Canine primary bronchial epithelial cell and application thereof in preparation of immortalized cells

A technology of bronchioles and epithelial cells, applied in the biological field, can solve the problems of changing the original characteristics of primary cells, carcinogenicity, inappropriate pathogenic mechanism, etc.

Active Publication Date: 2015-04-22
NANJING AGRICULTURAL UNIVERSITY
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Problems solved by technology

At present, the SV40 large T antigen is most widely used in the immortalization of cells. However, because the immortalized cells are carcinogenic and will change the original characteristics of the primary cells, the immortalized cells obtained by this method are used to study virus-infected cells. The pathogenic mechanism is inappropriate

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  • Canine primary bronchial epithelial cell and application thereof in preparation of immortalized cells
  • Canine primary bronchial epithelial cell and application thereof in preparation of immortalized cells
  • Canine primary bronchial epithelial cell and application thereof in preparation of immortalized cells

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Embodiment Construction

[0043] Below in conjunction with specific embodiment. The experimental method that does not indicate specific condition in the embodiment, generally adopts routine condition, for example " Molecular Cloning Laboratory Handbook " (New York: Cold Spring Harbor Laboratory Press, 1989) (Sambrook et al.) described condition, or according to The method recommended by the manufacturer. As for the isolation and cell culture of canine primary bronchiole tissue, since there is no report on the isolation and culture of canine related tissue cells, we refer to the isolation method of human bronchial epithelial cells [Vos JB, van Sterkenburg MA, Rabe KF, et al.Transcriptional response of bronchial epithelial cells to Pseudomonas aeruginosa: identification of early mediators of host defense. Physiol Genomics 2005,21(3):324-336].

[0044] (1) Isolation, primary cell culture and identification of canine bronchiole tissue

[0045] 1. Selection of Experimental Animals

[0046] The experiment...

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Abstract

The invention provides a canine primary bronchial epithelial cell and an application thereof in preparation of immortalized cells, and belongs to the technical field of biology. The canine primary bronchial epithelial cell is prepared by taking 45-day-old clean male beagles; separating canine tracheal tissues, cutting into pieces, adding collagenase and trypsin to digest, so as to obtain intact bronchiole tissues; cultivating the obtained cells, and further carrying out indirect immunofluorescence assay to detect keratin 18 expression, wherein an experiment proves that the canine primary bronchial epithelial cell is successfully obtained. A eukaryotic expression vector pEGFP-hTERT containing telomerase genes (hTERT) is built; after the canine primary bronchial epithelial cell is transfected, the hTERT gene is highly expressed; the bronchial epithelial cell can be stably subcultured for at least 20 generations. According to the canine primary bronchial epithelial cell provided by the invention, important biological materials are provided for research of infection mechanism of canine pathogens such as canine influenza viruses.

Description

technical field [0001] The invention relates to canine primary bronchiole epithelial cells and their application in preparing immortalized cells, belonging to the field of biotechnology. Background technique [0002] With the improvement of human civilization, the disease research of companion animals such as dogs has long been a focus of veterinary research. For dogs, infectious diseases mainly occur in the digestive tract and respiratory tract, and respiratory diseases are common throughout the year, and they are the most harmful to dogs. At present, microorganisms such as a variety of viruses and bacteria are considered to be the pathogens that cause canine respiratory diseases, such as the canine influenza virus that broke out in 2004 and was subsequently reported. However, compared with livestock and poultry pathogens, research on canine pathogens is relatively weak, especially what role these pathogens play in the occurrence of canine respiratory diseases and how the ...

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071C12N15/70C12N15/66
Inventor 刘永杰谢星庞茂达郁磊林焱赵艳兵梁姗马可
Owner NANJING AGRICULTURAL UNIVERSITY
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