE THEREOF > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 2025/0155432 A1 - Document ID: US20250155432A1 - Status: Published application - Kind: A1 - Application number: 18/884,570 - Filed: 2024-09-13 - Published: 2025-05-15 - Earliest claimed: 2023-11-09 - Assignees: Qingdao Agricultural University; Innovus Solarex Biotech Co., Ltd. - Applicants: Qingdao Agricultural University; Innovus Solarex Biotech Co., Ltd. - Inventors: Zhi CAO; Dehua YIN; Wenling LI; Lihong ZHANG; Siyu LI; Yixin ZHANG; Xiaoduo WANG; Shuo LI - Web page: https://patentgrep.com/patent/US20250155432A1 ## CPC - G01N 33/54388: {based on lateral flow} - C07K 16/116: Togaviridae (F); Matonaviridae (F); Flaviviridae (F) - G01N 33/56983: {Viruses} - G01N 33/6857: {Antibody fragments} - C07K 2317/22: from camelids, e.g. camel, llama or dromedary - C07K 2317/569: Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody® - G01N 2333/183: Flaviviridae, e.g. pestivirus, mucosal disease virus, bovine viral diarrhoea virus, classical swine fever virus (hog cholera virus) or border disease virus - G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q) - C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00) ## Abstract A nanobody classical swine fever virus (CSFV)-E0-Nb1 against a CSFV E0 protein, and an encoding gene and use thereof are provided, belonging to the technical field of biological detection. The nanobody CSFV-E0-Nb1 has an amino acid sequence shown in SEQ ID NO: 1 and can be expressed using an expression system. The nanobody is coupled with a quantum dot to obtain an immunochromatographic test strip for distinguishing antibodies against a CSFV E2 subunit vaccine strain from those of a wild strain infected on site, and there is a simple production process of the test strip. The immunochromatographic test strip can differentiate and diagnose the antibodies against the CSFV E2 subunit vaccine strain and the wild strain, and has the advantages of rapid, convenient, and instant detection, thus providing a new method for the detection of classical swine fever (CSF) purification. ## Claims ### Claim 1 A nanobody classical swine fever virus (CSFV)-E0-Nb1 against CSFV E0 protein, wherein the nanobody CSFV-E0-Nb1 has the amino acid sequence of SEQ ID NO: 1. ### Claim 2 (depends on 1) A nucleotide molecule encoding the nanobody CSFV-E0-Nb1 according to claim 1. ### Claim 3 (depends on 2) The nucleotide molecule according to claim 2, wherein the nucleotide molecule has the nucleotide sequence of SEQ ID NO: 2. ### Claim 4 (depends on 1) A method for expressing the nanobody CSFV-E0-Nb1 according to claim 1, comprising: constructing a recombinant expression vector by ligating a nucleotide molecule encoding the nanobody CSFV-E0-Nb1 to an expression vector, transforming the recombinant expression vector into a host cell to allow induced expression, and subjecting a resulting expressed product to purification to obtain the nanobody CSFV-E0-Nb1. ### Claim 5 (depends on 4) The method according to claim 4, wherein the nucleotide molecule has the nucleotide sequence of SEQ ID NO: 2. ### Claim 6 (depends on 4) The method according to claim 4, wherein the expression vector comprises a prokaryotic expression vector. ### Claim 7 (depends on 1) An immunochromatographic test strip for antibodies against a CSFV E2 subunit vaccine strain and a wild strain, comprising a gold-labeled pad, a test line, and a control line; wherein the gold-labeled pad is coated with a quantum dot-coupled CSFV E0 protein and a quantum dot-coupled CSFV E2 protein; and the quantum dot-coupled CSFV E0 protein and the quantum dot-coupled CSFV E2 protein are used for a test line T1 and a test line T2, respectively, and the nanobody CSFV-E0-Nb1 according to claim 1 and a CSFV E2 protein-specific nanobody are used for a control line C1 and a control line C2, respectively. ### Claim 8 (depends on 7) The immunochromatographic test strip according to claim 7, wherein a preparation process of a quantum dot-coupled protein comprises: incubating a water-soluble quantum dot with a 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl) solution and an N-hydroxysuccinimide (NHS) solution in the dark; incubating a resulting first incubated product with β-mercaptoethanol and a protein; and subjecting a resulting second incubated product to blocking, centrifuging a resulting blocked product, and re-dissolving a resulting precipitate to obtain the quantum dot-coupled protein. ### Claim 9 (depends on 8) The immunochromatographic test strip according to claim 8, wherein the protein and the quantum dot are coupled at a pH value of 6 to 8; the quantum dot and an activator are at a mass ratio of 1:2 to 1:8; and the protein and the quantum dot are coupled at a mass ratio of 1:1 to 1:15. ### Claim 10 (depends on 7) The immunochromatographic test strip according to claim 7, wherein the test line is labeled with 50 μg/line to 150 μg/line of the protein; and the control line is labeled with 75 μg/line to 175 μg/line of the nanobody. ### Claim 11 (depends on 7) The immunochromatographic test strip according to claim 7, wherein when the test line T1 is labeled with the quantum dot-coupled CSFV E0 protein, the test line T2 is labeled with the quantum dot-coupled CSFV E2 protein, the control line C1 is labeled with the CSFV-E0-Nb1 nanobody, and the control line C2 is labeled with the CSFV E2 protein-specific nanobody; if there is no band on each of the test line T1, the test line T2, the control line C1, and the control line C2, it is determined that detection is unsuccessful and needs to be repeated; if there is one or no band on the control line C1 or the control line C2, it is determined that the detection is unsuccessful and needs to be repeated; if there is a band on each of the test line T1, the control line C1, and the control line C2 but there is no band on the test line T2, it is determined that the detection is unsuccessful and needs to be repeated; if there is a band on each of the test line T1, the test line T2, the control line C1, and the control line C2, it is determined that there is an antibody produced by the wild strain; if there is a band on each of the test line T2, the control line C1, and the control line C2 but there is no band on the test line T1, it is determined that there is an antibody produced by the CSFV E2 subunit vaccine strain; and if there is a band on each of the control line C1 and the control line C2 but there is no band on each of the test line T1 and the test line T2, it is determined that a detection result is negative and no CSFV antibody is produced.