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IgY antibody for resisting botulinus toxin substrate peptide SubA as well as preparation method and application thereof

A technology of botulinum toxin and substrate peptide, which is applied to the preparation method of peptides, chemical instruments and methods, peptides, etc., can solve the problems that have not been seen in the detection and analysis of chicken yolk immunoglobulin botulinum toxin, and achieve convenient toxicity The effect of screening method, good stability and high analysis efficiency

Inactive Publication Date: 2010-02-17
MICROBE EPIDEMIC DISEASE INST OF PLA MILITARY MEDICAL ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] So far, there is no report on the detection and analysis of botulinum toxin by using SNAP-25 protein to prepare chicken yolk immunoglobulin (IgY antibody)

Method used

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  • IgY antibody for resisting botulinus toxin substrate peptide SubA as well as preparation method and application thereof
  • IgY antibody for resisting botulinus toxin substrate peptide SubA as well as preparation method and application thereof
  • IgY antibody for resisting botulinus toxin substrate peptide SubA as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Expression and purification of embodiment 1 recombinant SNAP-25

[0027] 1. Materials

[0028] 1.1 Strains

[0029] The engineering strain pET-SNAP25 / BL21 was constructed using molecular biology methods (Liu Hao et al., Journal of the Academy of Military Medical Sciences, 2009). According to the SNAP25 coding gene sequence reported in GenBank, specific primers were designed, and the whole brain of mice was obtained by RT-PCR. The SNAP25 gene was obtained from the total tissue RNA, and the full-length gene was cloned into the prokaryotic expression vector pET22b, and the obtained recombinant plasmid was transformed into E. coli BL21 to construct an engineering strain pET-SNAP25 / BL21 for the expression of exogenous protein SNAP25.

[0030] 1.2 Reagents

[0031] Ni affinity chromatography column HisTRAP FF TM Purchased from GE Company.

[0032] 2. Methods and Results

[0033] 2.1 Induced expression of recombinant SNAP-25 in Escherichia coli

[0034] Inoculate enginee...

Embodiment 2

[0040] Example 2 Preparation of IgY Antibody Specific Anti-Botulinum Toxin Substrate Peptide SubA

[0041] 1. Materials

[0042] Laying Laihang chickens were provided by Beijing Experimental Animal Center, and complete and incomplete adjuvants were purchased from Sigma. The sequence of the substrate peptide SubA is 188NKTRIDEANQ197, as shown in Sequence 1 in the sequence listing. SubA and its modified polypeptide KLP-SubA were synthesized by Shanghai Botai Biotechnology Co., Ltd. with a purity of 90%.

[0043] 2. Methods and Results

[0044] 2.1 Animal immunity

[0045] The synthetic short peptide KLP-SubA antigen was used to immunize SPF Laihang layer hens. For the first immunization, Freund's complete adjuvant was mixed with the short peptide at a ratio of 1:1, and injected at a dose of 1.0 mg / kg after emulsification. The 2nd, 3rd, and 4th immunizations were carried out at 3w, 6w, and 11w with complete adjuvant, respectively. Collect immunized eggs.

[0046] 2.2 Extract...

Embodiment 3I

[0061] Application of Example 3 IgY Antibody in the Detection of Type A Botulinum Toxin

[0062] 1. Materials

[0063] The SpectroPlus automatic enzyme-linked immunosorbent detection instrument was a product of MD Company, anti-IgY-HRP was purchased from SIGMA Company, and enzyme-linked plates and color reagents were purchased from Biokang Company.

[0064] 2. Methods and Results

[0065] 2.1 Analysis of standard botulinum toxin type A

[0066] The operation steps are:

[0067] Coat triple wells of SNAP-25 dissolved in Ni-NTA elution buffer (0.02M sodium phosphate, 0.5M sodium chloride, 0.4M imidazole, pH7.4) on an enzyme-linked plate, 0.4 μg per well , overnight at 4°C;

[0068] Pat off the coating solution, wash three times with 0.1% PBST, 200 μl per well, 3 min each time;

[0069] Block with 3% BSA, 200 μl per well, room temperature for 2 hours;

[0070] Take off the blocking solution, dissolve in cleavage reaction buffer (50mmol / L HEPES, 2mmol / LDTT, 10μmol / L ZnCl 2 , ...

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Abstract

The invention discloses an IgY antibody for resisting botulinus toxin substrate peptide, which can be prepared by the following steps: preparing SNAP25 short peptide subA by a chemical synthesis method or a gene recombination expression method; collecting eggs by subA immune laying chicken and extracting and purifying yelk immunoglobulin by a biochemical method. The antibody has the characteristics of high specificity of combining substrate linear short peptide subA, but not combining integral substrate SNAP25, has the capability of especially identifying zymolytic SNAP25 of A botulinus toxin,can be used as a detection reagent to be applied to the detection of the A botulinus toxin and the activity analysis of neurovirulence or endopeptidase as well as the poisoning diagnosis of clinicalkreotoxism, and has good market prospect and social significance.

Description

technical field [0001] The present invention relates to an IgY antibody, in particular to an anti-botulinum toxin substrate peptide IgY antibody, and to a preparation method and application of the antibody. Background technique [0002] Botulinum toxin (botulinum neurotoxin, BoNT) is the most toxic substance known in the world. It is produced by Clostridium botulinum under anaerobic conditions. There are 7 serotypes (A-G). The pathogenic types are mainly A, B, and E. And type A botulinum toxin can cause more severe symptoms of poisoning than other types, resulting in higher mortality. The half lethal dose LD50 of the human body is only 0.1-1ng / kg. Poisoning usually manifests as paralysis of eye and throat muscles, and then develops into general skeletal muscle paralysis, and finally dies due to respiratory failure. In recent years, botulism incidents have occurred frequently. Although botulism mainly occurs in the form of food contamination, wound infection, and infant int...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/12C07K16/02C07K1/14C12P21/06G01N33/53G01N33/543G01N33/569
Inventor 王慧史晶刘昊侯晓军王琴李涛
Owner MICROBE EPIDEMIC DISEASE INST OF PLA MILITARY MEDICAL ACAD OF SCI
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