宿主细胞CyPB在Orf病毒感染过程中功能的初步研究
摘要
羊传染性脓疱病毒（Orf virus, ORFV）是一种重要的人兽共患病病原，ORFV感染不仅给世界养羊业造成巨大的经济损失，而且严重威胁着人类的身体健康，已成为影响我国乃至世界公共卫生安全的一个重要隐患。之前针对ORFV致病机理的研究多从病原学角度出发，然而病毒感染是一个复杂的过程，涉及到病原与宿主两方面的相互作用。病毒感染后能够在不同的复制时期以不同的方式与宿主细胞的多种组分发生相互作用，引起宿主细胞内一些基因的差异表达及其所介导信号通路的改变，造成细胞的生理功能异常从而导致疾病的发生。因此，仅从病原的角度很难阐明其对宿主的感染机制。鉴于此，本研究从病毒与宿主细胞的相互作用角度入手，针对病毒早期感染阶段，采用抑制性消减杂交技术构建了ORFV早期感染宿主细胞差异表达基因文库。经质粒PCR、反向斑点杂交筛选及序列测定等方法对文库中差异基因进行筛选鉴定，共获得差异表达基因81条。应用在线生物学软件对差异基因进行功能预测，结果表明：差异表达基因所具有分子功能主要分为结构活性、结合功能、转录调节活性、转运功能、翻译调节活性、酶调节活性、催化活性；其参与的生物学过程包括代谢过程、运输过程、定位过程、免疫过程、细胞周期、发育过程、刺激反应过程、凋亡过程。
根据对差异基因的功能预测结果，我们选择参与细胞免疫生物学过程的上调差异表达基因亲环素B（Cyclophilin B,CyPB）作为研究对象。尽管许多疾病如肿瘤、免疫调节障碍以及血管异常等的发生都与CyPB的异常表达和分布有关，其在痘病毒、猪瘟病毒、人免疫缺陷病毒、丙型肝炎病毒、日本乙型脑炎病毒、牛水泡性口炎病毒、甲型流感病毒等多种病毒的感染过程中发挥着重要的调控作用，但是尚不清楚CyPB在ORFV感染过程中的作用，也未见相关报道。因此，本研究对CyPB在ORFV感染过程中功能进行验证。首先我们对ORFV感染前后宿主细胞中的CyPB基因进行RT-PCR扩增，测序结果显示，病毒感染后宿主细胞CyPB基因未发生碱基的突变或缺失；为进一步分析CyPB在ORFV感染过程中的表达变化情况，本研究利用Real-Time PCR与Western Blot检测方法分别从mRNA与蛋白水平对ORFV感染不同时间段（2h、4h、8h、12h、18h、24h）CyPB在宿主细胞中的表达情况进行监测，结果表明，CyPB基因在病毒感染早期表达量骤升，其中在感染后4h mRNA表达量最高，为正常细胞的106倍。为进一步明确CyPB在ORFV感染过程中的功能，本研究构建了PCDNA3.1/His A+CyPB真核表达质粒，通过G418加压筛选获得1株过量表达CyPB MDBK细胞系，此外，通过siRNA转染实现CyPB在MDBK细胞内的沉默表达。之后，将ORFV分别感染CyPB过表达及抑制表达MDBK细胞，利用Real-Time PCR与病毒滴度测定等方法对病毒的增殖情况进行检测，结果证实，过量表达CyPB能够促进病毒的复制增殖，病毒滴度提前12h即达到峰值；抑制CyPB基因表达后，病毒的复制增殖速度受到明显抑制；与此同时，应用MTT方法对CyPB抑制剂CsA的体外抗病毒活性进行检测，结果发现CsA在低剂量情况下能够有效抑制ORFV感染所引起的细胞毒性。
The infection of Orf virus (ORFV) causes enormous economic losses to the world'ssheep industry. As a zoonosis, this disease is a serious threat to human health, and hasalready become a significant hazard which has an important impact on our country andthe global public health security. Previous researches of ORFV are mainly about etiology,however, viral infection is the process of interaction between pathogen and host. Virusinfection is capable of replicating at different phases through different ways and interactswith the various components of the host cell, which can then cause some differences inthe gene expression in the host cell and change the mediated cellular signal pathway,consequently, it results in cell physiological dysfunction and leads to the disease.Therefore, it is difficult to understand the mechanism of viral infection just based on thestudy of etiology. From this study, we start from the interaction of the virus with the hostcell. We have finished gene library constructed from ORFV early infection in host cell byusing suppression subtractive hybridization. After plasmid PCR, reverse dot blothybridization screening, sequencing, we obtain81differential expressed genes in ourresearch. Online biologically applied software was used for predicting gene function, theresults indicate that functions of differential expressed genes are divided into structuralmolecule activity, binding, transcription regulator activity, transporter activity, translationregulator activity, regulation of enzyme activity, catalytic activity. The biologicalprocesses where these genes are involved include metabolic process, transportationprocess, localization, immune process, cellular process, development process, response tostimulus and the apoptotic process.
