Method for detecting duck IFN-beta promoter activity by using double luciferase reporter genes

Abstract

The invention provides a method for detecting duck IFN-beta promoter activity by using double luciferase reporter genes. The method comprises the following steps: A, constructing firefly luciferase recombinant plasmids containing a duck IFN-beta promoter target sequence; B, premixing the firefly luciferase recombinant plasmids containing the duck IFN-beta promoter target sequence with an irritant,internal reference plasmids and lipidosomes, and then carrying out transfection on target cells, wherein the internal reference plasmids are sea cucumber luciferase plasmids; and C, carrying out cracking after cultivation on the target cells after transfection, and detecting luciferase activity so as to obtain the duck IFN-beta promoter activity. The method is simple and practicable in operationand high in flux, and has relatively high repeatability and accuracy, and an effective detection tool is provided for researching duck IFN-beta passages.

Description

technical field [0001] The invention relates to the technical fields of molecular genetics and cell biology, in particular to a method for detecting duck IFN-β promoter activity with dual luciferase reporter genes. Background technique [0002] Duck is the main species of poultry farming. In recent years, it has been affected by highly pathogenic avian influenza, which has brought huge losses to farmers, and has also produced a serious threat to public safety. Interferon (Interferon, IFN) has anti-virus, anti-tumor activity and strong immune regulation effect, which is very important for innate immunity and triggering adaptive immunity of poultry. According to the difference of IFN amino acid sequence and its specific recognition receptor, IFN is divided into type I, type II and type III, and interferon beta (IFN-β) belongs to type I IFN. IFN-β is a highly active multifunctional protein produced by stimulating cells with interferon inducers such as microorganisms, dsRNA and...

AI Technical Summary

Problems solved by technology

The current IFN activity detection methods mainly include: antiviral activity detection method, commonly used vesicular stomatitis virus (VSV) detection, but the sensitivity and accuracy of this method are low, and have certain biological safety risks; ELISA detection method, detection It is convenient and fast, but it is limited to the detection of IFN antigenicity, and different IFNs require different monoclonal antibodies, which has low versatility; the fluorescent quantitative PCR detection method is based on the extraction of host cell mRNA, but the RNA is extremely unstable. have a certain impact on the accuracy of the test results

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