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Method of detecting biological activity of bovine interferon alpha by luciferase reporter gene process

A biological activity and reporter gene technology, applied in the field of interferon activity detection, can solve the problems of inconvenience for large-throughput sample detection, hidden dangers of live virus biological safety, and unsatisfactory sensitivity, so as to avoid biological safety hazards and shorten detection. time, the effect of improving repeatability

Inactive Publication Date: 2018-11-06
ANHUI JIUCHUAN BIOTECH
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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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  • Method of detecting biological activity of bovine interferon alpha by luciferase reporter gene process
  • Method of detecting biological activity of bovine interferon alpha by luciferase reporter gene process
  • Method of detecting biological activity of bovine interferon alpha by luciferase reporter gene process

Examples

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Embodiment 1

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

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

Embodiment 2

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

[0079] 20 samples of recombinant bovine interferon-α were detected simultaneously by luciferase reporter gene method and microcytopathic inhibition method, and whether the results of the two methods were correlated by linear regression analysis.

[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 bovine interferon-α specimen, dilute it with complete medium 1:100, and then dilute it with complete medium; inoculate MDBK subculture to 96-well cell culture plate, Wells were inoculated with 100 μl cell suspension (2×10 5 individual / mL); then add 100 μl of recombi...

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Abstract

The invention discloses a method of detecting biological activity of bovine interferon alpha by luciferase reporter gene process and application of the method. The method comprises the steps of performing PCR (polymerase chain reaction) amplification to obtain a gene fragment of pMx1 of bovine Mx1 protein; inserting the gene fragment to 5' end of pGL3-basic vector luc gene; performing PCR amplification to obtain a pMx1-luc fusion gene fragment; replacing gene fragments of pCMV and EGFP in pEGFP-N1 vector via the pMx1-luc fusion gene fragment so as to construct pMx1-luc plasmid, transfecting cells, and selecting a cell strain that is stably transfected; subjecting the screened cell strain that is stably transfected to cloning culture; using bovine interferon alpha standard gradiently diluted to prepare a standard curve, adding a bovine interferon alpha sample under detection into the cells that are subjected to cloning culture, allowing co-incubation, detecting fluorescence intensity, and evaluating titer of the bovine interferon alpha sample under detection through the standard curve.

Description

technical field [0001] The invention relates to a method for detecting the biological activity of bovine interferon alpha by luciferase reporter gene method, belonging 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-stim...

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 单雪芹凡玉芳许高涛李雅森徐文俊刘家炉蒋敏之赵雨
Owner ANHUI JIUCHUAN BIOTECH
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