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CN-122012403-A - Pigeon I-type paramyxovirus HN protein monoclonal antibody, preparation and application thereof

CN122012403ACN 122012403 ACN122012403 ACN 122012403ACN-122012403-A

Abstract

The invention discloses a pigeon I-type paramyxovirus HN protein monoclonal antibody, preparation and application thereof, wherein the monoclonal antibody is secreted by a hybridoma positive cell strain 4H3 which secretes pigeon I-type paramyxovirus HN protein, the hybridoma positive cell strain 4H3 is preserved in China academy of sciences microbiological study at 2023 8 and 3 days, the address is North Star Xiu No. 1, no. 3 in the Korean region of Beijing, and the preservation number is CGMCC No.45648. The invention solves the problem that HN protein specific monoclonal antibodies are absent in the current pigeon I-type paramyxovirus clinical detection and basic research process. The monoclonal antibody secreted by the 4H3 can specifically react with pigeon-derived newcastle disease virus and chicken-derived newcastle disease virus, but does not react with a clinical common vaccine strain Lasota, and the antibody 4H3 has high potency, is easy for mass production and has a very wide application prospect.

Inventors

  • LIANG LIBIN
  • LI JUNPING
  • YANG YUHAN
  • MA HAILI
  • ZHAO JINGJING
  • YANG HONGWEI

Assignees

  • 山西农业大学

Dates

Publication Date
20260512
Application Date
20231205

Claims (10)

  1. 1. A monoclonal antibody hybridoma positive cell strain 4H3 secreting pigeon I paramyxovirus HN protein is characterized in that the hybridoma positive cell strain 4H3 is preserved in the institute of microbiology of China academy of sciences at 2023, address: north Star Xiya No. 1 of the Korean region of Beijing, 3, preservation number: CGMCC No.45648, and the heavy chain subclass of the hybridoma positive cell strain 4H3 is IgG2a and the light chain subclass is kappa chain.
  2. 2. A pigeon type I paramyxovirus HN protein monoclonal antibody, wherein the monoclonal antibody is secreted by the pigeon type I paramyxovirus HN protein-secreting monoclonal antibody hybridoma positive cell line 4H3 of claim 1; Wherein, the amino acid sequence shown as SEQ ID NO.1 is selected as the immunogen of the pigeon I-type paramyxovirus HN protein.
  3. 3. The pigeon type I paramyxovirus HN protein monoclonal antibody of claim 1, wherein the epitope of the monoclonal antibody is on the pigeon type I paramyxovirus HN protein.
  4. 4. The pigeon type I paramyxovirus HN protein monoclonal antibody according to claim 1, wherein the immunogen of pigeon type I paramyxovirus HN protein is produced by the following method: (1) Synthesizing 198-390 amino acid sequences of codon-optimized pigeon I-type paramyxovirus HN proteins into a pET30a expression vector through EcoRI/XhoI enzyme cleavage site genes, transforming an escherichia coli BL21 expression strain, selecting a monoclonal from the transformed flat plate into an LB liquid culture medium containing kanamycin resistance, and culturing and activating at 37 ℃; (2) Inoculating the activated bacterial liquid into LB liquid culture medium containing kanamycin resistance, and culturing at 37 ℃; (3) Transferring the cultured bacterial liquid into LB liquid culture medium containing kanamycin resistance, culturing at 37 ℃ until the Optical Density (OD) is 0.6-0.8, and inducing overnight at 16 ℃ with the concentration of isopropyl-beta-D-thiogalactoside being 0.5mM; (4) Centrifuging and collecting the induced thalli overnight, discarding the supernatant, then performing ultrasonic sterilization, and purifying the protein after ultrasonic cleavage to obtain the immunogen of the pigeon I-type paramyxovirus HN protein.
  5. 5. The pigeon I-type paramyxovirus HN protein monoclonal antibody according to claim 4, wherein in the step (4), the bacterial harvesting is performed at 6000rpm for 8min, the ultrasonic bacterial disruption is performed by blowing off bacterial cells with a tris (hydroxymethyl) aminomethane hydrochloride solution with a pH of 8.0, 20-30 mL and 10mM, the power of the ultrasonic disruption is 500W, the times are 180 times, the time of each ultrasonic disruption is 5s, and the interval time between adjacent disruption is 5s.
  6. 6. The monoclonal antibody of the pigeon type I paramyxovirus HN protein according to claim 4, wherein the 37 ℃ culture is performed at 200rpm in steps (1) - (3).
  7. 7. Use of a pigeon type I paramyxovirus HN protein monoclonal antibody according to any one of claims 1-6 in the field of detection of newcastle disease virus.
  8. 8. The use according to claim 7, wherein the newcastle disease virus comprises pigeon-derived newcastle disease virus and/or chicken-derived newcastle disease virus.
  9. 9. The use according to claim 8, wherein the pigeon-derived newcastle disease virus is PG/SX/01 and/or PG/JX/812.
  10. 10. The use according to claim 8, wherein the chicken-derived newcastle disease virus is CK/SX/01 and/or CK/HuN/905.

