Chicken interferon alpha biological activity detection method

A biological activity, chicken interferon technology, applied in the field of interferon activity detection, can solve problems such as increased use cost, difficult development, etc., and achieve the effects of improving accuracy, improving repeatability, and having practicability

Inactive Publication Date: 2018-11-13
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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  • Chicken interferon alpha biological activity detection method
  • Chicken interferon alpha biological activity detection method
  • Chicken interferon alpha biological activity detection method

Examples

Experimental program
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Embodiment 1

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

[0049] According to the gene sequence of the chicken 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 CEF 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 As...

Embodiment 2

[0061] This example provides a comparative correlation experiment between the detection results of the chicken interferon-α 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 chicken 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 1mL chicken interferon α sample, dilute it with complete medium 1:100 and then dilute it with complete medium; inoculate CEF subculture to 96-well cell culture plate, inoculate each well 100μl cell suspension (2×10 5 individual / mL); then add 100 μl of recombinant chicken interferon α of different dilutions to each h...

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Abstract

The invention discloses a chicken interferon alpha biological activity detection method, and applications thereof. The chicken interferon alpha biological activity detection method comprises followingsteps: PCR amplification is adopted to obtain Mxp gene segments of chicken Mx protein; pCMV of pEGFP-N1 vector plasmid is removed; the Mxp gene segments of chicken Mx protein obtained through PCR amplification are subjected to construction of pEGFP-N1-Mxp plasmid through replacing of pCMV of orginal pEGFP-N1 vector plasmid with T4DNA ligase; the pEGFP-N1-Mxp plasmid is adopted for cell transfection, and a cell strain capable of realizing stable transfection is obtained through screening using neomycin; the screened cell strain capable of realizing stable transfection is subjected to cloning culture, chicken interferon alpha is added for co-incubation with the cell strain which is capable of realizing stable transfection and is treated throug cloning culture, so that Mx gene promoter activity is activated to promote intracellular EGFP expression, the intensity of fluorescence emitted by cells after excitation light source irradiation is positively related to chicken interferon alpha biological activity, so that quantitative evaluation of chicken interferon alpha biological activity is realized.

Description

technical field [0001] The invention relates to a method for detecting the biological activity of chicken 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): C12N15/85C12N15/65C12Q1/02
CPCC12N15/85C12N15/65C12N2503/02C12N2800/106G01N33/5008G01N2500/10
Inventor 单雪芹刘家炉徐文俊王亚男赵雨蒋敏之
Owner ANHUI JIUCHUAN BIOTECH
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