Establishment and evaluation of LLC-MK2 cell lines stably expressing transmembrane protease serine 2 (TMPRSS2)
10.3760/cma.j.cn112866-20250224-00042-1
- VernacularTitle:稳定表达TMPRSS2的LLC-MK2细胞系的构建和评价
- Author:
Yitong LIN
1
;
Hai LI
1
;
Yan ZHANG
1
;
Naiying MAO
1
;
Zhen ZHU
1
Author Information
1. 传染病溯源预警与智能决策全国重点实验室 国家卫生健康委医学病毒和病毒病重点实验室 WHO西太平洋地区麻疹/风疹参比实验室 中国疾病预防控制中心病毒病预防控制所，北京 102206
- Publication Type:Journal Article
- Keywords:
Transmembrane protease serine 2;
Human parainfluenza virus;
Virus isolation;
Transposon system
- From:
Chinese Journal of Experimental and Clinical Virology
2025;39(3):303-311
- CountryChina
- Language:Chinese
-
Abstract:
Objective:A monoclonal cell line that stably expresses transmembrane protease serine 2 (TMPRSS2) was constructed using the PiggyBac (PB) transposon system to establish a technique for isolating human parainfluenza viruses (HPIVs) without rely on exogenous trypsin.Methods:The pB513B/TMPRSS2 recombinant expression plasmid was synthetized based on the principle of PB transposition, and co-transfected into LLC-MK2, HEp-2, and Vero cells, together with the helper plasmid. Positive cells were then identified using the puromycin resistance and limiting dilution method. Stable TMPRSS2 expression was verified by PCR and western blot (WB). Furthermore, the viral replication capacity of the cell lines was evaluated using digital PCR and immunostaining plaque assays.Results:The successful construction of the recombinant plasmid pB513B/TMPRSS2 was confirmed through restriction enzyme digestion and DNA sequencing. Following transfection, the LLC-MK2/TMPRSS2 cell survival rate was higher than those of the HEp-2/TMPRSS2 and Vero/TMPRSS2 cells. Subsequent PCR and WB analysis results revealed that the LLC-MK2/TMPRSS2 cells maintained stable TMPRSS2 expression after 20 successive passages. Cell counting kit-8 (CCK-8) assays confirmed that there was no significant difference in proliferative activity between LLC-MK2/TMPRSS2 cells and LLC-MK2 cells. Viral replication capability testing showed that the laboratory-adapted strain of HPIV3 (ATCC strain) exhibited exponential growth in both LLC-MK2 and LLC-MK2/TMPRSS2 cells in culture without exogenous trypsin. Notably, LLC-MK2/TMPRSS2 cells significantly supported the replication of clinical HPIV3 isolates (circulating strain), whereas LLC-MK2 cells failed to sustain their infectivity effectively.Conclusions:In this study, we successfully generated the LLC-MK2/TMPRSS2 cell line using the PB transposon system to stably express TMPRSS2. Our TMPRSS2-transfected cell line enables the efficient isolation and propagation of HPIVs in vitro, eliminating the need for exogenous trypsin and providing a vital technical platform for isolating HPIVs and studying their pathogenicity.
