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MDCK (Madin-Darby canine kidney) cell line capable of stably expressing human-derived TIGAR (TP53-induced glycolysis and apoptosis regulator) gene

A stable expression and cell line technology, applied to genetically modified cells, cells modified by introducing foreign genetic material, animal cells, etc., can solve the complex digestion process, limited substrate surface area, and difficulty in large-scale cultivation of influenza vaccines in MDCK cells Industrial production and other issues, to achieve the effect of prolonging survival time, reducing adhesion performance, and improving anti-apoptosis ability

Active Publication Date: 2017-03-08
ZHONGCHONG XINNUO BIOTECH TAIZHOU CO LTD
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Problems solved by technology

The traditional culture method adopts two-dimensional monolayer culture [Frisch SM&Francis H (1994) Disruption of epithelial cell-matrix interactions induces apoptosis. The Journal of cell biology 124 (4): 619-626.], due to the limitation of the substrate surface area, in addition Difficult to achieve large-scale cultivation of MDCK cells and industrial production of influenza vaccine due to complex digestion process, long production time and high production cost

Method used

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  • MDCK (Madin-Darby canine kidney) cell line capable of stably expressing human-derived TIGAR (TP53-induced glycolysis and apoptosis regulator) gene
  • MDCK (Madin-Darby canine kidney) cell line capable of stably expressing human-derived TIGAR (TP53-induced glycolysis and apoptosis regulator) gene
  • MDCK (Madin-Darby canine kidney) cell line capable of stably expressing human-derived TIGAR (TP53-induced glycolysis and apoptosis regulator) gene

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Experimental program
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Effect test

Embodiment Construction

[0027] 1. Construction of MDCK-TIGAR cell line

[0028] The eukaryotic expression plasmid containing the open reading frame of the human TIGAR gene was purchased from Genecopoeia Company (article number: EX-W1314-M02-5-5); the GenBank accession numbers of the human TIGAR gene are NM_020 375, respectively. The primer sequences used are listed in Table 1. at 35mm 2 Inoculate 2×10 cells in a culture dish 5 MDCK cells in good growth state were placed at 37°C, 6% CO 2 cultured in a constant temperature incubator. When the cells grow to 70%-80%, they are ready for transfection. Equilibrate the plasmid and transfection reagent Lipofectamine2000Regeant to room temperature, take 4.3 μL of the plasmid with a concentration of 470 ng / mL in a sterilized finger tube, add 100 μL of anti-blood-free medium, mix well and add 5 μL of transfection reagent, Act at room temperature for 15-20 minutes to form DNA-liposome complexes. Then this complex was added dropwise to the MDCK cells to be u...

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Abstract

The invention belongs to the technical field of biology, and particularly relates to an MDCK (Madin-Darby canine kidney) cell line capable of stably expressing a human-derived TIGAR (TP53-induced glycolysis and apoptosis regulator) gene. The cell line is an MDCK cell line TG-418-E5 capable of stably expressing the human-derived TIGAR gene and contains a TIGAR coding gene sequence, and the preservation number of the cell line is CGMCC NO:12983. Through stable expression of the TIGAR gene in MDCK cells, anti-apoptosis capacity of the cells is improved, and the survival time of recombinant cells is prolonged. The method not only lays the foundation for research of application of MDCK serum-free fully-suspended culture in mass production of cell culture vaccines, but also increases breeding titer of avian influenza vaccine strains and saves the cost of a production process. Besides, the cell line TG-418-E5 has better application prospect in aspects of screening of anti-avian influenza virus drugs, screening of vaccine strains and production of the cell culture vaccines.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a MDCK cell line stably expressing a human TIGAR gene, and the cell line can improve cell anti-apoptosis ability, reduce MDCK cell adhesion performance, and be used for the screening of anti-avian influenza virus drugs, in the and antibody assays as well as low-serum culture for MDCK cells to produce cell culture vaccines on a large scale. Background technique [0002] Avian influenza virus (AIV) belongs to the Orthomyxoviridae family Influenzavirus A, and there are 16 and 9 surface glycoproteins hemagglutinin (HA) and neuraminidase (NA), respectively [Webster RG, Bean WJ, Gorman OT , Chambers TM, & Kawaoka Y (1992) Evolution and ecology of influenza A viruses. Microbiological reviews 56(1):152-179.]. Among them, highly pathogenic avian influenza (subtypes H5 and H7) has caused huge economic losses to the poultry industry worldwide, and at the same time poses a serious t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N5/02C12Q1/02C12N7/00
CPCC12N5/0686C12N7/00C12N2503/02C12N2510/00C12N2760/16151G01N33/5044G01N2500/10
Inventor 刘秀梵孙中涛刘晓文许靖李群辉王晓泉胡娇顾敏胡顺林
Owner ZHONGCHONG XINNUO BIOTECH TAIZHOU CO LTD
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