宿主SRC在鸡传染性喉气管炎病毒感染中作用的研究
发布时间：2018-12-14 16:07
【摘要】：鸡传染性喉气管炎(AILT)是由传染性喉气管炎病毒(ILTV)感染引发的一种鸡的呼吸系统疾病。ILTV属于疱疹病毒科(Herpesviridae family)传喉炎病毒属(Iltovirus genus)。不同病毒株依据其毒力不同可以造成鸡不同程度的上呼吸道疾病,对世界养禽业造成巨大经济损失。当前,AILT防治主要依赖于疫苗接种引发的机体免疫应答发挥作用。然而,由于免疫接种不能清除宿主细胞中潜伏感染的病毒和抑制病毒在细胞内的复制,难以长效防控疫情。因此,探索新策略对AILT的防控具有重要意义。本研究用感染了ILTV LJS09毒株的LMH细胞作为体外实验的模型,探讨了宿主细胞应答ILTV感染的分子机制。为了更好的阐述病毒感染宿主细胞的相关分子事件,本试验首先通过ILTV特异性实时定量PCR和TCID50等方法对感染LMH细胞过程中ILTV LJS09的生物学特性进行了测定。然后,通过基因芯片技术在全基因组范围内检测了宿主基因的转录水平,并结合生物信息学分析预测宿主SRC在LMH细胞应答ILTV感染的过程中可能发挥重要作用。SRC是第一个被鉴定的原癌基因,可以参与真核生物细胞粘附、增殖、骨架重建、细胞分裂及凋亡等多种生命活动。蛋白免疫印迹结果显示,在ILTV感染后第二天LMH细胞中SRC的磷酸化作用增强,说明ILTV感染可以活化宿主细胞SRC。而LMH细胞的生长曲线显示在病毒感染后第三天,病毒感染组的细胞数量大大减少。鉴于SRC被广泛报道可以影响细胞增殖,我们用流式细胞术对细胞周期做了分析,发现随着感染时间的增加,病毒感染使处于G1期的宿主细胞显著减少,凋亡细胞不断增加,然而进入S/M期和G2期的细胞比例并没有降低。为了深入了解SRC在ILTV感染中的具体作用,本研究应用小分子抑制剂和特异性靶向鸡SRC基因的小干扰片段对SRC在LMH细胞应答ILTV侵染过程中的生物学功能进行了检测。ILTV特异性实时定量PCR检测显示,SRC被抑制后LMH细胞中的病毒含量显著降低。流式细胞术结果表明,SRC抑制可以促进病毒介导的细胞死亡。因此,SRC对维持病毒的增殖是不可或缺的,但ILTV介导的细胞死亡并不依赖SRC。以上结果提示,SRC在ILTV感染的细胞中是病毒毒力和复制的关键决定因素,ILTV感染后,宿主细胞通过SRC提高病毒诱发细胞死亡的阈值进而延缓宿主细胞的死亡。通过检测SRC特异性小分子抑制剂处理的SPF鸡胚接种ILTV后的死亡情况和尿囊膜及尿囊液中的病毒含量,我们在鸡胚试验中也证实了SRC对ILTV感染的影响。为了探究SRC影响ILTV感染LMH细胞的生物学机制,我们进一步分析了转录组学的数据并构建了表达差异基因的蛋白质互作网络。分析结果预测局部粘着斑激酶(FAK)可能也参与了SRC调节LITV感染的过程。应用小分子抑制剂和RNA干扰等功能学研究,我们在细胞和鸡胚中同时证实了这一推论,并进一步发现SRC与FAK间存在蛋白磷酸化的正反馈调节作用。随后的功能学研究显示这一过程对于SRC调节ILTV感染至关重要。本研究初步探讨了宿主细胞应答ILTV感染的分子机制,拓展了当前对于ILTV与宿主互作分子机制的认知,也为建立更为安全、长效的AILT防治方法提供一定的理论支持。
[Abstract]:Chicken infectious laryngotracheitis (AILT) is a respiratory disease of a chicken caused by infectious laryngotracheitis virus (ILTV) infection. ILTV is a virus genus of the family of Herpesvirus family. The different virus strains can cause the upper respiratory tract diseases of different degrees of the chicken according to different virulence, and can cause great economic loss to the poultry industry in the world. At present, the prevention and control of AILT mainly depends on the immune response of the body induced by the vaccination. However, it is difficult for long-term prevention and control of the disease due to the inability of the immunization to remove the virus that is latent in the host cell and to inhibit the replication of the virus within the cell. Therefore, it is of great significance to explore new strategies for prevention and control of AILT. In this study, LMH cells infected with ILTV LJS09 strain were used as a model of in vitro experiments, and the molecular mechanism of host cell response to ILTV infection was discussed. The biological characteristics of ILTV LJS09 in the infected LMH cells were determined by ILTV-specific real-time quantitative PCR and TCID50. Then, the transcription level of the host gene is detected by the gene chip technology in the whole genome range, and the host SRC is predicted to play an important role in the process of the LMH cell response to the ILTV infection by combining the bioinformatics analysis. SRC is the first to be identified protooncogene and can be involved in many life activities such as the adhesion, proliferation, skeleton reconstruction, cell division and apoptosis of the true nuclear biological cells. Western blot showed that the phosphorylation of SRC in LMH cells increased after ILTV infection, indicating that ILTV infection could activate the host cell SRC. The growth curve of LMH cells showed a significant decrease in the number of cells in the viral infection group on the third day after the virus infection. Because SRC is widely reported to affect cell proliferation, we have analyzed the cell cycle by flow cytometry, and it was found that with the increase of the infection time, the host cells at the G1 phase were significantly reduced and the apoptotic cells increased continuously. However, the proportion of cells entering the S/ M phase and G2 phase was not reduced. In order to understand the specific role of SRC in ILTV infection, the biological function of SRC in LMH cell response to ILTV infection was detected by using small molecule inhibitor and small interfering fragment of specific target chicken SRC gene. The detection of ILTV-specific real-time quantitative PCR showed that the virus content in LMH cells was significantly reduced after SRC was inhibited. Flow cytometry showed that SRC inhibition could promote viral-mediated cell death. Therefore, SRC is essential for maintaining the proliferation of the virus, but the ILTV-mediated cell death does not rely on SRC. The above results suggest that SRC is a key determinant of virus virulence and replication in ILTV-infected cells. After ILTV infection, host cells increase the threshold of virus-induced cell death by SRC and delay the death of host cells. The effect of SRC on ILTV infection was also confirmed in the chicken embryo test by detecting the death of the SPF chicken embryo treated with SRC-specific small-molecule inhibitor and the virus content in the allantoic membrane and allantoic fluid. In order to explore the biological mechanism of SRC on LMH cells infected by ILTV, we further analyze the data of transcriptome and construct a protein cross-network for differentially expressed genes. The results of the analysis predict that the local adhesion kinase (FAK) may also be involved in the process of SRC regulation of the LITV infection. In the study of the function of small molecule inhibitor and RNA interference, this deduction was also confirmed in the cell and chick embryo, and the positive feedback regulation of protein phosphorylation between SRC and FAK was further found. Subsequent functional studies show that this process is critical to the SRC regulation of ILTV infection. In this study, the molecular mechanism of the host cell response to ILTV infection is discussed, and the current cognition of the interaction between the ILTV and the host is expanded, and a certain theoretical support for establishing a more safe and long-acting AILT control method is also provided.
