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

A technology of Shiga toxin, type I, applied in the preparation of the antibody, the field of IgY antibody specific for type I Shiga toxin, can solve problems such as no reports, and achieve the effect of inhibiting the toxicity of Shiga toxin and having a good application prospect.

Active Publication Date: 2012-06-13
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] Example 1 Expression and purification of recombinant Stx1B

[0019] 1. Material

[0020] 1.1 strain

[0021] The engineered 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) were purchased from GE Company.

[0024] 2. Methods and results

[0025] 2.1 Induced expression of recombinant StxB in E. coli

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

Embodiment 2

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

[0033] 1. Material

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

[0035] 2. Methods and results

[0036] 2.1 Animal immunity (Table 1)

[0037] Mix the purified recombinant Stx1B with Freund’s adjuvant (lanolin: liquid paraffin = 1:3, autoclaved for later use) in equal volume, stir well and immunize SPF-2 layer hens, add 1mg / ml BCG vaccine for the first immunization (China Institute for the Control of Pharmaceutical and Biological Products).

[0038] Table 1 Animal Immunization Implementation

[0039] Immunization time

Immunization dose

Immune adjuvant

Immune pathway

0d

1mg / piece

Freund's complete adjuvant

Subcutaneous and intramuscular injections

14d

1mg / piece

Freund's incomplete adjuvant

Subcutaneous and intramuscular injections

28d

0.5mg / piece

Freund's incomplete adjuvant

Subcutaneous and ...

Embodiment 3

[0046] Example 3 Application of IgY antibody in Shiga toxin detection

[0047] 1. Material

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

[0049] 2. Methods and results

[0050] 2.1 IgY antibody and Stx1B binding specificity (ELISA)

[0051] Enzyme-linked plates are coated with antigens such as type I Shiga toxin (Stx1), type II Shiga toxin (Stx2), tetanus toxin (Tet), Staphylococcus aureus enterotoxin B (SEB), 10μg per well, ELISA method To detect the binding activity of IgY, the secondary antibody is HRP-labeled rabbit anti-chicken antibody (SIGMA). The results showed that IgY antibody can specifically recognize type I Shiga toxin and have obvious binding effect, but does not bind to other toxin antigens (tetanus toxin-Tet, Staphylococcus aureus enterotoxin B-SEB), especially type II Shiga The toxin (Stx2...

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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 type I Shiga toxin, and also relates to a preparation method and application of the antibody. Background technique [0002] Shiga Toxin (Stx) is a type of enterotoxic, cytotoxic and neurotoxic bacterial exotoxin produced by enteric pathogens. It is the most emerging enteric pathogens such as acute bacillary dysentery, cholera O139, O157:H7, etc. The key viral potency factor. Clinically, it causes serious complications such as hemorrhagic colitis (HC), thrombotic thrombocytopenic purpura (TTP), and hemolytic uremic syndrome (HUS) with a fatality rate of 80 to 90%. Millions of people worldwide suffer from such diseases. [0003] Currently, there are no specific and effective preventive products and emergency treatment drugs for Shiga toxin. Antibiotic treatments commonly used in clinical practice mainly target toxin-producing pathogens, which have caused the emerg...

Claims

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Application Information

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Patent Type & Authority Patents(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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