Primary porcine tracheal epithelial cells (PTECs) are an appropriate model for studying the molecular mechanism of various porcine respiratory diseases, including swine-origin mycoplasmas, which are isolated from respiratory tract of pigs and mainly found on the mucosal surface surrounding swine trachea. However, the short proliferation ability of primary PTECs greatly limits their lifespan. In this study, primary PTECs were carefully isolated and cultured, and immortal PTECs were constructed by transfecting primary PTECs with the recombinant constructed plasmid pEGFP-hTERT containing human telomerase reverse transcriptase (hTERT). Immortal PTECs (hTERT-PTECs) maintained both the morphological and functional characteristics of primary PTECs, as indicated by the expression of cytokeratin 18, cell-cycle analysis, proliferation assay, Western blotting, telomerase activity assay, karyotype analysis and quantitative RT-PCR. Compared to primary PTECs, hTERT-PTECs had an extended replicative lifespan, higher telomerase activity, and enhanced proliferative activity. In addition, this cell line resulted in a lack of transformed and grown tumors in nude mice, suggesting that it could be safely applied in further studies. Moreover, hTERT-PTECs were vulnerable to all swine-origin mycoplasmas through quantitative analysis as indicated by 50％ color changing unit (CCU50) calculation, and no significant differences of adhesion ability between primary and immortal PTECs were observed. For the representative swine mycoplasma Mycoplasma hyopneumoniae (Mhp), except for DNA copies quantitative real-time PCR assay, indirect immunofluorescence assay and Western blotting analysis also depicted that hTERT-PTECs was able to adhere to different Mhp strains of different virulence. In summary, like primary PTECs, hTERT-PTECs could be widely used as an adhesion cell model for swine-origin mycoplasmas and in infection studies of various porcine respiratory pathogens.

XIE Xing;HAO Fei;WANG Hai-yan;PANG Mao-da;GAN Yuan;LIU Bei-bei;ZHANG Lei;WEI Yan-na;CHEN Rong;ZHANG Zhen-zhen;BAO Wen-bin;BAI Yun;SHAO Guo-qing;XIONG Qi-yan;FENG Zhi-xin

Key Laboratory for Veterinary Bio-Product Engineering of Ministry of Agriculture and Rural Affairs,Institute of Veterinary Medicine,Jiangsu Academy of Agricultural Sciences,Nanjing 210014,P.R.China;Jiangsu Key Laboratory for Food Quality and Safety-State Key Laboratory Cultivation Base of Ministry of Science and Technology,Institute of Food Safety and Nutrition,Jiangsu Academy of Agricultural Sciences,Nanjing 210014,P.R.China;College of Animal Science and Technology,Yangzhou University,Yangzhou 225009,P.R.China

农业科学学报（英文）

[1]XIE Xing;HAO Fei;WANG Hai-yan;PANG Mao-da;GAN Yuan;LIU Bei-bei;ZHANG Lei;WEI Yan-na;CHEN Rong;ZHANG Zhen-zhen;BAO Wen-bin;BAI Yun;SHAO Guo-qing;XIONG Qi-yan;FENG Zhi-xin-.Construction of a telomerase-immortalized porcine tracheal epithelial cell model for swine-origin mycoplasma infection)[J].农业科学学报（英文）,2022(02):504-520

PTECs,mycoplasmas,Immortal,CCU50,hyopneumoniae

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