【学位授予单位】：中国农业科学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S858.31
本文编号：2378921
[Abstract]:Chicken infectious laryngotracheitis (AILT) is a respiratory disease of a chicken caused by infectious laryngotracheitis virus (ILTV) infection. ILTV is a virus genus of the family of Herpesvirus family. The different virus strains can cause the upper respiratory tract diseases of different degrees of the chicken according to different virulence, and can cause great economic loss to the poultry industry in the world. At present, the prevention and control of AILT mainly depends on the immune response of the body induced by the vaccination. However, it is difficult for long-term prevention and control of the disease due to the inability of the immunization to remove the virus that is latent in the host cell and to inhibit the replication of the virus within the cell. Therefore, it is of great significance to explore new strategies for prevention and control of AILT. In this study, LMH cells infected with ILTV LJS09 strain were used as a model of in vitro experiments, and the molecular mechanism of host cell response to ILTV infection was discussed. The biological characteristics of ILTV LJS09 in the infected LMH cells were determined by ILTV-specific real-time quantitative PCR and TCID50. Then, the transcription level of the host gene is detected by the gene chip technology in the whole genome range, and the host SRC is predicted to play an important role in the process of the LMH cell response to the ILTV infection by combining the bioinformatics analysis. SRC is the first to be identified protooncogene and can be involved in many life activities such as the adhesion, proliferation, skeleton reconstruction, cell division and apoptosis of the true nuclear biological cells. Western blot showed that the phosphorylation of SRC in LMH cells increased after ILTV infection, indicating that ILTV infection could activate the host cell SRC. The growth curve of LMH cells showed a significant decrease in the number of cells in the viral infection group on the third day after the virus infection. Because SRC is widely reported to affect cell proliferation, we have analyzed the cell cycle by flow cytometry, and it was found that with the increase of the infection time, the host cells at the G1 phase were significantly reduced and the apoptotic cells increased continuously. However, the proportion of cells entering the S/ M phase and G2 phase was not reduced. In order to understand the specific role of SRC in ILTV infection, the biological function of SRC in LMH cell response to ILTV infection was detected by using small molecule inhibitor and small interfering fragment of specific target chicken SRC gene. The detection of ILTV-specific real-time quantitative PCR showed that the virus content in LMH cells was significantly reduced after SRC was inhibited. Flow cytometry showed that SRC inhibition could promote viral-mediated cell death. Therefore, SRC is essential for maintaining the proliferation of the virus, but the ILTV-mediated cell death does not rely on SRC. The above results suggest that SRC is a key determinant of virus virulence and replication in ILTV-infected cells. After ILTV infection, host cells increase the threshold of virus-induced cell death by SRC and delay the death of host cells. The effect of SRC on ILTV infection was also confirmed in the chicken embryo test by detecting the death of the SPF chicken embryo treated with SRC-specific small-molecule inhibitor and the virus content in the allantoic membrane and allantoic fluid. In order to explore the biological mechanism of SRC on LMH cells infected by ILTV, we further analyze the data of transcriptome and construct a protein cross-network for differentially expressed genes. The results of the analysis predict that the local adhesion kinase (FAK) may also be involved in the process of SRC regulation of the LITV infection. In the study of the function of small molecule inhibitor and RNA interference, this deduction was also confirmed in the cell and chick embryo, and the positive feedback regulation of protein phosphorylation between SRC and FAK was further found. Subsequent functional studies show that this process is critical to the SRC regulation of ILTV infection. In this study, the molecular mechanism of the host cell response to ILTV infection is discussed, and the current cognition of the interaction between the ILTV and the host is expanded, and a certain theoretical support for establishing a more safe and long-acting AILT control method is also provided.
【学位授予单位】：中国农业科学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S858.31
【参考文献】
相关期刊论文 前2条
1 黄金华,李成,谷守林;鸡传染性喉气管炎病毒形态发生的研究进展[J];中国畜禽传染病;1997年04期
2 郑世军,甘孟侯;间接法Dot—ELISA检测传染性喉气管炎病毒的研究[J];中国兽医杂志;1991年03期
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