Detection method for skin mucus functional protein of marine fish in high temperature environment

Abstract

A detection method for skin mucus functional protein of marine fish in high temperature environment comprises the following steps: firstly collecting skin mucus of fish, then crudely extracting skin mucus protein; obtaining a skin mucus protein pattern by two-dimensional electrophoresis technology; performing image analysis by an image analysis software of Imagemaster 2D Platinum 6.0 to locate a protein spot with significant difference; performing MALDI-TOF-TOF mass spectrometry, identifying the protein spot by PMF technology and retrieval tools provided by MASCOT, NCBI websites so as to obtain functional protein related to high temperature stress. The invention uses a simple and aseptic plastic pipette to collect mucus, and the method is quick, simple, and is applicable to quick split charging; the mucus can be preserved for more than one year at a temperature below -80 DEG C; The two-dimensional electrophoresis isoelectric focusing electrophoresis procedure in the invention can completely remove salt ions, and the obtained protein spot is clear and smooth, and can be used to locate and quantify the skin mucus functional protein more accurately. The invention is applicable to detection technology such as the differential expression and the functional protein determination of skin mucus protein of marine fish in high temperature environment.

Description

Technical field: [0001] The invention belongs to the technology of comparative proteomics, and is a method for detecting mucus functional proteins on the body surface of seawater fish under high-temperature environment. Background technique [0002] The mucus on the surface of fish is the first protective barrier for the body to defend against the invasion of external pathogenic microorganisms, and a variety of functional proteins with immune functions contained in the mucus on the body surface play a vital role. It will be a new and faster way of temperature-resistant breeding to determine the temperature-related biological indicator protein. [0003] Before the present invention is made, Optimization of sample preparation from skin mucus of a neotropical fish for two-dimensional substrate gel electrophoresis published by Cristiane et al. (Analytical Biochemistry 2006) has reported that the body surface mucus protease of tropical fish Tambacu can be established by using two...

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Problems solved by technology

[0003] Before the present invention is made, Optimization of sample preparation from skin mucus of a neotropical fish for two-dimensional substrate gel electrophoresis published by Cristiane et al. (Analytical Biochemistry 2006) has reported that the body surface mucus protease of tropical fish Tambacu can be established by using two-dimensional electrophoresis technology Atlas, but did not apply comparative proteomics techniques to identify functional proteins associated with certain traits; A proteomic analysis of green and white surgeon larvae exposed to heat stress and selenium published by Frédéric Silvestre et al. (Science of the Total Environment 2010) , has reported the use of two-dimensional electrophoresis technology to compare and analyze the effect of high temperature stress on the tissue proteins of sturgeon larvae, but this technology has not been applied to the rapid detection of functional proteins in body surface mucus

To sum up, the method of using two-dimensional electrophoresis technology to detect mucus functional proteins on the surface of seawater fish under high temperature environment has not been reported at home and abroad.

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