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Cell line for detecting activity of human interferon-kappa and construction and detection method thereof

A construction method and activity detection technology, which is applied in the field of detection of human interferon-κ activity, can solve the problems of rare hIFN-κ rapid detection activity, and achieve the effect of improving activity release efficiency, large signal window, and short detection time

Active Publication Date: 2021-05-07
SHANDONG RUIYING PIONEER PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, in the prior art, research on methods for rapid detection of hIFN-κ activity is relatively rare

Method used

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  • Cell line for detecting activity of human interferon-kappa and construction and detection method thereof
  • Cell line for detecting activity of human interferon-kappa and construction and detection method thereof
  • Cell line for detecting activity of human interferon-kappa and construction and detection method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0080] Example 1: Monoclonal preparation of stably transfected cell lines

[0081] 1. Recovery culture of HEK293 cells

[0082] HEK293 cells were taken out of the liquid nitrogen tank, melted and recovered in a water bath at 37°C, and then cultured in a complete medium (high-glucose DMEM medium (Gbico) containing 10% fetal bovine serum) and placed in a 37°C, 5% carbon dioxide atmosphere. Cultivate in a cell incubator; the cells are normally passaged for 3 generations to ensure that the cells are in good condition.

[0083] 2. Transfection of reporter gene plasmid

[0084] 1) Take HEK293 cells in the logarithmic growth phase, digest with trypsin, stop the digestion with complete medium, add to a centrifuge tube after pipetting, centrifuge at 1000rpm for 5 minutes, and adjust the cell concentration to 3x10 with complete medium 5 cells / ml;

[0085] 2) Inoculate the cells into a 6-well plate, 100 μl per well, and continue culturing for 24 hours;

[0086] 3) According to the ...

Embodiment 2

[0121] Example 2: Detection of hIFN-κ using HEK293-ISRE-Luc cell line

[0122] The positive HEK293-ISRE-Luc monoclonal cell line with the best signal prepared in Example 1 was continuously passaged in the screening medium for stability testing, and some cells were replaced at P5, P10, P15 and P20 respectively, according to Example 1 Steps 1)-8) in the process of "6. Monoclonal function test and passage stability test" measure the interferon-κ activity in the same way as the steps, the results are as follows in Table 1 and figure 2 As shown, and the process flow of hIFN-κ detection is as follows image 3 shown.

[0123] Table 1

[0124] pass on P5 P10 P15 P20 EC50(ng / ml) 26.55 33.69 16.71 42.26

[0125] It can be seen from the above examples that the HEK293-ISRE-Luc cell line of the present invention can effectively detect human interferon-κ activity when it is subcultured to the fifth generation. When the cell line of the present invention was...

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Abstract

The invention discloses a cell line for detecting the activity of human interferon-kappa and a construction and detection method thereof. The cell line is obtained by transfecting eukaryotic cells with ISRE (interferon-sensitive response element) luciferase reporter genes and stably screening monoclonal cell lines. The cell line constructed by the invention is easy to culture and amplify and good in passage stability, and has the advantages of high sensitivity, short detection time, completion within 4-6 hours, and capability of greatly improving the activity release efficiency of the human interferon-kappa in the production process.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to detecting the activity of human interferon-κ using a stably transfected cell line. Background technique [0002] Interferon (Interferon, IFN) is a class of cytokines with broad-spectrum anti-virus, anti-tumor, and immunomodulatory effects. It can exert antiviral activity by binding to cognate receptor complexes on target cells. [0003] Interferon kappa (interferon-κ, IFN-κ) is a new IFN member discovered in recent years, in which IFN-κ consists of 207 amino acid residues, including a signal peptide of 27 amino acid residues at the N-terminal, and I Other members of type IFN have about 30% homology, and the IFN-κ member is slightly larger than other type I IFNs, with an insertion of 12 amino acid residues between the C and D helices. The IFN-κ gene is located on the short arm of the ninth pair of chromosomes, adjacent to other type I IFN genes, and the 3' non...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/85C12N15/53C12Q1/02G01N21/64C12R1/91
CPCC12N5/0686C12N15/85C12N9/0069G01N33/5044G01N21/6428C12N2510/00C12N2503/02Y02A50/30
Inventor 彭继先柴辉郭灵华闫韵秋钱文正于海勤
Owner SHANDONG RUIYING PIONEER PHARMA
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