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Method for detecting biological activity of dog interferon alpha

A technology of biological activity and canine interferon, which is applied in the field of detection of canine interferon α biological activity, can solve the problems of increasing the cost of use and difficulty in development, and achieve the effects of improving accuracy, improving repeatability, and having practicability

Inactive Publication Date: 2018-03-30
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 dog interferon alpha
  • Method for detecting biological activity of dog interferon alpha
  • Method for detecting biological activity of dog interferon alpha

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Experimental program
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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 embodiment provides a comparative correlation experiment between the detection results of the method for detecting the biological activity of canine interferon-alpha of the present invention and the detection results of the trace cytopathic inhibition method.

[0062] 20 recombinant canine interferon-α specimens 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 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 each / mL); then add different dilutions of recombinant canine interferon α 10...

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Abstract

The invention discloses a method for detecting the biological activity of canine interferon alpha and its application. The method comprises the following steps: using PCR amplification to obtain the Mxp gene fragment of the dog Mx protein; removing the pCMV of the pEGFP-N1 carrier plasmid; replacing the original pEGFP with the Mxp gene fragment of the dog Mx protein obtained by PCR amplification by T4DNA ligase pCMV in the ‑N1 vector plasmid, construct pEGFP‑N1‑Mxp plasmid; transfect cells with pEGFP‑N1‑Mxp plasmid, and select stable transfection cell lines by neomycin; After cloning culture, adding canine interferon α and co-incubating with the stable transfected cell line after cloning culture will activate the Mx gene promoter activity and promote the expression of EGFP in the cells. The biological activity of canine interferon α is positively correlated, so the biological activity of canine interferon α can be quantitatively evaluated.

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 subjected to the effects of 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 N...

Claims

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Application Information

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IPC IPC(8): C12Q1/6897C12N15/85
CPCC12Q1/6897C12N15/85
Inventor 赵俊王明丽赵雨王利利梅志强蒋敏之
Owner ANHUI JIUCHUAN BIOTECH