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Detection method for biological activity of sheep interferon tau 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 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: 2018-10-26
WUHU YINGTE FEIER BIOLOGICAL PROD IND RES INST CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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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 sheep interferon tau through luciferase reporter gene method
  • Detection method for biological activity of sheep interferon tau through luciferase reporter gene method
  • Detection method for biological activity of sheep interferon tau through luciferase reporter gene method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] This embodiment provides a luciferase reporter gene method for detecting the biological activity of sheep interferon τ, which is an application for quantitative detection of the biological activity of recombinant sheep interferon τ, and the detection method of this embodiment can also be Correspondingly be used for the activity detection of natural sheep interferon tau, it comprises the following steps:

[0058] According to the gene sequence of the sheep Mx1 protein published in Genebank, select the promoter region containing the ISRE response element at the 5' end (interferon response element is shown in bold), 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 DNA is extracted from sheep kidney cells by the phenol-chloroform-isoamyl alcohol method as a template, using the above PCR primers ...

Embodiment 2

[0078] This example provides a comparative correlation experiment between the detection results of the method for detecting the biological activity of sheep interferon τ by the luciferase reporter gene method of the present invention and the detection results of the microcytopathic inhibition method.

[0079] The luciferase reporter gene method and the microcytopathic inhibition method were used to detect 20 recombinant sheep interferon τ 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 sheep interferon τ sample, dilute it with complete medium 1:100, and then dilute it with complete medium; inoculate sheep kidney subculture into 96-well cell culture plate, 100 μl of cell suspension (2×10 5 each / mL); then add...

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Abstract

The invention discloses a detection method for the biological activity of a sheep interferon tau through a luciferase reporter gene method and application of the detection method. The method comprisesthe following steps: obtaining a gene segment of pMx1 of sheep 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 cultureon the screened stably-transfected cell line; preparing a standard curve by taking a sheep interferon tau standard product which is subjected to gradient dilution; adding a sheep interferon tau sampleto 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 sheep interferontau sample to be detected.

Description

technical field [0001] The invention relates to a method for detecting the biological activity of sheep interferon tau by a luciferase reporter gene method, and belongs to the technical field of interferon activity detection. Background technique [0002] Interferon τ 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 tau in cells has been studied thoroughly, mainly through the activation of JAK-STAT signal cascade. Interferon tau 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
CPCC12Q1/66C12N15/65C12N15/66C12N15/85G01N2333/555
Inventor 张振韬单雪芹刘家炉王亚男李雅森徐高涛蒋敏之赵雨
Owner WUHU YINGTE FEIER BIOLOGICAL PROD IND RES INST CO LTD
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