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Anti-I type Shiga toxin IgY antibody as well as preparation method and use thereof

A technology of Shiga toxin and antibody, which is applied in the preparation of the antibody and the field of IgY antibody specific to type I Shiga toxin, can solve the problems that have not been reported, achieve the effect of inhibiting the toxicity of Shiga toxin and have a good application prospect

Active Publication Date: 2009-11-04
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

At present, there is no report on the IgY antibody against Shiga toxin at home and abroad

Method used

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  • Anti-I type Shiga toxin IgY antibody as well as preparation method and use thereof
  • Anti-I type Shiga toxin IgY antibody as well as preparation method and use thereof
  • Anti-I type Shiga toxin IgY antibody as well as preparation method and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Expression and purification of embodiment 1 recombinant Stx1B

[0019] 1. Materials

[0020] 1.1 Strains

[0021] The engineering strain pBV220-stx1b / DH5α was constructed by our laboratory for the expression of foreign protein Stx1B.

[0022]1.2 Reagents

[0023] Ion Exchange Chromatography Column Sepharose TM HP HTrap TM And Sephacry S-100 gel column (26 / 100) was purchased from GE Company.

[0024] 2. Methods and results

[0025] 2.1 Induced expression of recombinant StxB in Escherichia coli

[0026] Inoculate the engineered strain pBV220-stx1b / DH5α into LB liquid medium containing 50 μg / mL ampicillin, shake overnight at 37°C, inoculate into LB liquid medium at a ratio of 1:100 the next day, and continue shaking culture at 37°C until the bacterial liquid Concentration reaches OD 600 0.8 ~ 1.0, quickly raise the temperature to 41 ℃, continue to cultivate 5h. Centrifuge at 5000g for 10min at 4°C, wash the bacteria once with pre-cooled PBS, collect the bacteria ...

Embodiment 2

[0032] Example 2 Preparation of anti-Shiga toxin type I IgY antibody

[0033] 1. Materials

[0034] Laying Laihang chickens were provided by Beijing Experimental Animal Center, and complete and incomplete adjuvants were purchased from Sigma.

[0035] 2. Methods and results

[0036] 2.1 Animal immunization (Table 1)

[0037] Mix the purified recombinant Stx1B with Freund's adjuvant (lanolin: liquid paraffin = 1:3, autoclaved for later use), mix well, and then immunize SPF-2 laying hens, and add 1 mg / ml BCG for the first immunization (National Institute for the Control of Pharmaceutical and Biological Products).

[0038] Table 1 Implementation of animal immunization

[0039] immune time

immune dose

immune adjuvant

immune pathway

0d

1mg / piece

Freund's complete adjuvant

Subcutaneous and intramuscular injection

14d

1mg / piece

Freund's incomplete adjuvant

Subcutaneous and intramuscular injection

28d

0.5mg...

Embodiment 3I

[0046] Example 3 Application of IgY Antibody in Shiga Toxin Detection

[0047] 1. Materials

[0048] BIAcore 3000 biosensor system, CM5 chip, coupling reagents, working solution, etc. are all products of GE Company, and anti-IgY-HRP was purchased from SIGMA Company. Enzyme-linked plates and chromogenic reagents were purchased from Biocom.

[0049] 2. Methods and results

[0050] 2.1 Binding specificity of IgY antibody to Stx1B (ELISA)

[0051] Coat enzyme-linked plates with Shiga toxin type I (Stx1), Shiga toxin type II (Stx2), tetanus toxin (Tet), Staphylococcus aureus enterotoxin B (SEB) and other antigens, 10 μg per well, ELISA method The binding activity of IgY was detected, and the secondary antibody was HRP-labeled rabbit anti-chicken antibody (SIGMA Company). The results show that the IgY antibody can specifically recognize type I Shiga toxin, and has a significant binding effect, but does not bind to other toxin antigens (tetanus toxin-Tet, Staphylococcus aureus en...

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Abstract

The invention discloses an anti-I type Shiga toxin IgY antibody, and the antibody can be prepared by the following method: non-toxic Shiga toxin immune antigen is prepared by the method of chemical synthesis or gene recombinant expression, egg-laying hens are immunized, eggs are collected, and the biological chemical method is applied in extracting and purifying egg yolk immunoglobulin (IgY antibody). The antibody has the effects of neutralizing Shiga toxin and effectively inhibiting the toxicity of the Shiga toxin, can be used as an oral antitoxin for preventing and treating complications caused by toxin-producing Shigella, entero-hemorrhagic Escherichia coli O157, vibrio cholerae and the like and can be simultaneously applied in the detection and the infection diagnosis of type I Shiga toxin and pathogen thereof.

Description

technical field [0001] The present invention relates to an IgY antibody, in particular to an IgY antibody specific for resisting type I Shiga toxin, and also relates to a preparation method and application of the antibody. Background technique [0002] Shiga toxin (Stx) is a kind of bacterial exotoxin with enterotoxicity, cytotoxicity and neurotoxicity produced by enteropathogenic bacteria, and it is the most common cause of new enteropathogens such as acute bacillary dysentery, cholera O139, O157:H7, etc. A key potent virulence factor. Clinically, it causes serious complications such as hemorrhagic colitis (hemorrhagic colitis, HC), thrombotic thrombocytopenic purpura (TTP), and hemolytic uremic syndrome (HUS) with a mortality rate as high as 80-90%. Millions of people around the world suffer from these diseases. [0003] At present, there are no specific and effective preventive products and emergency treatment drugs for Shiga toxin. The commonly used antibiotic treatme...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/12C07K16/02C07K1/14C12P21/08A61K39/40A61P39/02G01N33/53G01N33/569
CPCY02A50/30
Inventor 王慧侯晓军王琴包士中蔡昆史晶荫俊
Owner MICROBE EPIDEMIC DISEASE INST OF PLA MILITARY MEDICAL ACAD OF SCI
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