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Porcine bronchial epithelial cell line, preparation method and application thereof

A bronchial epithelium, cell line technology, applied in the biological field, can solve the problems of lack of host cell model, unclear, weak research on porcine pathogens, etc., to achieve the effect of improving screening efficiency, reducing damage, and saving digestion time

Active Publication Date: 2020-08-28
JIANGSU ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, compared with humans and poultry, the research on porcine pathogens is still relatively weak, especially how these pathogens play a role in the generation of porcine respiratory diseases and how the pathogenic mechanism is still unclear
[0003] At present, the research on porcine respiratory pathogens mainly focuses on the infection in pigs, and there is still a lack of suitable host cell models, such as bronchial epithelial cell lines

Method used

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  • Porcine bronchial epithelial cell line, preparation method and application thereof
  • Porcine bronchial epithelial cell line, preparation method and application thereof
  • Porcine bronchial epithelial cell line, preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1 Separation of Porcine Bronchial Tissue, Culture and Identification of Primary Cells

[0028] 1. Isolation of Porcine Bronchial Tissue

[0029] (1) Sumianxin (purchased from Foshan Shuangping Pet Products Co., Ltd.), 50-day-old clean-grade male Dudu hybrid pigs (purchased from Nanjing Zhoubang Biotechnology Co., Ltd.) were killed by intravenous anesthesia of the forelimbs After fixation, use a set of surgical instruments (two scissors, two tweezers, one ophthalmic scissors, one ophthalmic forceps) to bleed the abdominal incision after the epidermis tissue to be treated is disinfected with alcohol.

[0030] (2) The surgical scissors cut the skin longitudinally from the throat to the last rib of the chest, and cut the skin horizontally along the ribs on both sides to separate the skin.

[0031] (3) Another set of surgical instruments (two scissors, two tweezers, one ophthalmic scissors, one ophthalmic tweezers), separate the throat muscles, expose the trachea, u...

Embodiment 2

[0061] Example 2 Establishment of porcine bronchial epithelial cell line hTERT-PBEC

[0062] 1. Test material

[0063] Human telomerase plasmid PCI-neo-hTERT (carrying human telomerase reverse transcriptase gene fragment) and fluorescent eukaryotic expression vector pIRES2-EGFP were purchased from Biovector China Plasmid Vector Strain Cell Line Gene Collection Center.

[0064] 2. Construction of human telomerase eukaryotic expression vector pEGFP-hTERT

[0065] The human telomerase recombinant plasmid PCI-neo-hTERT carries the human telomerase reverse transcriptase gene. Since this plasmid only has neomycin G418 resistance, the direct transfection of PCI-neo-hTERT into porcine primary bronchial epithelial cells can only Blindly passed the G418 screening and it is very easy to form drug resistance to G418, so the present invention has been improved as follows.

[0066] Human telomerase recombinant plasmid PCI-neo-hTERT and blank plasmid pIRES2-EGFP were purchased and kept in ...

Embodiment 3

[0082] Example 3 Detection of porcine bronchial epithelial cell line hTERT-PBEC

[0083] 1. Determination of cell growth curve

[0084] The 60th passage cells of porcine bronchial epithelial cell line hTERT-PBEC and the 4th passage cells of porcine primary bronchial epithelial cells contained 2×10 4 Cells were inoculated into 24-well cell culture plates at the same time point every day, and the cells in three wells were collected by trypsinization at the same time point every day, and the cell growth curve was measured, referring to the method reported in the literature (Hong HX, Zhang Y M, Xu H, Su Z Y , Sun P. Immortalization of swine umbilical veinendothelial cells with human telomerase reverse transcriptase. Mol Cells, 2007, 24(3): 358-63) for cell counting, which continued for 9 days. The result is as Figure 4 As shown in A, both the porcine primary bronchial epithelial cells and the porcine bronchial epithelial cell line hTERT-PBEC showed a trend of increasing first a...

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Abstract

The invention provides a porcine bronchial epithelial cell line and a preparation method and application thereof, and belongs to the field of biological technology. The porcine bronchial epithelial cell line is hTERT-PBEC, and the preservation number is CCTCCNO:C201749. The preparation method of the cell line hTERT-PBEC includes constructing of eukaryotic expression vector pEGFP-hTERT carrying green fluorescent protein gene and human telomerase reverse transcriptase gene; using of Lipofectamin3000 for transfection of primary porcine bronchial epithelial cells with the eukaryotic expression vector pEGFP-hTERT, and G418 screening to obtain the porcine bronchial epithelial cell line hTERT-PBEC. The screening efficiency is remarkably improved through ingenious method, and the obtained porcine bronchial epithelial cell line can steadily pass over 60 generations, retains the characteristics of the primary cells, and can be applied to study on the pathogenic mechanism of porcine respiratory tract pathogen infection.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a porcine bronchial epithelial cell line, a preparation method and an application thereof. Background technique [0002] Porcine infectious diseases are a major health threat to the breeding industry and human beings all over the world, and the research on pig diseases has already become a focus of veterinary research. For pigs, infectious diseases mainly occur in the digestive tract and respiratory tract, and respiratory diseases are common throughout the year and are the most harmful to pigs. At present, a variety of viruses, bacteria, parasites and other microorganisms are considered to be pathogens that can cause respiratory diseases in pigs, such as Mycoplasma hyopneumoniae, porcine blue ear disease, type 2 circovirus, swine influenza virus, etc., which are common in pig farms. However, compared with humans and poultry, the research on porcine pathogens is still rel...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/10C12N15/85C12R1/91
CPCC12N5/0625C12N9/1276C12N15/85C12N2510/00C12Y207/07049
Inventor 谢星邵国青冯志新甘源庞茂达张磊刘蓓蓓王海燕于岩飞陈蓉熊祺琰刘茂军韦艳娜华利忠吴猛
Owner JIANGSU ACAD OF AGRI SCI
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