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Method for analyzing aeromonas hydrophila infected macrobrachium nipponense blood cell differential expression protein based on proteomics quantitative technology

A technology of Aeromonas hydrophila and proteomics, applied in the field of freshwater aquaculture, can solve the problems of high cost, large demand and inability to label animal models.

Active Publication Date: 2020-06-12
JINING UNIV
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Problems solved by technology

Disadvantages: The repeatability requirements for mass spectrometry are relatively high; the ability to analyze complex samples is not as good as labeling quantitative methods
However, there are also the following disadvantages: for tissue samples commonly used in biomedical research, body fluid samples, etc. cannot be analyzed; for animal models, the cost of labeling is too high to achieve
Disadvantages: suitable for proteins with high abundance, and not suitable for hydrophobic, extremely basic or extremely acidic proteins; the demand for samples is relatively large, in principle, each sample should not be less than 1mg

Method used

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  • Method for analyzing aeromonas hydrophila infected macrobrachium nipponense blood cell differential expression protein based on proteomics quantitative technology
  • Method for analyzing aeromonas hydrophila infected macrobrachium nipponense blood cell differential expression protein based on proteomics quantitative technology
  • Method for analyzing aeromonas hydrophila infected macrobrachium nipponense blood cell differential expression protein based on proteomics quantitative technology

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Embodiment 1

[0051] 1. Determination of bacterial infection concentration: Macrobrachium japonica is from Weishan Lake in Jining, with a body length of 6-8cm. Healthy Macrobrachium japonica is kept in the laboratory for a week. Prepare Aeromonas hydrophila suspension and set three concentration gradients (1×10 4 CFU / ml, 1×10 5 CFU / ml, 1×10 6 CFU / ml) is used for intramuscular injection of Macrobrachium japonicus, 50 μl / tail, and the negative control group is injected with the same amount of 0.85% (w / v) NaCl solution (pH=7.0), 50 shrimps in each group, three parallels, and the cumulative Mortality curve to determine the optimal infection concentration.

[0052] After Macrobrachium japonicus was infected with Aeromonas hydrophila, the death of Macrobrachium prawn was counted every day, and the cumulative mortality curve was drawn (see figure 1 ). in 1×10 4 In the CFU / ml infection group, the cumulative mortality rate reached 10%±0.82 on day 1, 16.67%±0.47 and 30%±1.63 on day 2 and day 3 r...

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Abstract

The invention belongs to the technical field of freshwater aquaculture, and relates to a method for analyzing aeromonas hydrophila infected macrobrachium nipponense hemocyte differential expression protein based on a proteomics quantitative technology. The method comprises the following steps of (1) extracting the hemolymph of macrobrachium nipponense infected with aeromonas hydrophila, separatingthe blood cells, and cracking the cells to extract protein; (2) carrying out trypsin enzymolysis on the protein obtained in the step (2), and carrying out iTRAQ labeling on an enzymolysis peptide fragment; (3) carrying out purification, liquid chromatographic separation and mass spectrometric analysis on the peptide fragment marked in the step (3); and (4) carrying out protein identification, quantitative analysis and bioinformatics analysis on the mass spectrum analysis result obtained in the step (3), and analyzing to obtain differentially expressed protein. The invention is based on the rapid proteomics research of the infected macrobrachium nipponense by iTRAQ, and provides a reference for further researching the effect of immune-related proteins.

Description

technical field [0001] The invention belongs to the technical field of freshwater aquaculture, and relates to a method for analyzing proteins differentially expressed in hemocytes of Macrobrachium japonicus infected by Aeromonas hydrophila based on proteomics quantitative technology. Background technique [0002] At present, scholars at home and abroad have made great progress in the research on the biological characteristics, epidemiological characteristics, rapid identification, typing and proteomics of Aeromonas hydrophila. Research on the immune mechanism of shrimp is mainly concentrated in marine cultured shrimp, and research on the immune genes and immune-related proteins of marine shrimp such as Penaeus sinensis, Penaeus monodon, and Litopenaeus vannamei has achieved rich results. The research results of seawater shrimp can certainly provide a reference for the study of the immune mechanism of freshwater shrimp. However, freshwater cultured shrimp and seawater culture...

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Application Information

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IPC IPC(8): G01N30/02G01N30/06G01N33/68
CPCG01N30/02G01N30/06G01N33/6848
Inventor 常彦红宋阳刘锡叶秀华田璐
Owner JINING UNIV
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