Detection method for biological activity of porcine interferon alpha through luciferase reporter gene method

A biological activity and reporter gene technology, applied in the field of interferon activity detection, can solve the problems of inconvenient large-throughput sample detection, potential biological safety hazards of live viruses, and unsatisfactory sensitivity, so as to avoid biological safety hazards and shorten detection time. time, increasing the effect of repeatability

Inactive Publication Date: 2018-10-26
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 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.

Method used

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  • Detection method for biological activity of porcine interferon alpha through luciferase reporter gene method
  • Detection method for biological activity of porcine interferon alpha through luciferase reporter gene method
  • Detection method for biological activity of porcine interferon alpha through luciferase reporter gene method

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Experimental program
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Effect test

Embodiment 1

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

[0058] According to the gene sequence of the pig Mx1 protein published in Genebank, select the promoter region containing the ISRE response element at the 5' end (see the bold part for the interferon response element), design PCR primers), and introduce Kpn I enzyme into 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 pk-15 cells by the phenol-chloroform-isoamyl alcohol method as a template, using the above PCR ...

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 porcine interferon α and the detection results of the trace cytopathic inhibition method.

[0079] 20 samples of recombinant porcine interferon α were detected simultaneously by luciferase reporter gene method and trace cytopathic inhibition method, and 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 porcine interferon-α specimen, dilute it with complete medium 1:100, and then dilute it with complete medium; inoculate pk-15 subculture to 96-well cell culture plate , each well was inoculated with 100 μl cell suspension (2×10 5 each / mL); then ...

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Abstract

The invention discloses a detection method for the biological activity of a porcine interferon alpha through a luciferase reporter gene method and application of the detection method. The method comprises the following steps: obtaining a gene segment of pMx1 of porcine Mx1 protein by adopting PCR (Polymerase Chain Reaction) amplification; inserting the gene segment of the pMx1 into a 5' end of a pGL3-basic vector luc gene; then carrying out the PCR amplification to obtain a pMx1-luc fused gene segment; replacing gene segments of pCMV (Porcine Cytomegalovirus) and EGFP (Enhanced Green Fluorescent Protein) in a pEGFP-N1 vector by utilizing the pMx1-luc fused gene segment; constructing a pMx1-luc plasmid and transfecting a cell; selecting a stably-transfected cell line; carrying out clonal culture on the screened stably-transfected cell line; preparing a standard curve by taking a porcine interferon alpha standard product which is subjected to gradient dilution; adding a porcine interferon alpha sample to be detected into cells subjected to the clonal culture for co-incubating; then detecting the fluorescence intensity and combining the standard curve to evaluate the valence of the porcine interferon alpha sample to be detected.

Description

technical field [0001] The invention relates to a method for detecting the biological activity of porcine 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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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/66C12N15/85C12N15/65C12N15/66
CPCC12Q1/66C12N15/65C12N15/66C12N15/85G01N2333/56
Inventor 单雪芹李雅森刘家炉凡玉芳许高涛赵雨蒋敏之
Owner ANHUI JIUCHUAN BIOTECH
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