宿主SRC在雞傳染性喉氣管炎病毒感染中作用的研究
發(fā)布時間：2018-12-14 16:07
【摘要】：雞傳染性喉氣管炎(AILT)是由傳染性喉氣管炎病毒(ILTV)感染引發(fā)的一種雞的呼吸系統(tǒng)疾病。ILTV屬于皰疹病毒科(Herpesviridae family)傳喉炎病毒屬(Iltovirus genus)。不同病毒株依據(jù)其毒力不同可以造成雞不同程度的上呼吸道疾病,對世界養(yǎng)禽業(yè)造成巨大經(jīng)濟損失。當(dāng)前,AILT防治主要依賴于疫苗接種引發(fā)的機體免疫應(yīng)答發(fā)揮作用。然而,由于免疫接種不能清除宿主細(xì)胞中潛伏感染的病毒和抑制病毒在細(xì)胞內(nèi)的復(fù)制,難以長效防控疫情。因此,探索新策略對AILT的防控具有重要意義。本研究用感染了ILTV LJS09毒株的LMH細(xì)胞作為體外實驗的模型,探討了宿主細(xì)胞應(yīng)答ILTV感染的分子機制。為了更好的闡述病毒感染宿主細(xì)胞的相關(guān)分子事件,本試驗首先通過ILTV特異性實時定量PCR和TCID50等方法對感染LMH細(xì)胞過程中ILTV LJS09的生物學(xué)特性進行了測定。然后,通過基因芯片技術(shù)在全基因組范圍內(nèi)檢測了宿主基因的轉(zhuǎn)錄水平,并結(jié)合生物信息學(xué)分析預(yù)測宿主SRC在LMH細(xì)胞應(yīng)答ILTV感染的過程中可能發(fā)揮重要作用。SRC是第一個被鑒定的原癌基因,可以參與真核生物細(xì)胞粘附、增殖、骨架重建、細(xì)胞分裂及凋亡等多種生命活動。蛋白免疫印跡結(jié)果顯示,在ILTV感染后第二天LMH細(xì)胞中SRC的磷酸化作用增強,說明ILTV感染可以活化宿主細(xì)胞SRC。而LMH細(xì)胞的生長曲線顯示在病毒感染后第三天,病毒感染組的細(xì)胞數(shù)量大大減少。鑒于SRC被廣泛報道可以影響細(xì)胞增殖,我們用流式細(xì)胞術(shù)對細(xì)胞周期做了分析,發(fā)現(xiàn)隨著感染時間的增加,病毒感染使處于G1期的宿主細(xì)胞顯著減少,凋亡細(xì)胞不斷增加,然而進入S/M期和G2期的細(xì)胞比例并沒有降低。為了深入了解SRC在ILTV感染中的具體作用,本研究應(yīng)用小分子抑制劑和特異性靶向雞SRC基因的小干擾片段對SRC在LMH細(xì)胞應(yīng)答ILTV侵染過程中的生物學(xué)功能進行了檢測。ILTV特異性實時定量PCR檢測顯示,SRC被抑制后LMH細(xì)胞中的病毒含量顯著降低。流式細(xì)胞術(shù)結(jié)果表明,SRC抑制可以促進病毒介導(dǎo)的細(xì)胞死亡。因此,SRC對維持病毒的增殖是不可或缺的,但I(xiàn)LTV介導(dǎo)的細(xì)胞死亡并不依賴SRC。以上結(jié)果提示,SRC在ILTV感染的細(xì)胞中是病毒毒力和復(fù)制的關(guān)鍵決定因素,ILTV感染后,宿主細(xì)胞通過SRC提高病毒誘發(fā)細(xì)胞死亡的閾值進而延緩宿主細(xì)胞的死亡。通過檢測SRC特異性小分子抑制劑處理的SPF雞胚接種ILTV后的死亡情況和尿囊膜及尿囊液中的病毒含量,我們在雞胚試驗中也證實了SRC對ILTV感染的影響。為了探究SRC影響ILTV感染LMH細(xì)胞的生物學(xué)機制,我們進一步分析了轉(zhuǎn)錄組學(xué)的數(shù)據(jù)并構(gòu)建了表達(dá)差異基因的蛋白質(zhì)互作網(wǎng)絡(luò)。分析結(jié)果預(yù)測局部粘著斑激酶(FAK)可能也參與了SRC調(diào)節(jié)LITV感染的過程。應(yīng)用小分子抑制劑和RNA干擾等功能學(xué)研究,我們在細(xì)胞和雞胚中同時證實了這一推論,并進一步發(fā)現(xiàn)SRC與FAK間存在蛋白磷酸化的正反饋調(diào)節(jié)作用。隨后的功能學(xué)研究顯示這一過程對于SRC調(diào)節(jié)ILTV感染至關(guān)重要。本研究初步探討了宿主細(xì)胞應(yīng)答ILTV感染的分子機制,拓展了當(dāng)前對于ILTV與宿主互作分子機制的認(rèn)知,也為建立更為安全、長效的AILT防治方法提供一定的理論支持。
[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.
【學(xué)位授予單位】：中國農(nóng)業(yè)科學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S858.31
本文編號：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.
【學(xué)位授予單位】：中國農(nóng)業(yè)科學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S858.31
【參考文獻(xiàn)】
相關(guān)期刊論文 前2條
1 黃金華,李成,谷守林;雞傳染性喉氣管炎病毒形態(tài)發(fā)生的研究進展[J];中國畜禽傳染病;1997年04期
2 鄭世軍,甘孟侯;間接法Dot—ELISA檢測傳染性喉氣管炎病毒的研究[J];中國獸醫(yī)雜志;1991年03期
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