Protein yolk antibody for immunologic diagnosis of acute hepatopancreas necrosis syndrome, and preparation process and application thereof

A technology of egg yolk antibody and syndrome, which is applied in the direction of immunoglobulin, antibacterial immunoglobulin, immunoglobulin from eggs, etc., can solve the problems of rapid diagnosis of unfavorable epidemics and inability to diagnose toxin expression, and achieve low cost, Strong resistance and good effect

Inactive Publication Date: 2017-06-20
深圳市广慈生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The shortcomings of the current diagnostic methods mainly lie in three aspects: (1) Gene diagnosis requires PCR equipment and complex operation process, and the time is generally more than 4 hours, which is not conducive to the rapid diagnosis of the epidemic at the grassroots level and on the spot
Moreover, there is no commercial product of this diagnostic method i

Method used

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  • Protein yolk antibody for immunologic diagnosis of acute hepatopancreas necrosis syndrome, and preparation process and application thereof
  • Protein yolk antibody for immunologic diagnosis of acute hepatopancreas necrosis syndrome, and preparation process and application thereof
  • Protein yolk antibody for immunologic diagnosis of acute hepatopancreas necrosis syndrome, and preparation process and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Embodiment 1: Preparation of pirA and pirB proteins

[0058] (1) Cloning of Vibrio parahaemolyticus pirA and pirB genes, construction and identification of expression vectors

[0059] (a) primers are designed according to the sequence of Vibrio parahaemolyticus pirA and pirB genes:

[0060] The PCR amplification primers of the pirA gene sequence are:

[0061] Upstream primer: 5'- CGCGGATCC ATGAGTAACAATATAAAACATGAAAC-3' (SEQ ID NO.3), wherein the underlined part is the BamH I restriction site;

[0062] Downstream primer: 5'- CCCGCGGCCGC TTAGTGGTAATAGATTGTACAGAAA-3' (SEQ ID NO.4), wherein the underlined part is the Not I restriction site.

[0063] The primers for PCR amplification of the pirB gene sequence are:

[0064] Upstream primer: 5'- CGCGGATCC ATGACTAACGAATACGTTGTAACAA-3' (SEQ ID NO.5), wherein the underlined part is the BamH I restriction site;

[0065] Downstream primer: 5'- CCCGCGGCCGC CTACTTTTTCTGTACCAAATTCATCG-3' (SEQ ID NO.6), wherein the underl...

Embodiment 2

[0097] Embodiment 2: Immunization laying hens

[0098] For immunization, the pirA and pirB proteins were mixed as antigens with an equal volume of Fischer's adjuvant, and multi-point subcutaneous injection was used to immunize laying hens;

[0099] Specifically, measure the concentration of the purified toxin protein, adjust the concentration of the purified recombinant protein to 0.01-0.1 mg / ml with PBS, mix the adjusted recombinant protein with the adjuvant at a ratio of 1:1 and follow the company's special Laying hens were immunized with five times of two-way immunization. For the first immunization, 1ml recombinant protein was fully mixed with 1ml Freund's complete adjuvant and then injected into the chest muscle at four points. Two weeks later, 0.5ml recombinant protein was fully mixed with 0.5ml Freund's incomplete adjuvant and then the chest muscle was injected at four points. . Repeat the above operation two weeks later, but the total dose of immunization is changed ...

Embodiment 3

[0100] Example 3: Extraction of anti-acute hepatopancreatic necrosis syndrome toxin protein egg yolk antibody

[0101] (1) Preliminary separation of egg yolk antibody: mix the egg yolk liquid with the acidified water buffer solution of acetic acid-sodium acetate at a ratio of 1:10, let stand at 4 degrees overnight, centrifuge at 10000g for 20min, and save the supernatant. Slowly add saturated ammonium sulfate to a final concentration of 35%, and stand at 4°C until the layers are clearly separated. Centrifuge the mixture at 10000g for 20min, remove the supernatant, and dissolve the precipitate with 30ml of PBS. Slowly add saturated ammonium sulfate to a final concentration of 35%, and stand at 4°C until the layers are clearly separated. Centrifuge at 10000g for 20min, remove the supernatant, and dissolve the precipitate with PBS;

[0102] (2) Immunoaffinity chromatography of anti-PEDV specific egg yolk antibody: the recombinant Pri toxin protein was coupled with HiTrap to pre...

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Abstract

The invention relates to a protein yolk antibody for immunologic diagnosis of acute hepatopancreas necrosis syndrome, and a preparation process and application thereof. The protein yolk antibody uses pirA and pirB protein as antigen immune egg laying poultry and is extracted from the yolk of eggs; the specific binding with the pirA and pirB protein can be realized. The prepared protein yolk antibody has good activity, good specificity, good stability and high hypertonicity resistance. Meanwhile, a preparation method provided by the invention has the characteristics of high benefit, safety, low cost, environment-friendly effect, high prevention performance and the like; the method provides an effective path for prevention, treatment and detection of the acute hepatopancreas necrosis syndrome.

Description

technical field [0001] The invention belongs to the technical field of bioengineering and aquatic disease detection, and specifically relates to an anti-acute hepatopancreatic necrosis syndrome toxin protein egg yolk antibody, its preparation method and application. Background technique [0002] Since 2009, Early Mortality Syndrome (EMS), also known as Acute Hepatopancreatic Necrosis Syndrome (AHPNS), has had an unprecedented impact on the shrimp farming industry in Asia, especially Southeast Asia and China. In addition to the large-scale production reduction, the disease has also brought many negative problems, such as: shrimp farming employment problems, social welfare problems, shrimp supply and demand problems, and global shrimp overall price problems and so on. [0003] In May 2013, the University of Arizona Aquatic Pathology Laboratory (UAZ-APL) defined the pathogen of acute hepatopancreatic necrosis syndrome (AHPNS) as a specific strain of Vibrio parahaemolyticus, whi...

Claims

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Application Information

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IPC IPC(8): C07K16/12C07K16/02A61K39/40A61P31/04G01N33/569
CPCA61K39/00C07K16/02C07K16/1239C07K2317/35
Inventor 祁振强肖宏学
Owner 深圳市广慈生物科技有限公司
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