According to the prediction of the function of differential expressed genes, wechoose the cyclophilin B (CyPB) as study object, which involved in immuneprocess.Many diseases like tumors, immune regulation, and blood vessel formation areassociated with abnormal expression and distribution of CyPB. CyPB plays an importantrole in the infection of vaccinia virus, swine fever virus, human immunodeficiency virus,hepatitis C virus, Japanese encephalitis virus, bovine vesicular stomatitis virus, influenza virus etc. However, there is no report about CyPB in ORFV infection. In this study, westudy the function of CyPB during ORFV infection. First, we analyze the changes insequence of CyPB in ORFV infection, there is no mutation of CyPB gene found in thehost cells after ORFV infection. By using Real-Time PCR and Western Blot to monitorCyPB expression in mRNA and protein levels respectively within24h（2h,4h,8h,12h,18h,24h）after ORFV infection we found that the level of CyPB geneexpression increases rapidly in the early viral infection. The number of mRNA of CyPBin ORFV infected cells is106times more than that in normal cells at4h after infection. Inthis research, we construct PCDNA3.1/His A+CyPB eukaryotic expression plasmid, andobtain1CyPB over-expressing MDBK cells by G418selection, and inhibit CyPBexpression in MDBK cells using siRNA. After the virus infection, the results from viraltiter and Real-Time PCR assays demonstrate that CyPB plays a key role in the viralreplication: the over-expressed CyPB accelerates viral DNA replication. Virus titer reacha peak ahead of12hours. After the inhibition of CyPB expression, the viral replication issignificantly blocked. The MTT assay reveals that CsA (an inhibitors of CyPB) at losedose can effectively inhibit the cytotoxicity caused by ORFV infection.
根据对差异基因的功能预测结果，我们选择参与细胞免疫生物学过程的上调差异表达基因亲环素B（Cyclophilin B,CyPB）作为研究对象。尽管许多疾病如肿瘤、免疫调节障碍以及血管异常等的发生都与CyPB的异常表达和分布有关，其在痘病毒、猪瘟病毒、人免疫缺陷病毒、丙型肝炎病毒、日本乙型脑炎病毒、牛水泡性口炎病毒、甲型流感病毒等多种病毒的感染过程中发挥着重要的调控作用，但是尚不清楚CyPB在ORFV感染过程中的作用，也未见相关报道。因此，本研究对CyPB在ORFV感染过程中功能进行验证。首先我们对ORFV感染前后宿主细胞中的CyPB基因进行RT-PCR扩增，测序结果显示，病毒感染后宿主细胞CyPB基因未发生碱基的突变或缺失；为进一步分析CyPB在ORFV感染过程中的表达变化情况，本研究利用Real-Time PCR与Western Blot检测方法分别从mRNA与蛋白水平对ORFV感染不同时间段（2h、4h、8h、12h、18h、24h）CyPB在宿主细胞中的表达情况进行监测，结果表明，CyPB基因在病毒感染早期表达量骤升，其中在感染后4h mRNA表达量最高，为正常细胞的106倍。为进一步明确CyPB在ORFV感染过程中的功能，本研究构建了PCDNA3.1/His A+CyPB真核表达质粒，通过G418加压筛选获得1株过量表达CyPB MDBK细胞系，此外，通过siRNA转染实现CyPB在MDBK细胞内的沉默表达。之后，将ORFV分别感染CyPB过表达及抑制表达MDBK细胞，利用Real-Time PCR与病毒滴度测定等方法对病毒的增殖情况进行检测，结果证实，过量表达CyPB能够促进病毒的复制增殖，病毒滴度提前12h即达到峰值；抑制CyPB基因表达后，病毒的复制增殖速度受到明显抑制；与此同时，应用MTT方法对CyPB抑制剂CsA的体外抗病毒活性进行检测，结果发现CsA在低剂量情况下能够有效抑制ORFV感染所引起的细胞毒性。
The infection of Orf virus (ORFV) causes enormous economic losses to the world'ssheep industry. As a zoonosis, this disease is a serious threat to human health, and hasalready become a significant hazard which has an important impact on our country andthe global public health security. Previous researches of ORFV are mainly about etiology,however, viral infection is the process of interaction between pathogen and host. Virusinfection is capable of replicating at different phases through different