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Detection method for biological activity of pig 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 inconvenient large-throughput sample detection, hidden dangers of live virus biological safety, and long detection process, so as to avoid biological safety hazards and shorten detection time. Time, the effect of improving accuracy

Inactive Publication Date: 2018-03-27
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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  • Detection method for biological activity of pig interferon alpha by luciferase report gene method
  • Detection method for biological activity of pig interferon alpha by luciferase report gene method
  • Detection method for biological activity of pig interferon alpha by luciferase report 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 porcine interferon alpha, which is an application for quantitatively detecting the biological activity of recombinant porcine interferon alpha. The detection method of this embodiment can also Correspondingly, it is used to detect the activity of natural porcine interferon alpha, which includes the following steps:

[0058] According to the gene sequence of porcine Mx1 protein published in Genebank, select the promoter region containing ISRE response element at the 5'end (for interferon response element see the bolded part), design PCR primer), and introduce Kpn I enzyme into the upstream primer Cut site, the downstream primer is introduced into the Hind III restriction site (the underlined part is the position of the upstream and downstream primers), and DNA is extracted from pk-15 cells as a template by the phenol-chloroform-isoamyl alcohol method, using the above PCR prime...

Embodiment 2

[0078] This example provides a comparative correlation experiment between the detection results of the luciferase reporter gene method for porcine interferon alpha biological activity detection 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 porcine interferon α samples simultaneously, and linear regression analysis showed 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 microcytopathic inhibition method is as follows: Take 1 mL of recombinant porcine interferon alpha specimen, dilute it with complete medium 1:100 and then dilute it with complete medium times; inoculate pk-15 subculture into 96-well cell culture plate , Inoculate 100μl cell suspension (2×10 5 Pieces / mL); then add 100μl of recombinant p...

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Abstract

The invention discloses a detection method for the biological activity of a pig interferon alpha by a luciferase report gene method and application method of the detection method. The method comprisesthe following steps: carrying out PCR (Polymerase Chain Reaction) amplification to obtain a gene segment of pMx1 of pig Mx1 protein; inserting the gene segment of the pMx1 into a 5' end of a luc geneof a pGL3-basic vector and 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 (plasmid Enhanced Green Fluorescent Protein-N1) vector with the pMx1-luc fused gene segment, so as to construct a pMx1-luc plasmid; transfecting cells and selecting stably transfected cell strains; carrying out colonized culture on the screened stably transfected cell strains; preparing a standard curve by adopting a pig interferon alpha standard product which is diluted in agradient manner; adding a pig interferon alpha sample to be detected into the cells subjected to the colonized culture and carrying out common incubation; then detecting the fluorescence intensity and combining the standard curve to evaluate the efficiency of the pig interferon alpha sample to be detected.

Description

Technical field [0001] The invention relates to a method for detecting the biological activity of pig interferon alpha by a luciferase reporter gene method, and belongs to the technical field of interferon activity detection. Background technique [0002] Interferon alpha is widely used in the treatment of diseases against viral infections and immune dysfunction. As a cytokine, it activates a series of signal transduction pathways in cells by binding to specific receptors indicated by target cells, and exhibits antiviral effects. Or immune regulation. At present, the signal transduction pathways of interferon-α in cells have been studied thoroughly, mainly by activating the JAK-STAT signal cascade. Interferon α binds to the receptor and activates the receptor-related JAK1 and TYK2, phosphorylates STAT1 and STAT2 tyrosine, and the phosphorylated STAT1 and STAT2 form a dimer and transfer to the nucleus, and form the assembly of interferon regulatory factor 9 (IRF9) 3-mer interfer...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/66G01N21/64
CPCC12Q1/66G01N21/6428G01N2333/56
Inventor 赵俊王明丽甘霖王利利赵雨梅志强蒋敏之
Owner ANHUI JIUCHUAN BIOTECH
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