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Application of haemophilus parasuis CdtB hybridoma cell and monoclonal antibody

A monoclonal antibody, Haemophilus suis technology, applied in the direction of antibodies, antibacterial drugs, antibacterial immunoglobulin, etc., can solve the problem of no vaccine immunity and wild virus infection, the prevention and treatment of Haemophilus parasuis disease Diagnosis and treatment difficulties, poor protection and other problems

Active Publication Date: 2020-10-20
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the large number of serotypes of Haemophilus parasuis, the inactivated vaccine prepared by using one or several serotype strains has poor protection against different serotype strains, and even has cross protection against isolates of the same serotype in different regions. are very limited, and there is no reliable method to distinguish vaccine immunity from wild virus infection. The lack of these detection methods makes the prevention, diagnosis and treatment of Haemophilus parasuis difficult.

Method used

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  • Application of haemophilus parasuis CdtB hybridoma cell and monoclonal antibody
  • Application of haemophilus parasuis CdtB hybridoma cell and monoclonal antibody
  • Application of haemophilus parasuis CdtB hybridoma cell and monoclonal antibody

Examples

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Effect test

Embodiment 1

[0037] Example 1 Screening and Application of Monoclonal Antibody 4F10

[0038] 1. Cloning of the CdtB gene of Haemophilus parasuis

[0039] According to the CdtB gene sequence of Haemophilus parasuis SH0165 strain published by GenBank, two pairs of PCR amplification primers CdtB30a-F, CdtB30a-R and CdtB6p1-F, CdtB6p1-R were designed by using Primer 5 software, and included in the upstream and downstream primers respectively. EcoRI and XhoI restriction sites were introduced, and the primers were synthesized by Nanjing GenScript Biotechnology Co., Ltd.

[0040] Use the extracted genomic DNA of Haemophilus parasuis SH0165 as a template to amplify the target gene by PCR. The PCR reaction system is: Prime Star 25 μL, upstream and downstream primers 1 μL, template 1 μL, ddH 2 O to make up to 50 μL. The PCR reaction program was: pre-denaturation at 98°C for 3 min; denaturation at 98°C for 10 s, annealing at 60°C for 10 s, extension at 72°C for 15 s, 33 cycles; and finally extensio...

Embodiment 2

[0077] Example 2 Identification of Monoclonal Antibody 4F10 Subclass and Its Recognized Antigen Epitope

[0078] 1. Subclass identification of monoclonal antibody 4F10

[0079] The monoclonal antibody subtype rapid identification kit (Roche Company) was used for identification, and the operation process was referred to the kit manual. The monoclonal antibody 4F10 diluted with phosphate buffer was incubated with the latex particles for 30 minutes and then added to the test strip to react with it for 5-10 minutes. The results showed that the subclass of the antibody was IgG1 and the light chain was κ.

[0080] 2. Identification of epitopes recognized by monoclonal antibody 4F10

[0081] 1) According to software analysis (http: / / tools.iedb.org / main / bcell / ), the complete sequence of CdtB protein (SEQ ID NO.1) is divided into three overlapping segments A1 (SEQ ID NO.2), A2 (SEQ ID NO.3), A3 (SEQ ID NO.4) for truncated expression ( Figure 11 ), and the monoclonal antibody 4F10 w...

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Abstract

The invention discloses application of a haemophilus parasuis CdtB hybridoma cell and a monoclonal antibody, and belongs to the field of detection, diagnosis and treatment of animal infectious diseases. The preservation number of the haemophilus parasuis CdtB hybridoma cell is CCTCC NO:C201910, the subclass of the monoclonal antibody generated by the haemophilus parasuis CdtB hybridoma cell is IgG1, the light chain type is kappa, and the recognized epitope amino acid sequence is highly conservative in haemophilus parasuis. The monoclonal antibody disclosed by the invention is high in titer, can identify all serotype haemophilus parasuis, and also has neutralizing activity against CdtB. The monoclonal antibody and the epitope amino acid sequence recognized by the monoclonal antibody can bewidely applied to etiological diagnosis of haemophilus parasuis, serological diagnosis, immunological detection, disease prevention and treatment, wild virus infection and vaccine distinguishing and the like of haemophilus parasuis, and are used for preparing products related to the application.

Description

technical field [0001] The invention relates to the field of detection, diagnosis and treatment of animal infectious diseases, in particular to the application of a Haemophilus parasuis CdtB hybridoma cell and a monoclonal antibody. Background technique [0002] Haemophilus parasuis (HPS) is a Gram-negative bacterium belonging to the genus Haemophilus of Pasteurellaceae. It is a pathogen of porcine respiratory diseases. A common respiratory commensal bacterium. In cases of disease, it can cause Glasser's disease in porcine, characterized by polyserositis, meningitis, arthritis in pigs, mainly in piglets of 5-8 weeks of age. Currently, Haemophilus parasuis disease in pigs has caused serious economic losses worldwide. [0003] The traditional diagnostic methods of Haemophilus parasuis mainly include: autopsy of dead pigs and isolation and identification of bacteria, biochemical identification, serological identification, and detection and identification based on PCR. Howeve...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/12C12N5/20G01N33/68G01N33/577G01N33/569A61K39/395A61P31/04
CPCA61K2039/505A61P31/04C07K16/1242G01N33/56911G01N33/577G01N33/68G01N2410/00
Inventor 王湘如陈启超
Owner HUAZHONG AGRI UNIV
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