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Specific antibody of campylobacter jejuni specific multi-epitope artificial polypeptide and coated immunomagnetic beads and application thereof

A technology of Campylobacter jejuni and immune magnetic beads, applied in the direction of anti-bacterial immunoglobulin, application, peptide, etc., can solve the problem of small sampling volume, inability to distinguish live bacteria from dead bacteria, and the sensitivity and detection rate of traditional separation methods are reduced To achieve the effect of improving the detection rate, enrichment effect and efficiency, and improving sensitivity

Inactive Publication Date: 2012-07-04
ANIMAL AND PLANT & FOOD DETECTION CENTER JIANGSU ENTRY EXIT INSPECTION AND QUARANTINE BUREAU
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The traditional isolation and cultivation method of C.jejuni takes less samples. After the streaking cultivation, some miscellaneous bacteria quickly absorb the effective nutrients in the medium, which seriously interferes with the isolation and cultivation of C.jejuni, resulting in the sensitivity and detection of traditional isolation methods. The detection rate is reduced; enzyme-linked immunosorbent assay (ELESA), nucleic acid probe hybridization, polymerase chain reaction (PCR), real-time quantitative PCR (Real-time PCR) and other methods are sensitive, specific, and have a high detection rate, but there are Some non-specific reaction, inability to distinguish between live and dead bacteria

Method used

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  • Specific antibody of campylobacter jejuni specific multi-epitope artificial polypeptide and coated immunomagnetic beads and application thereof
  • Specific antibody of campylobacter jejuni specific multi-epitope artificial polypeptide and coated immunomagnetic beads and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] Example 1 Preparation of Campylobacter jejuni-specific multi-epitope artificial polypeptide

[0018] 1. Design and synthesis of artificial polypeptides (also known as Campylobacter jejuni multi-epitope antigen, or C.jejuni multi-epitope antigen recombinant protein, the same below) prepared after tandem expression of multiple epitope specific surface proteins of Campylobacter jejuni And clone

[0019] Use Epitope Conservancy Analysis software to predict the epitopes of 4 specific proteins of Campylobacter jejuni (gi numbers: 6967637, 6678894, 6968218, and 6968454) located on the surface of the bacteria, and select a strong antigenicity from each protein. Epitope, artificially designed a polypeptide, its encoded amino acid sequence is: SEQ ID No.1, length is 53 amino acids; combined with the degeneracy of the prokaryotic expression vector E.coli codon, the designed artificial polypeptide nucleic acid sequence is: SEQ ID No.2, the length is 159bp, and BamH I and Hind III restr...

Embodiment 2

[0024] Example 2 Identification of reactogenicity and immunogenicity of multi-epitope antigen of Campylobacter jejuni

[0025] 1. Immunoblot

[0026] After SDS-PAGE electrophoresis of the C.jejuni multi-epitope antigen recombinant protein purified in Example 1, the gel was electro-transferred to PVNF membrane (Osmonics) using a semi-dry transfer device (Bio-Rad). Seal the transferred PVNF membrane with 5% skimmed milk powder solution, add 1:200 diluted rabbit anti-C.jejuni whole bacteria positive serum, wash the membrane after 1.5h at 37°C; add 1:10000 diluted horseradish peroxidation HRP-labeled rabbit anti-goat IgG, washed at 37°C for 1 hour, and washed with 3,3-diaminobenzidine (DAB) for 5 minutes, showing a single specific band, similar in size to the recombinant protein , Indicating that the prepared C.jejuni multi-epitope antigen recombinant protein has good reactogenicity.

[0027] 2. Rabbit immunity test

[0028] The C.jejuni multi-epitope antigen recombinant protein purifie...

Embodiment 3

[0032] Example 3 Preparation of immunomagnetic beads coated with specific antibodies of Campylobacter jejuni

[0033] 1. Take 165μL of magnetic bead suspension (containing 5mg of magnetic beads) into a centrifuge tube, and use a magnetic stand to precipitate the magnetic beads (hereinafter referred to as magnetic precipitation. The specific operation steps are to put the centrifuge tube on the magnetic stand, and wait until the upper liquid becomes After clarification, discard the supernatant, keep the magnetic beads at the bottom, and remove the magnetic stand);

[0034] 2. Add 1mL magnetic bead coupling buffer, resuspend the magnetic beads, and magnetically precipitate;

[0035] 3. Add 75.7μL of magnetic bead coupling buffer and 74.3μL of the purified Campylobacter jejuni specific antibody (containing 100μg of antibody) prepared in Example 2 and mix well, then add 100μL of treatment solution and mix well, and place on a shaker at 37°C After 18 hours of incubation, magnetic precipi...

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Abstract

The invention discloses a specific antibody of a campylobacter jejuni specific multi-epitope artificial polypeptide and coated immunomagnetic beads and an application thereof. The amino acid sequence of the artificial polypeptide prepared after tandem expression of a campylobacter jejuni specific surface protein multi-antigen epitope is SEQ ID No.1. A nucleotide sequence for encoding the campylobacter jejuni specific surface protein multi-antigen epitope is SEQ ID No.2. A kit for enriching campylobacter jejuni comprises immunomagnetic beads containing a specific antibody coated with the campylobacter jejuni specific multi-epitope artificial polypeptide. By applying the immunomagnetic beads or the kit, campylobacter jejuni can be enriched from a sample pre-enrichment liquid, and the sensitivity and detectable rate of subsequent separate culturing or PCR (Polymerase Chain Reaction) detection are increased greatly; and the specific antibody has the advantages of high specificity, high sensitivity, easiness, convenience, rapidness and suitability for large-scale sample detection.

Description

Technical field [0001] The invention belongs to the field of biological detection, and relates to a specific antibody of Campylobacter jejuni-specific multi-epitope artificial polypeptide and its coated immunomagnetic beads and applications. Background technique [0002] Campylobacter jejuni (C.jejuni) exists in large numbers in the intestines of various wild or domestic animals. Contaminated water or food can cause an outbreak of diarrhea. Infected animals often have no obvious symptoms and carry it for a long time. bacteria. At present, C.jejuni infection has become one of the most common causes of acute gastroenteritis worldwide. In developing countries, it is an important cause of death from children’s diarrhea and the most common cause of traveler’s diarrhea. In the United States and other industrialized countries, diarrhea caused by Campylobacter jejuni infection is Salmonella, Shigella or E. coli (O157: H7) Diarrhea caused by 2 to 7 times. The bacteria can also cause Gu...

Claims

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Application Information

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IPC IPC(8): C07K14/205C12N15/31C07K16/12C12Q1/24C12Q1/04C12Q1/68C12R1/01
Inventor 唐泰山祝长青蒋原张睿张常印薛峰栾军王凯民姜焱
Owner ANIMAL AND PLANT & FOOD DETECTION CENTER JIANGSU ENTRY EXIT INSPECTION AND QUARANTINE BUREAU
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