猪伪狂犬病病毒感染对PK-15细胞增殖和热休克蛋白27、70和90表达的影响

doi:10.11843/j.issn.0366-6964.2019.07.013

摘要/Abstract

摘要： 研究猪伪狂犬病病毒（PRV）-YY株感染对宿主细胞增殖和热休克蛋白（HSP）27、70和90表达的影响。用Reed-Muench法测定PRV-YY滴度后，用不同病毒量感染PK-15细胞，采用iCELLigence实时无标记细胞功能分析仪监测细胞的增殖，并用实时荧光定量PCR（RT-qPCR）和免疫印迹试验（Western blot）分别检测感染后细胞中病毒核酸含量及三种HSPs mRNA和蛋白质表达的变化。结果表明，当用80 TCID₅₀和100 TCID₅₀的病毒量（滴度为10⁶TCID₅₀·0.1 mL^-1）感染细胞时，在所监测的时间内细胞指数均显著低于对照组；当感染60 h时，除10 TCID₅₀感染组外，其余各组的细胞指数均极显著低于对照组（P<0.01）；用10 TCID₅₀病毒感染细胞60 h时，细胞中病毒核酸含量达峰值；当病毒黏附结束即感染0 h时，HSP70和90 mRNA的转录升高（P<0.01），两者分别在感染6、12 h后表达降低（P<0.01），而HSP27在感染0 h升高（P<0.05），3 h时极显著高于对照组（P<0.01），但在感染48 h后其表达降低；PRV感染细胞后三种HSP蛋白的变化趋势基本与其mRNA水平的变化类似（部分时间点不同）。结果表明，一定滴度的PRV可显著抑制PK-15细胞的增殖，PRV感染可诱导PK-15细胞迅速发生应激反应，使HSP27、70和90表达快速升高，三种HSP的快速应答可能在保护细胞免受PRV-YY的感染损伤起着重要作用。

Abstract: In present study we investigated the effects of porcine pseudorabies virus (PRV) infection on proliferation and heat shock protein 27,70 and 90 expression of host cells in vitro. PK-15 cells were infected with different amounts of PRV-YY strain after the titer was determined by Reed-Muench method; cell proliferation was monitored by an iCELLigence Real-Time Labeled Cell Function Analyzer after infection; levels of viral nucleic acid and the mRNA and protein expression of HSP27, 70 and 90 in PRV-infected cells were detected using Real-time fluorescence quantitative PCR (qPCR) and Western blot assays, respectively. Results were as follows:The cell indexes were decreased when cells were infected with 80 TCID₅₀ and 100 TCID₅₀ of virus (10⁶ TCID₅₀·0.1 mL^-1) within the time of detection. Sixty hours after infection, cell indexes of all treatment groups, except the group of 10 TCID₅₀ virus infection, were significantly lower than that of the control group (P<0.01). Levels of viral nucleic acid reached a peak after cells were infected with 10 TCID₅₀virus for 60 h. The transcription of HSP70 and 90 mRNA increased significantly at 0 h after infection (P<0.01), and significantly decreased at 6 and 12 h after infection (P<0.01), respectively; and the transcription of HSP27 increased significantly at 0 (P<0.05) and 3 h after infection (P<0.01), and expression of its protein significantly decreased at 48 h post of infection (P<0.01). The change trends of the three HSP proteins were similar to those of their mRNAs after PRV infection other than partial time points. The results indicate that PRV-YY infection can inhibit the proliferation of PK-15 cells, and induce rapid stress response in PK-15 cells with increasing expression of HSP27, 70 and 90. The rapid response of the three HSPs in host cells upon PRV infection may play important roles in protecting cells from virus infection.

中图分类号:

S852.659.1

方娟, 陈指龙, 黎陈, 张逢, 伍小松, 杨青. 猪伪狂犬病病毒感染对PK-15细胞增殖和热休克蛋白27、70和90表达的影响[J]. 畜牧兽医学报, 2019, 50(7): 1441-1448.

FANG Juan, CHEN Zhilong, LI Chen, ZHANG Feng, WU Xiaosong, YANG Qing. Effect of Porcine Pseudorabies Virus Infection on Proliferation of PK-15 Cell and Expression of Heat Shock Protein 27,70 and 90[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2019, 50(7): 1441-1448.

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