Detection method for biological activity of chicken interferon alpha according to luciferase reporter gene method and application of detection method

Abstract

The invention discloses a detection method for biological activity of chicken interferon alpha according to a luciferase reporter gene method and application of the detection method. The detection method comprises the following steps: obtaining a pMx1 gene fragment of chicken protein Mx1 through PCR amplification; inserting the pMx1 gene fragment into the 5' end of a pGL3-basic vector luc gene, and then performing PCR amplification to obtain a pMx1-luc fusion gene fragment; using the pMx1-luc fusion gene fragment to replace the pCMV and EGFP gene fragments in a pEGFP-N1 vector, constructing apMx1-luc plasmid, transfecting cells, and selecting stably transfected cell lines; performing cloning culture on the selected stably transfected cell lines; preparing a standard curve by using a chicken interferon alpha standard subjected to gradient dilution, adding a to-be-detected chicken interferon alpha sample to the cells subjected to cloning culture, for co-incubation, then detecting the fluorescence intensity, and evaluating the titer of the to-be-detected chicken interferon alpha sample according to the standard curve.

Description

technical field [0001] The invention relates to a method for detecting the biological activity of chicken interferon alpha by a luciferase reporter gene method, and belongs 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...

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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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