ways and interactswith the various components of the host cell, which can then cause some differences inthe gene expression in the host cell and change the mediated cellular signal pathway,consequently, it results in cell physiological dysfunction and leads to the disease.Therefore, it is difficult to understand the mechanism of viral infection just based on thestudy of etiology. From this study, we start from the interaction of the virus with the hostcell. We have finished gene library constructed from ORFV early infection in host cell byusing suppression subtractive hybridization. After plasmid PCR, reverse dot blothybridization screening, sequencing, we obtain81differential expressed genes in ourresearch. Online biologically applied software was used for predicting gene function, theresults indicate that functions of differential expressed genes are divided into structuralmolecule activity, binding, transcription regulator activity, transporter activity, translationregulator activity, regulation of enzyme activity, catalytic activity. The biologicalprocesses where these genes are involved include metabolic process, transportationprocess, localization, immune process, cellular process, development process, response tostimulus and the apoptotic process.
According to the prediction of the function of differential expressed genes, wechoose the cyclophilin B (CyPB) as study object, which involved in immuneprocess.Many diseases like tumors, immune regulation, and blood vessel formation areassociated with abnormal expression and distribution of CyPB. CyPB plays an importantrole in the infection of vaccinia virus, swine fever virus, human immunodeficiency virus,hepatitis C virus, Japanese encephalitis virus, bovine vesicular stomatitis virus, influenza virus etc. However, there is no report about CyPB in ORFV infection. In this study, westudy the function of CyPB during ORFV infection. First, we analyze the changes insequence of CyPB in ORFV infection, there is no mutation of CyPB gene found in thehost cells after ORFV infection. By using Real-Time PCR and Western Blot to monitorCyPB expression in mRNA and protein levels respectively within24h（2h,4h,8h,12h,18h,24h）after ORFV infection we found that the level of CyPB geneexpression increases rapidly in the early viral infection. The number of mRNA of CyPBin ORFV infected cells is106times more than that in normal cells at4h after infection. Inthis research, we construct PCDNA3.1/His A+CyPB eukaryotic expression plasmid, andobtain1CyPB over-expressing MDBK cells by G418selection, and inhibit CyPBexpression in MDBK cells using siRNA. After the virus infection, the results from viraltiter and Real-Time PCR assays demonstrate that CyPB plays a key role in the viralreplication: the over-expressed CyPB accelerates viral DNA replication. Virus titer reacha peak ahead of12hours. After the inhibition of CyPB expression, the viral replication issignificantly blocked. The MTT assay reveals that CsA (an inhibitors of CyPB) at losedose can effectively inhibit the cytotoxicity caused by ORFV infection.
引文
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