Method for detecting biological activity of swine interferon alpha

A technology of biological activity and porcine interferon, which is applied in the field of detection of porcine interferon α biological activity, can solve problems such as difficulty in development and increase in use costs, and achieve the effects of improving accuracy, improving repeatability, and reducing costs

Inactive Publication Date: 2018-11-20
ANHUI JIUCHUAN BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, this method is based on the transient transfection of plasmids. It is necessary to prepare internal reference plasmids each time and ensure consistent plasmid transfection efficiency to obtain accurate detection results. Therefore, it is difficult to carry out in general laboratories, and luciferase substrates are required. material, increasing the cost of use

Method used

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  • Method for detecting biological activity of swine interferon alpha
  • Method for detecting biological activity of swine interferon alpha
  • Method for detecting biological activity of swine interferon alpha

Examples

Experimental program
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Embodiment 1

[0048] This embodiment provides a method for detecting the biological activity of porcine interferon α, which is an application for detecting the biological activity of recombinant porcine interferon α, and the detection method in this embodiment can also be used for natural porcine interferon correspondingly The activity detection of α, it comprises the steps:

[0049] According to the gene sequence of the porcine Mx1 protein published in Genebank, the promoter region containing the ISRE response element at the 5' end was selected, PCR primers were designed, and DNA was extracted from pk-15 cells by the phenol-chloroform-isoamyl alcohol method as a template. Use above-mentioned PCR primer and Ex-Taq enzyme to carry out PCR amplification (underlined part is upstream and downstream primer position);

[0050]

[0051] The product obtained by PCR amplification was double-digested by AseI and AgeI and then recovered and purified by gel cutting (introduce the AseI restriction si...

Embodiment 2

[0061] This example provides a comparative correlation experiment between the detection results of the method for detecting the biological activity of porcine interferon α of the present invention and the detection results of the micro-cytopathic inhibition method.

[0062] EGFP reporter gene method and trace cytopathic inhibition method were used to detect 20 recombinant porcine interferon-alpha samples at the same time, and linear regression was used to analyze whether the results of the two methods were correlated.

[0063] The detection process of the EGFP reporter gene method is shown in Example 1.

[0064] The micro-cytopathic inhibition method is operated as follows: take 1 mL of 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, Wells were inoculated with 100 μl cell suspension (2×10 5 each / mL); then add 100 μl of recombinant porcine interferon α in dif...

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Abstract

The invention discloses a method for detecting biological activity of swine interferon alpha and application thereof. The method comprises the following steps: acquiring pMx1 gene segments of swine Mx1 proteins by adopting PCR (Polymerase Chain Reaction) amplification; removing pCMV of a vector plasmid pEGFP-N1; replacing pCMV of the original vector plasmid pEGFP-N1 with the pMx1 gene segments ofthe swine Mx1 proteins acquired by adopting the PCR amplification by virtue of T4DNA ligase, and constructing a plasmid pEGFP-N1-pMx1; transfecting cells by using the plasmid pEGFP-N1-pMx1, and screening stable transfected cell strains by virtue of neomycin; and performing cloning culture on the screened stable transfected cell strains, and adding the swine interferon alpha to co-incubate with thestable transfected cell strains subjected to cloning culture. The Mx1 gene promoter activity is activated to promote expression of EGFP in cells, and the intensity of fluorescence emitted by cells after irradiation of an excitation light source is positive correlation to biological activity of the swine interferon alpha. Therefore, the biological activity of theswine interferon alpha can be quantitatively evaluated.

Description

technical field [0001] The invention relates to a method for detecting the biological activity of porcine interferon alpha, belonging to the technical field of interferon activity detection. Background technique [0002] Interferon (IFN) is a broad-spectrum antiviral glycoprotein secreted by recipient cells after cells and organisms are infected by viruses, or affected by nucleic acids, bacterial endotoxins, and mitogens. According to the source and physical and chemical properties of interferon, it can be divided into type I, type II and III interferon. Type I interferons include IFN-α, IFN-β, and IFN-τ; type II interferon is IFN-γ; III interferon is IL-28A, IL-28B and IL-29. [0003] IFN-α has four main functions. First, it can make the virus-infected cells and their surrounding cells enter the endogenous anti-viral state to limit the spread of the virus; second, maintain the natural immune response in a balanced state, promote antigen presentation and NK cell function...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N33/533C12N15/85C12N15/66
CPCC12N15/66C12N15/85C12Q1/6897G01N21/6428G01N33/533G01N33/6866G01N2333/56
Inventor 单雪芹王亚男徐文俊赵雨蒋敏之何志远郭志燕
Owner ANHUI JIUCHUAN BIOTECH
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