Method for detecting biological activity of canine interferon alpha

A biological activity, canine interferon technology, applied in the field of canine interferon alpha biological activity detection, can solve the problems of increased use cost and difficult development, and achieve the effects of improving accuracy, improving repeatability, and reducing costs

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, 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 canine interferon alpha
  • Method for detecting biological activity of canine interferon alpha
  • Method for detecting biological activity of canine interferon alpha

Examples

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Embodiment 1

[0048] This embodiment provides a method for detecting the biological activity of canine interferon α, which is an application for detecting the biological activity of recombinant canine interferon α. ​​The detection method in this embodiment can also be used for natural canine interferon α. The active detection of element alpha, it comprises the steps:

[0049] According to the gene sequence of the canine Mx 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 MDCK cells by the phenol-chloroform-isoamyl alcohol method as a template, using the above PCR primers and Ex-Taq enzyme for PCR amplification (the underlined part is the upstream and downstream primer positions);

[0050]

[0051] The product obtained by PCR amplification was double-digested by Ase I and Age I, and then recovered and purified by gel cutting (the upstream PCR primer introduced the Ase I...

Embodiment 2

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

[0062] EGFP reporter gene method and trace cytopathic inhibition method were used to detect 20 recombinant canine interferon-α 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 canine interferon-α specimen, dilute it with complete medium 1:100, and then dilute it with complete medium; inoculate MDCK subculture to 96-well cell culture plate, and inoculate each well 100μl cell suspension (2×10 5 individual / mL); then add different dilutions of recombinant canine interferon α 1...

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Abstract

The invention discloses a method for detecting the biological activity of canine interferon alpha and application of the method. The method includes: using PCR amplification to acquire the gene segment of the Mxp of canine Mx protein; removing the pCMV of pEGFP-N1 vector plasmids; using the obtained gene segment to replace the pCMV in the pEGFP-N1 vector plasmids through T4DNA ligase to build pEGFP-N1-Mxp plasmids; using the pEGFP-N1-Mxp plasmids to transfect cells, and using neomycin to screen out stably transfected cell strains; subjecting the screened stably transfected cell strains to cloning culture, adding the canine interferon alpha into the stably transfected cell strains after the cloning culture to perform co-incubation so as to activate the activity of an Mx gene promoter to promote the expression of EGFP in the cells, and quantitatively evaluating the biological activity of the canine interferon alpha according to the fact that the intensity of fluorescent light emitted bythe cells irradiated by an excitation light source is in positive correlation with the biological activity of the canine interferon alpha.

Description

technical field [0001] The invention relates to a method for detecting the biological activity of canine 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
CPCC12N15/66C12N15/85C12Q1/6897G01N33/533G01N33/6866G01N2333/56
Inventor 单雪芹凡玉芳许高涛赵雨蒋敏之何志远郭志燕
Owner ANHUI JIUCHUAN BIOTECH
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