猪源G3BP1基因的稳定敲低PK-15细胞系构建及生物信息学分析

Construction and bioinformatics analysis of a PK-15 cell line with stable knockdown of porcine G3BP1 gene

摘要:

【背景】Ras-GTP酶活化蛋白SH3结构域结合蛋白1 (Ras-GTPase-activating protein SH3 domain binding protein 1, G3BP1)是应激颗粒(stress granule, SG)的重要组成部分和标志性蛋白，在宿主抵抗病原体入侵过程中发挥重要作用。【目的】构建G3BP1稳定敲低的PK-15细胞系，探究敲低G3BP1基因对水泡性口炎病毒(vesicular stomatitis virus, VSV)、塞内卡病毒(seneca virus, SVV)和痘苗病毒(vaccinia virus, VACV)复制的影响和对肿瘤坏死因子-α (tumour necrosis factor α, TNF-α)、白细胞介素6 (interleukin 6, IL-6)、抗黏病毒蛋白1 (myxovirus resistance protein 1, Mx1)、干扰素刺激基因54 (IFN-stimulated gene 54, ISG54)和干扰素β (interferon β, IFN-β)表达的影响，并对该基因进行生物信息学分析。【方法】利用慢病毒载体构建稳定敲低G3BP1基因的PK-15细胞系，利用逆转录实时定量PCR (reverse transcription quantitative real-time PCR, RT-qPCR)和实时定量PCR (quantitative real-time PCR, qPCR)分析敲低G3BP1基因对VSV、SVV和VACV复制的影响，利用RT-qPCR分析感染VSV、SVV和VACV后敲低G3BP1基因对TNF-α、IL-6、Mx1、ISG54和IFN-β的影响，通过在线工具对G3BP1基因进行生物信息学分析。【结果】RT-qPCR和免疫印迹(Western blotting, WB)结果显示成功构建稳定敲低G3BP1的PK-15细胞系；RT-qPCR和qPCR结果显示敲低G3BP1基因可极显著促进VSV和SVV的复制(P<0.000 1)，但对VACV的复制无显著影响(P>0.05)；RT-qPCR结果显示VSV和SVV感染可显著或极显著促进TNF-α、IL-6、Mx1、ISG54和IFN-β的mRNA表达水平(P<0.05，P<0.000 1)，敲低G3BP1基因可极显著抑制上述细胞因子的mRNA表达(P<0.01，P<0.000 1)，RT-qPCR结果显示VACV感染可以极显著抑制上述细胞因子的mRNA表达(P<0.01，P<0.000 1)，敲低G3BP1基因对VACV抑制的细胞因子mRNA表达无显著影响(P>0.05)；理化性质预测结果显示，猪源G3BP1蛋白存在30个磷酸化位点，具有不稳定性和亲水性，属于非跨膜和非分泌蛋白；蛋白质高级结构预测显示，该蛋白的二级结构主要由无规则卷曲(52.47%)组成，主要定位于细胞质和细胞核中，分别占34.38%和31.25%。【结论】本研究获得了猪源G3BP1基因稳定敲低的PK-15细胞系，敲低该基因可极显著促进VSV和SVV的复制，但对VACV的复制无显著影响；敲低该基因可显著或极显著抑制TNF-α、IL-6、Mx1、ISG54和IFN-β的产生；预测该蛋白存在30个磷酸化位点，为非跨膜和非分泌蛋白，具有不稳定性和亲水性。这些结果可为进一步研究G3BP1在病毒感染过程中的作用机制提供工具和参考依据。

Abstract:

[Background] Ras-GTPase-activating protein SH3 domain binding protein 1 (G3BP1), an important component and iconic protein of stress granules (SGs), plays a role in host resistance to pathogen invasion. [Objective] To construct a PK-15 cell line with stable knockdown of G3BP1 gene, and to explore the effects of knocking down G3BP1 gene on the replication of vesicular stomatitis virus (VSV), seneca virus (SVV), and vaccinia virus (VACV) and the expression of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), myxovirus resistance protein 1 (Mx1), interferon-stimulated gene 54 (ISG54), and interferon-β (IFN-β). [Methods] The lentiviral vector was used to construct a PK-15 cell line with stable knockdown of porcine G3BP1 gene. Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) and quantitative real-time polymerase chain reaction (qPCR) were carried out to analyze the effects of G3BP1 knockdown on the replication of VSV, SVV, and VACV and the expression of TNF-α, IL-6, Mx1, ISG54, and IFN-β after infection with VSV, SVV and VACV. The bioinformatics tools were used to characterize the G3BP1 gene. [Results] A PK-15 cell line with stable knockdown of G3BP1 was successfully constructed. RT-qPCR and qPCR results showed that knockdown of G3BP1 gene promoted the replication of VSV and SVV (P<0.000 1), while it had no significant effect on the replication of VACV (P>0.05). RT-qPCR results showed that VSV and SVV infections up-regulated the mRNA levels of TNF-α, IL-6, Mx1, ISG54, and IFN-β (P<0.05, P<0.000 1), and knocking down G3BP1 gene down-regulated the mRNA levels of these cytokines (P<0.01, P<0.000 1). RT-qPCR results showed that VACV infection inhibited the production of these cytokines (P<0.01, P<0.000 1), and knocking down G3BP1 gene had no significant effect on the expression of the cytokines inhibited by VACV (P>0.05). The deduced porcine G3BP1 protein was an unstable, hydrophilic, and non-secretory protein with 30 phosphorylation sites, with no transmembrane region. The secondary structure of the protein was mainly composed of random coils (52.47%). The protein was mainly located in the cytoplasm (34.38%) and nucleus (31.25%). [Conclusion] In this study, a PK-15 cell line with stable knockdown of porcine G3BP1 gene was obtained. Knocking down this gene promoted the replication of VSV and SVV but had no significant effect on the VACV replication. Furthermore, it significantly inhibited the production of TNF-α, IL-6, Mx1, ISG54, and IFN-β. The protein had 30 phosphorylation sites and was an unstable, hydrophilic, and non-secretory protein with no transmembrane region. The results help to further reveal the role of G3BP1 in viral infection.