稳定表达脯氨酰寡肽酶基因的PK15细胞系的建立及其对口蹄疫病毒增殖的影响

A PK15 cell line stably expressing prolyl oligopeptidase (POP) and its effect on the proliferation of foot-and-mouth disease virus

摘要:

目的 利用PiggyBac转座子系统构建稳定表达脯氨酰寡肽酶(prolyl oligopeptidase, POP)基因的猪肾PK15细胞系，并系统评估POP蛋白表达对口蹄疫病毒(foot and mouth disease virus, FMDV)增殖的影响。方法 构建重组表达质粒，转染PK15细胞后通过Western blotting检测目的蛋白表达；采用有限稀释法筛选获得稳定表达POP的单克隆细胞系，并利用RT-qPCR和Western blotting检测POP基因和蛋白表达的稳定性；通过Cell Counting Kit-8试验评估筛选细胞系的细胞活力；综合运用Western blotting、RT-qPCR以及病毒滴度测定等方法，系统分析FMDV在稳定表达细胞系中的复制。结果 成功构建了稳定表达POP蛋白的PK15单克隆细胞系；与对照细胞系相比，该细胞系的细胞活力无显著差异；单克隆细胞系传至P30代时，POP蛋白表达水平保持稳定；接种病毒实验结果显示，FMDV在稳定表达POP的PK15细胞系中的增殖水平显著高于对照组，并在不同代次细胞系中保持稳定。结论 本研究成功构建了稳定表达POP蛋白的PK15细胞系，并证实该细胞系可显著促进FMDV的复制，为进一步解析POP蛋白促进FMDV复制的分子机制提供了实验材料。

Abstract:

Objective To construct a PK15 cell line stably expressing the prolyl oligopeptidase (POP) by using the PiggyBac transposon system and systematically investigate its regulatory role in the proliferation of foot-and-mouth disease virus (FMDV).Methods A recombinant PiggyBac vector carrying the POP gene was constructed and transfected into PK15 cells, and the expression of the target protein was detected by Western blotting. The monoclonal cell line stably expressing POP was isolated via the limiting dilution method. The stability of mRNA and protein levels of POP was verified by RT-qPCR and Western blotting, respectively. The viability of the selected cell line was assessed by the Cell Counting Kit-8 (CCK-8) assay. FMDV replication kinetics in the stable cell line were comprehensively analyzed by Western blotting, RT-qPCR, and the 50% tissue culture infective dose (TCID₅₀) assay.Results A PK15 cell line stably expressing POP was established. No significant differences in cell viability were observed between the stable cell line and the control cell line. The protein level of POP remained stable in the established cell line after 30 passages. The results of the viral infection assay demonstrated that the FMDV proliferation level in the PK15 cell line stably expressing POP was significantly higher than that in the control group, with this stimulatory effect being maintained across multiple passages.Conclusion We successfully constructed a PK15 cell line stably expressing POP. The results reveal that POP overexpression enhances FMDV replication in a passage-independent manner. These findings provide a valuable experimental model for elucidating the molecular mechanism underlying the role of POP in FMDV replication.