Bovine interferon alpha biological activity detection method

A biological activity, bovine interferon technology, applied in the field of bovine 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 shortening detection time

Active Publication Date: 2018-11-02
WUHU YINGTE FEIER BIOLOGICAL PROD IND RES INST CO LTD
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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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  • Bovine interferon alpha biological activity detection method
  • Bovine interferon alpha biological activity detection method
  • Bovine interferon alpha biological activity detection method

Examples

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Embodiment 1

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

[0049] According to the gene sequence of the bovine 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 MDBK 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 bovine interferon-α biological activity detection method of the present invention and the detection results of the trace cytopathic inhibition method.

[0062] 20 samples of recombinant bovine interferon α were detected simultaneously by EGFP reporter gene method and trace cytopathic inhibition method, 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 bovine interferon-α specimen, dilute it with complete medium 1:100, and then double-dilution with complete medium; inoculate MDBK subculture to 96-well cell culture plate, and inoculate each well 100μl cell suspension (2×10 5 individual / mL); then add 100 μl of recombinant bovine interferon α in different dilutio...

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Abstract

The invention discloses a bovine interferon alpha biological activity detection method and application thereof. The method comprises the following steps: performing PCR amplification to obtain a genesegment of Mxp of bovine Mx protein; removing pCMV of pEGFP-N1 vector plasmid; replacing pCMV in original pEGFP-N1 vector plasmid by using the gene segment of the Mxp of the bovine Mx protein obtainedthrough PCR amplification and through T4DNA connecting enzyme and constructing pEGFP-N1-Mxp plasmid; transfecting cells by the pEGFP-N1-Mxp plasmid and screening out a stable transfected cell strainthrough neomycin; and performing colonized culture on the screened stable transfected cell strain, adding bovine interferon alpha, performing co-incubation on the bovine interferon alpha and the stable transfected cell strain after colonized culture, and activating the activity of a Mx gene promoter to promote expression of EGFP in the cells, wherein the intensity of fluorescence emitted by cellsand the biological activity of the bovine interferon alpha are in positive correlation after irradiation of excitation light source, so the biological activity of the bovine interferon alpha can be quantitatively evaluated.

Description

technical field [0001] The invention relates to a bovine interferon alpha biological activity detection method, which belongs 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 while i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/64
CPCC12N15/85C12Q1/6897G01N21/6486
Inventor 张振韬蒋敏之单雪芹凡玉芳王亚男徐文俊许高涛
Owner WUHU YINGTE FEIER BIOLOGICAL PROD IND RES INST CO LTD
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