Detection method for biological activity of pig interferon alpha by luciferase report gene method

Abstract

The invention discloses a method for detecting the biological activity of porcine interferon α by a luciferase reporter gene method and an application thereof. The method comprises the following steps: using PCR amplification to obtain the pMx1 gene fragment of the porcine Mx1 protein; inserting the pMx1 gene fragment into the 5' end of the pGL3-basic vector luc gene, and then using PCR amplification to obtain the pMx1-luc fusion gene fragment; Replace the pCMV and EGFP gene fragments in the pEGFP-N1 vector with pMx1-luc fusion gene fragments, construct pMx1-luc plasmids, transfect cells, and select stable transfected cell lines; Cloning culture; prepare a standard curve with the porcine interferon-α standard diluted in gradient, add the porcine interferon-α sample to be tested into the cells after cloning culture for co-incubation, then detect the fluorescence intensity, and evaluate the porcine interferon to be tested in combination with the standard curve Titer of alpha sample.

Description

Technical field [0001] The invention relates to a method for detecting the biological activity of pig interferon alpha by a luciferase reporter gene method, and belongs to the technical field of interferon activity detection. Background technique [0002] Interferon alpha is widely used in the treatment of diseases against viral infections and immune dysfunction. As a cytokine, it activates a series of signal transduction pathways in cells by binding to specific receptors indicated by target cells, and exhibits antiviral effects. Or immune regulation. At present, the signal transduction pathways of interferon-α in cells have been studied thoroughly, mainly by activating the JAK-STAT signal cascade. Interferon α binds to the receptor and activates the receptor-related JAK1 and TYK2, phosphorylates STAT1 and STAT2 tyrosine, and the phosphorylated STAT1 and STAT2 form a dimer and transfer to the nucleus, and form the assembly of interferon regulatory factor 9 (IRF9) 3-mer interfer...

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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