Sheep interferon Tau biological activity detection method and application thereof

A technology of biological activity and detection method, which is applied in the field of sheep interferon τ biological activity detection, can solve the problems of increased use cost and difficulty in development, and achieve the effects of improving accuracy, improving repeatability, and shortening detection time

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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  • Sheep interferon Tau biological activity detection method and application thereof
  • Sheep interferon Tau biological activity detection method and application thereof
  • Sheep interferon Tau biological activity detection method and application thereof

Examples

Experimental program
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Embodiment 1

[0048] This embodiment provides a method for detecting the biological activity of sheep interferon τ, which is an application for detecting the biological activity of recombinant sheep interferon τ, and the detection method of this embodiment can also be used correspondingly for natural sheep interferon The activity detection of element tau, it may further comprise the steps:

[0049] According to the gene sequence of the sheep 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 sheep kidney cells by the phenol-chloroform-isoamyl alcohol method as a template. The above PCR primers and Ex-Taq enzymes are used for PCR amplification (the underlined part is the position of the upstream and downstream primers);

[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...

Embodiment 2

[0061] This example provides a comparative correlation experiment between the detection results of the sheep interferon τ biological activity detection method of the present invention and the detection results of the trace cytopathic inhibition method.

[0062] EGFP reporter gene method and trace cytopathic inhibition method were used to detect 20 recombinant sheep interferon τ 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 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, each well Inoculate 100 μl cell suspension (2×10 5 each / mL); then add different dilutions of recombinant sheep interferon τ100 μl per well, ...

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Abstract

The invention discloses a sheep interferon Tau biological activity detection method and application thereof. The method includes following steps: adopting PCR amplification to acquire a gene segment of Mxp of sheep Mx protein; removing pCMV of pEGFP-N1 carrier plasmid; using the gene segment of Mxp of the sheep Mx protein obtained through PCR amplification to replace pCMV in original pEGFP-N1 carrier plasmid through T4DNA ligase to build pEGFP-N1-Mxp plasmid; using the pEGFP-N1-Mxp plasmid to transfect cells, and screening out a stable transfection cell line through neomycin; performing cloning culture on the screened stable transfection cell line, and adding sheep interferon Tau to co-incubate with the stable transfection cell line after going through cloning culture. In this way, Mx genepromoter activity can be activated to promote expression of EGFP in cells, and intensity of fluorescence emitted by the cells after being irradiated by an excitation light source is in positive correlation with biological activity of the sheep interferon Tau, so that the biological activity of the sheep interferon Tau can be evaluated quantitatively.

Description

technical field [0001] The invention relates to a method for detecting the biological activity of sheep interferon tau, 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-τ, IFN-β, and IFN-τ; type II interferon is IFN-γ; III interferon is IL-28A, IL-28B and IL-29. [0003] IFN-τ 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 whi...

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 单雪芹刘家炉赵雨蒋敏之李雅森许高涛何志远凡玉芳
Owner ANHUI JIUCHUAN BIOTECH
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