Luciferase reporter gene dog interferon alpha biological activity detection method

Abstract

The invention discloses a luciferase reporter gene dog interferon alpha biological activity detection method and application thereof. The method comprises the following steps: performing PCR amplification, and obtaining pM*1 gene segments of M*1 proteins; inserting the pM*1 gene segments into a 5' end of a pGL3-basic carrier luc gene, then performing PCR amplification, and obtaining pM*1-luc fusedgene segments; using the pM*1-luc fused gene segments to substitute pCMV and EGFP gene segments in a pEGFP-N1 carrier, building pM*1-luc plasmids, transfecting cells, and selecting a stable transfected cell line; performing cloning culture on the selected stable transfected cell line; and preparing a standard curve by adopting a gradient diluted dog interferon alpha standard product, co-incubating a dog interferon alpha sample to be detected in the cloning cultured cell, detecting a fluorescence intensity, and evaluating titer of the dog interferon alpha sample to be detected according to thestandard curve.

Description

technical field [0001] The invention relates to a luciferase reporter gene method for detecting the biological activity of canine interferon alpha, belonging to the technical field of interferon activity detection. Background technique [0002] Interferon α is widely used in the treatment of diseases in antiviral infection and immune dysfunction. As a cytokine, it can activate a series of intracellular signal transduction pathways by binding to specific receptors indicated by target cells, showing antiviral effects. or immunomodulatory effects. At present, the signal transduction pathway of interferon α in cells has been studied thoroughly, mainly through the activation of JAK-STAT signal cascade. Interferon α binds to receptors to activate receptor-related JAK1 and TYK2, phosphorylate STAT1 and STAT2 tyrosine, phosphorylated STAT1 and STAT2 form dimers and transfer into the nucleus, and assemble interferon regulatory factor 9 (IRF9) to form 3-mer interferon-stimulating fa...

AI Technical Summary

Problems solved by technology

However, these three methods are prone to large errors, poor repeatability, and low accuracy.

As an improvement, the recombinant VSV virus expressing GFP was used to replace the wild-type VSV virus. The expression of fluorescent protein can reflect the degree of virus infection and replication, and then determine the degree of inhibition of IFN on virus replication. The sensitivity and repeatability are significantly improved. However, there are still many deficiencies such as the detection process takes too long, it is not convenient for large-throughput sample detection, the sensitivity is not ideal, and there are hidden dangers to the biological safety of live viruses.

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