Recombinant expression method of grass carp tumor necrosis factor-alpha gene

Abstract

The invention relates to a competitive inhibition enzyme-linked immunosorbent assay (ELISA) method for grass carp tumor necrosis factors alpha. The method comprises the following steps of: 1, determining the concentration of coating antigens and the optimal dilution ratio of polyclonal antibodies; 2, establishing the competitive inhibition ELISA method, and coating an ELISA plate of grass carp tumor necrosis factor alpha protein to obtain antigens immobilized on the ELISA plate; mixing enzyme-labeled antibodies and an antigen mixed solution, and performing competitive binding on the solution and the antigens immobilized on the ELISA plate; washing the ELISA plate and developing the color of the substrate; and reading holes of the ELISA plate to obtain the light absorption value of each hole, calculating the inhibition rates of a standard antigen and a sample to be detected according to the read light absorption values respectively, and calculating the concentration of the sample to be detected according to the inhibition rates. The method has the advantages that a quick and efficient detection means is provided for detecting the protein expression level of the grass carp tumor necrosis factors alpha; and the method is stable, reliable and low in cost.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to techniques such as cloning, recombinant expression, separation and purification of expression products, and activity identification of grass carp (Ctenopharyngodon idella) tumor necrosis factor alpha (TNF-α) gene. Background technique [0002] Tumor necrosis factor alpha (tumor necrosis factor-alpha, TNF-α) is an important cytokine. As early as more than 30 years ago, Carswell et al. discovered a protein substance present in serum that can inhibit and kill certain tumor cells and cause hemorrhage and necrosis of tumors in vivo, which was named tumor necrosis factor (TNF). ). Shalaby et al named the tumor necrosis factor secreted by activated monocytes, macrophages and T cells as TNF-α; and named the lymphotoxin secreted by activated T lymphocytes and NK cells as TNF-β . TNF-α precursor is a type II transmembrane protein, which is cleaved by TNF-α converting enzyme (TAC...

AI Technical Summary

Problems solved by technology

At the same time, due to the large gap in structure and function between fish tumor necrosis factor alpha and mammalian and amphibian tumor necrosis factor alpha molecules, it is not possible to replace fish tumor necrosis factor alpha proteins and antibodies with other races α research, coupled with the lack of fish tumor necrosis factor α antibodies, the research on the functional status of TNF α is a bottleneck

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