Description

Pigeon I-type paramyxovirus HN protein monoclonal antibody, preparation and application thereof Technical Field The invention relates to a hybridoma cell strain of a monoclonal antibody, in particular to a pigeon I-type paramyxovirus HN protein monoclonal antibody, and preparation and application thereof. Background The pigeon newcastle disease is an acute and septic infectious disease caused by pigeon paramyxovirus type I (Pigeon Paramyxovirus, PPMV-1), can cause nervous symptoms, enteritis, diarrhea and other digestive tract symptoms of pigeons, and is different from chicken newcastle disease Virus (NEWCASTLE DISEASE Virus, NDV) in that PPMV-1 hosts are often limited to pigeons and wild birds. In recent years, the newcastle disease of the pigeons has outbreaks in the multiple provinces of meat pigeons and racing pigeons in China, and causes great harm to the pigeon raising industry. The pigeon paramyxovirus type I is a cyst membrane virus, the virus diameter is about 200-300 nm, the genome is a single-strand negative-strand RNA which is not segmented, and the genome length is 15192nt, similar to the newcastle disease virus. The pigeon paramyxovirus type I genome includes a 3 '-end 55nt leader sequence and a trailer' -end 114nt sequence, and the intermediate coding region encodes 6 essential proteins, including nucleocapsid protein (Nucleocapsidprotein, NP), phosphoprotein (Phosphorprotein, P), matrix protein (Matrixprotein, M), fusion protein (Fusionprotein, F), hemagglutinin-neuraminidase protein (Heamagglutinin-Neuraminidase protein, HN) and macromolecular protein (Large protein, L). Wherein the NP structural protein participates in viral genome replication and forms a viral nucleocapsid together with L protein, P protein and RNA of viral genome, and HN protein and F protein are two glycoproteins on the viral surface. HN protein plays a plurality of important functions in the life cycle of I-type pigeon paramyxovirus, and mainly comprises that HN protein can be combined with sialic acid receptor on the surface of cells to promote the adsorption of viruses and host cells, HN protein can enhance the fusion activity of F protein to promote the invasion of viruses, and HN protein also has neuraminidase activity to cut sialic acid on a sugar side chain and plays a role in the release stage of progeny virus particles from the surface of infected cells. And HN protein is one of main protective antigens of pigeon newcastle disease virus and plays an important role in the pathogenic process of the virus. NDV has only one serotype, but there is a large difference between their genomes, and NDV is currently divided into two major classes, classI and ClassII. Wherein, the ClassII type gene II, gene IV type and gene VII type NDV strains are separated in pigeon bodies, but the type VI gene is popular in pigeon, the type VI gene comprises the subtype VIa-VIk and the like, and the literature 1 (Pei Yo and the like, genome sequence and pathogenicity analysis of 4 pigeon newcastle disease viruses [ J ], virology report, 2022,38 (2): 402-413) indicates that the pigeon group separation strain in China is mainly of the type VIb. At present, the main epidemic gene VII type Newcastle disease in chicken flocks in China has weak virus removal capability and poor immune effect due to mismatching of the commonly used Lasota vaccine strain and the gene VII type Newcastle disease virus genotype and antigenicity. Along with the creation and popularization of the gene VII type Newcastle disease inactivated vaccine, document 2 (Liu Xiufan, creation of the gene VII type new vaccine and prevention and control progress of the Newcastle disease in China [ J ], veterinarian guide, 2020 (19): 4-5) shows that the gene VII type Newcastle disease inactivated vaccine plays an important promotion role in prevention and control of the poultry Newcastle disease. However, as the pigeon newcastle disease does not have commercial vaccine at present, the applied vaccine for chickens such as Lasota and the like can not provide effective protection for the pigeon newcastle disease, and the pigeon newcastle disease is aggravated in the pigeon crowd, which can cause serious harm to the pigeon industry. Disclosure of Invention The invention aims to provide a pigeon I-type paramyxovirus HN protein monoclonal antibody, and preparation and application thereof, and solves the problem that HN protein specific monoclonal antibodies are absent in the current pigeon I-type paramyxovirus clinical detection and basic research processes. In order to achieve the aim, the invention provides a monoclonal antibody hybridoma positive cell strain 4H3 secreting pigeon I paramyxovirus HN protein, wherein the hybridoma positive cell strain 4H3 is preserved in the institute of microbiology of China academy of sciences at 8 month 3 of 2023, the address is North Star Xiya No. 1 of Beijing, the preservation number is CGMCC No.45648, the heavy chain subclass of the hybridoma posit