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

A biological activity and reporter gene technology, applied in the field of interferon activity detection, can solve the problems of biological safety hazards, poor repeatability, and low accuracy of live viruses, so as to avoid biological safety hazards, simplify the detection process, and shorten the detection time. the effect of time

Inactive Publication Date: 2018-03-27
ANHUI JIUCHUAN BIOTECH
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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 o

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

Examples

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Embodiment 1

[0057] This embodiment provides a method for detecting the biological activity of chicken interferon-alpha by luciferase reporter gene method, which is an application for quantitative detection of the biological activity of recombinant chicken interferon-alpha. The detection method of this embodiment can also be Correspondingly be used for the activity detection of natural chicken interferon alpha, it comprises the steps:

[0058] According to the gene sequence of the chicken Mx1 protein published in Genebank, select the promoter region containing the ISRE response element at the 5' end (the interferon response element is shown in bold), design PCR primers), and introduce Kpn I enzyme in the upstream primer Cutting site, the downstream primer introduces the Hind III restriction site (the underlined part is the position of the upstream and downstream primers), and the DNA is extracted from CEF cells by the phenol-chloroform-isoamyl alcohol method as a template, using the above P...

Embodiment 2

[0078] This example provides a comparative correlation experiment between the detection results of the luciferase reporter gene method of the present invention for detecting the biological activity of chicken interferon α and the detection results of the trace cytopathic inhibition method.

[0079] The luciferase reporter gene method and the micro-cytopathic inhibition method were used to detect 20 recombinant chicken interferon-α samples at the same time, and the linear regression analysis was used to show whether the results of the two methods were correlated.

[0080] The detection process of the luciferase reporter gene method is shown in Example 1.

[0081] The micro-cytopathic inhibition method is operated as follows: take 1 mL of recombinant chicken interferon-α specimen, dilute it with complete medium 1:100, and then dilute it with complete medium; inoculate CEF subculture to 96-well cell culture plate, Wells were inoculated with 100 μl cell suspension (2×10 5 individ...

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Abstract

The invention discloses a method for detecting the biological activity of chicken interferon alpha 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 chicken 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 chicken interferon-α standard diluted in gradient, add the chicken interferon-α sample to be tested into the cells after cloning culture and co-incubate, then detect the fluorescence intensity, and evaluate the chicken 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 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...

Claims

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Application Information

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IPC IPC(8): C12Q1/66G01N21/64
CPCC12Q1/66G01N21/6428G01N2333/56
Inventor 赵俊王明丽王利利甘霖赵雨梅志强蒋敏之
Owner ANHUI JIUCHUAN BIOTECH
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