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Delayed hypersensitivity epitope polypeptide of mycobacterium bovis cfp-10 and its application

A CFP-10, Mycobacterium bovis technology, applied in the application, fusion polypeptide, peptide and other directions, can solve the problem of poor specificity in diagnosing tuberculosis, and achieve the effects of easy mass production, good activity and short production cycle

Active Publication Date: 2018-07-31
NORTHEAST AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] At present, the most commonly used diagnostic method for tuberculosis infection clinically is PPD intradermal TST method, and it is also the only standard method for diagnosing bovine tuberculosis recommended by OIE. However, since PPD is a mixture containing many mycobacterial protein antigens, therefore , which has poor specificity for diagnosing tuberculosis

Method used

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  • Delayed hypersensitivity epitope polypeptide of mycobacterium bovis cfp-10 and its application
  • Delayed hypersensitivity epitope polypeptide of mycobacterium bovis cfp-10 and its application
  • Delayed hypersensitivity epitope polypeptide of mycobacterium bovis cfp-10 and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0088] Identification of the Delayed Hypersensitivity Epitope of Example 1 Mycobacterium bovis CFP-10

[0089] method:

[0090] 1. Identification of delayed-type allergic reaction of Mycobacterium bovis CFP-10

[0091] 1.1 Primer Design for the Amplification of Mycobacterium bovis CFP-10 Gene

[0092] Design a pair of primers according to the CFP-10 gene sequence of Mycobacterium bovis H37Rv strain (NC_000962.3) registered in GenBank, introduce a protective base and an EcoRI restriction site at the 5' end of the forward upstream primer, and introduce a reverse downstream primer A protective base, a HindIII restriction site and a stop codon were introduced into the 5' end (see Table 1). The underlined part is the restriction endonuclease restriction site, and the primers were synthesized by Beijing Huada Gene Company.

[0093] Table 1 Mycobacterium bovis CFP-10 gene amplification primers

[0094]

[0095] 1.2 PCR reaction and PCR product recovery

[0096] Amplify using g...

Embodiment 2

[0216] Example 2 Preparation and preliminary application of bovine tuberculosis new skin test diagnostic antigen

[0217] method:

[0218] 1. Design, optimization and synthesis of skin test diagnostic antigen gene

[0219] The identified DTH antigen peptide C-P C (shown in SEQ ID NO.1, 20aa) and C-P B7 (shown in SEQ ID NO.2, 25aa) and M-P 11 (shown in SEQ ID NO.5, 25aa) is connected in series, as image 3 As shown, at the B position, that is, the Linker (amino acid sequence is GGGGS) is connected between the antigen peptides, and the Linker is also introduced at the end of the tandem peptide (C site), but the difference is that it encodes the last two amino acids of the Linker (GS) The nucleotide sequence must be a BamHI restriction site. In addition, a BglII restriction site is introduced at the front end (site A) of the tandem peptide nucleotide sequence, an XhoI restriction site is introduced at the end (E site), and a stop codon is introduced before the XhoI restricti...

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Abstract

The invention discloses delayed-type allergic reaction antigen epitope polypeptides of mycobacterium bovis CFP-10 and an application thereof. With utilization of an overlapped short peptide piecewise expression technology, identification of the delayed-type allergic reaction antigen peptides is carried out on the CFP-10, and antigen peptides C-PC and C-PB7 having delayed-type allergic reaction are finally identified. The identified antigen peptides C-PC and C-PB7 are connected with M-P11 through Linker by means of genetic engineering, after genetic optimization, a tandem peptide gene CCM is obtained, and simultaneously, a multicopy tandem peptide gene containing multiple CCMs is constructed. The identified antigen peptides and the tandem peptides are subjected to delayed-type allergic reaction identification, and a result show that the C-PB7, the C-PC, the CCM and the 2CCM all can produce relatively strong delayed-type allergic reaction. A problem of poor PPD specificity in a tuberculin skin test (TST) diagnosis is improved, and in addition, the diagnostic sensitivity is increased by increasing the types of the tandem peptides. Therefore, new ideas are put forward for development of bovine tuberculosis skin test diagnostic preparations, and a good application prospect is provided.

Description

technical field [0001] The invention relates to an antigen epitope polypeptide, in particular to a delayed-type allergy antigen epitope polypeptide of mycobacterium bovis CFP-10, and also relates to the application of the epitope polypeptide in detecting mycobacterium bovis infection. The invention belongs to the field of biotechnology. Background technique [0002] Bovine tuberculosis (Bovine tuberculosis) is mainly caused by Mycobacterium bovis (Mycobacterium bovis), a chronic consumptive zoonosis, which is characterized by the formation of tuberculous nodules, granulomas, and caseous Necrotic and calcified lesions. The incubation period of bovine tuberculosis varies and is usually chronic. In the early stage of infection, there are generally no typical symptoms. The main manifestations are loss of spirit and appetite, gradual weight loss, messy and dull coat, inelastic skin, and gradually reduced milk production. Due to the infection site and The degree of damage to the...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/35C07K19/00C12N15/31G01N33/68G01N33/569
CPCC07K14/35C07K2319/00G01N33/5695G01N33/68
Inventor 王君伟刘洪涛高明春
Owner NORTHEAST AGRICULTURAL UNIVERSITY
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