Method for relative quantitative analysis of broiler chicken heat shock proteins HSPA5

A technology for quantitative analysis of heat shock proteins, applied in the field of relative quantitative analysis of broiler heat shock protein HSPA5, can solve the problems of long monoclonal control cycle, low success rate, high cost, etc., to improve protein detection throughput and efficiency, specificity Strong, eliminating the effect of cumbersome steps

Active Publication Date: 2018-05-15
INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The preparation of HSPA5 monoclonal antibody can improve the specificity of the antibody, reduce non-specific binding and cross-reaction, but the monoclonal control period is long and the cost is high; if the preparation of HSPA5 polyclonal antibody can reduce the cost, but the non-specific cross-reaction is more serious, very low success rate

Method used

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  • Method for relative quantitative analysis of broiler chicken heat shock proteins HSPA5
  • Method for relative quantitative analysis of broiler chicken heat shock proteins HSPA5
  • Method for relative quantitative analysis of broiler chicken heat shock proteins HSPA5

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Embodiment 1

[0036] Example 1 A method for relative quantitative analysis of broiler heat shock protein HSPA5

[0037] In this embodiment, the method for the relative quantitative analysis of broiler heat shock protein HSPA5 comprises the following steps:

[0038] (1) HSPA5 protein extraction

[0039] Take 0.1 g of broiler liver tissue from the control group and the heat treatment group respectively (the liver samples of the heat treatment group are from broiler chickens treated at 33°C for 24 hours, and the liver samples of the control group are from broiler chickens treated at 25°C for 24 hours during the same period), according to 1: Add protein extraction buffer (50mM Tris-HCl, 0.5M NaCl, 10mM dithiothreitol, 1% dodecyl maltoside, pH 8.0) at a ratio of 5 (mass ratio), and extract in a water bath at 90-100°C The HSPA5 protein in the broiler tissue was homogenized for 15-30 minutes, centrifuged at 20,000 g at 4°C for 30 minutes, and the supernatant was taken, and the total protein in th...

Embodiment 2

[0047] Example 2 A method for relative quantitative analysis of broiler heat shock protein HSPA5

[0048] In this embodiment, the method for the relative quantitative analysis of broiler heat shock protein HSPA5 comprises the following steps:

[0049] (1) HSPA5 protein extraction

[0050] Take 0.1 g of broiler liver tissue from the control group and the heat treatment group respectively (the liver samples of the heat treatment group come from broilers treated at 33°C for 18 hours, and the liver samples of the control group come from broilers treated at 25°C for 18 hours in the same period), according to 1: Add protein extraction buffer (100mM Tris-HCl, 0.1M NaCl, 20mM dithiothreitol, 2wt% dodecyl maltoside, pH 7.2) at a ratio of 5 (mass ratio), and extract in a water bath at 90-100°C The HSPA5 protein in the broiler tissue under different treatments was homogenized for 15-30 minutes, centrifuged at 20,000 g at 4°C for 30 minutes, and the supernatant was taken, and the total p...

Embodiment 3

[0058] Example 3 A method for relative quantitative analysis of broiler chicken heat shock protein HSPA5

[0059] In this embodiment, the method for the relative quantitative analysis of broiler heat shock protein HSPA5 comprises the following steps:

[0060] (1) HSPA5 protein extraction

[0061] Take 0.1 g of broiler liver tissue from the control group and the heat treatment group respectively (the liver samples of the heat treatment group come from broilers treated at 33°C for 30 hours, and the liver samples of the control group come from broilers treated at 25°C for 30 hours in the same period), according to 1: Add protein extraction buffer (80mM Tris-HCl, 0.3M NaCl, 15mM dithiothreitol, 1wt% dodecyl maltoside, pH 7.5) at a ratio of 5 (mass ratio), and extract in a water bath at 90-100°C The HSPA5 protein in the broiler tissue under different treatments was homogenized for 15-30 minutes, centrifuged at 20,000 g at 4°C for 30 minutes, and the supernatant was taken, and the ...

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Abstract

The invention discloses a method for relative quantitative analysis of broiler chicken heat shock proteins HSPA5. The method includes the steps: firstly, extracting the broiler chicken heat shock proteins HSPA5; secondly, performing enzyme digestion on the broiler chicken heat shock proteins HSPA5 to obtain specific peptide fragments; thirdly, detecting parent and daughter ion pairs of the specific peptide fragments by a liquid chromatograph-mass spectrometer to obtain the total signal intensity of daughter ions of the specific peptide fragments, acquiring the signal intensity of the broiler chicken heat shock proteins HSPA5 according to the total signal intensity of the daughter ions of the specific peptide fragments, and relatively quantifying the broiler chicken heat shock proteins HSPA5 in different treated broiler chicken samples by comparing the signal intensity of the broiler chicken heat shock proteins HSPA5 in the different treated broiler chicken samples. Amino acid sequencesof three specific peptide fragments of the broiler chicken heat shock proteins HSPA5 are as shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3. Compared with an antibody-based Western Blot method, therelative quantitative method is stronger in specificity, the cumbersome step of antibody preparation is omitted, and chicken-origin HSPA5 protein detection flux and efficiency are improved.

Description

technical field [0001] The invention relates to the technical field of food detection, in particular to a method for relatively quantitatively analyzing broiler heat shock protein HSPA5. Background technique [0002] Under the conditions of global warming and intensive farming, the breeding density of broilers is gradually increasing, and heat stress has become a common problem in the process of broiler farming. Heat stress is a common in vitro stress during the growth of animals. When the temperature of the animal's growth environment exceeds the upper limit of its metabolic stability zone, and the body's physical adjustment cannot maintain thermal balance, the body will produce non-specific responses to the high temperature of the environment. Response response. Heat stress can cause reduced feed intake, slower growth, lower body weight gain, and lower feed conversion in broilers. Chronic heat stress has been reported to significantly reduce feed intake (-16.4%), body we...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/02G01N30/06
CPCG01N30/02G01N30/06
Inventor 孟庆石高杰夏冰张宏福唐湘方
Owner INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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