HCV RNA复制过程中5'UTR与3'UTR 作用的初步探讨

构建具有不同类型和长度的5'非编码区(UTR)及3'UTR的丙型肝炎病毒(HCV)全长cDNA克隆,体外转录制备RNA,转染肝癌细胞HepG2,研究不同RNA转录体在细胞内的复制情况。通过RT-PCR与Westernblot等方法证明:蛋白编码区来源于1a基因型并具有1b型5'UTR及3'UTR(全长或缺失98nt保守区)的2种嵌合型HCVRNA,都可以在病毒易感细胞中复制和表达;而以脑心肌炎病毒(EMCV)内核糖体进入位点(IRES)替代HCV5'UTR的嵌合型RNA转染细胞,检测不到病毒正、负链RNA与蛋白表达。以上结果说明HCV1b型5'UTR与3'UTR可以支持1a型病毒RNA的正常复制,3'UTR保守区的存在与否,对病毒复制影响不大。     

正链RNA病毒基因组的复制

许多正链RNA病毒是严重危害人类健康的病原体,是造成经济植物动物死亡的致病因子.正链RNA病毒的基因组为正链RNA,其复制酶是依赖RNA的RNA聚合酶,非编码区是病毒基因组复制的主要调控位点,3’非编码区是复制酶的第一结合位点,正链RNA病毒基因组大多可能按copy-back模型进行复制.瘟病毒基因组的复制过程出现正链复制本的数量大于负链复制本的数量,这可能是以RF中间体的负链RNA为模板、正链RNA被置换的形式进行复制的结果.本文概述了HCV细胞培养系统的研究进展.     

丙型肝炎病毒NS3基因真核质粒的构建及其在人肝细胞中的诱导表达

目的：进行丙型肝炎病毒非结构蛋白3基因真核表达载体的构建,并分析其在体外培养的人肝细胞中的表达。方法：从含有丙肝病毒全长基因的重组质粒pBRTM／HCV1-3011表达载体中PCR扩增出HCVNS3基因片段,将其与表达载体pcDNA3．1（-）重组,得到重组的真核表达载体pcDNA3．1（-）／NS3。然后采用阳离子多聚体将其转染人肝细胞QSG7701,以免疫组织化学SP法及Western Blotting检测HCVNS3蛋白的表达。结果：所得到的NS3片段正确,序列正确,所构建的真核质粒成功转染QSG7701细胞并表达蛋白,表达的NS3蛋白相对分子质量为70000。结论：成功构建了丙型肝炎病毒非结构蛋白3基因的真核表达载体pcDNA3．1（-）／NS3,并且该载体在体外培养的人肝细胞中能有效表达特异性HCVNS3蛋白。     

HCV感染通过Erk信号的激活上调PTTG1的表达

 为探讨丙型肝炎病毒(HCV)感染导致肝细胞癌发生的分子机制,利用前期研究建立的体外HCV细胞培养体系,将HCV JFH-1 RNA转染CD81-Huh7细胞,确定能够获得感染性HCV后,收集HCV转染后10,50,100和150d的细胞,采用Real time PCR及Western Blot法检测不同时间点细胞内垂体瘤转化基因1(PTTG1)基因和蛋白水平的表达情况,同时检测总MAPK/Erk1/2,磷酸化Erk1/2(p-Erk1/2)蛋白的表达。随后,用干扰素抑制HCV的感染,再检测PTTG1及MAPK/Erk1/2的表达情况,以及用MAPK/Erk抑制剂(PD98059)阻断MAPK/Erk 1/2的磷酸化后,检测PTTG1的表达情况。结果显示,HCV转染的细胞与未转染细胞比较,PTTG1的mRNA和蛋白表达均显著增加,p-Erk1/2蛋白显著升高(P＜0.05);抑制HCV感染显著降低PTTG1的表达和MAPK/Erk1/2的磷酸化(P＜0.05);MAPK/Erk1/2抑制剂显著降低了PTTG1基因和蛋白的表达(P＜0.05)。体外HCV感染可以导致MAPK/Erk信号通路的磷酸化,进一步导致原癌基因PTTG1的表达增加,这可能是慢性HCV感染导致HCV相关性肝细胞癌发生的分子机制之一。     

拟南芥全基因组范围的non-polyA lncRNA检测

长非编码RNA(lncRNA)是一类长度大于200nt不编码蛋白质的RNA,它们在生物体内发挥重要的调控作用。Non-polyA lncRNA是lncRNA的一个重要类型。为系统鉴定植物全基因组范围的non-polyA lncRNA,该文优化了拟南芥基因组范围鉴定新型non-polyA lncRNA的RNA样本纯化和测序方法,并且比较了3种不同的方法。结果表明:链特异性的non-polyA RNA长序列测序检测灵敏度更高,可以检测到更多低表达的lncRNA和更高比率的长序列lncRNA。因此,non-polyA RNA长序列测序适用于拟南芥全基因组范围的non-polyA lncRNA检测。该方法也可用于其它物种中non-polyA lncRNA的鉴定,为探究lncRNA功能提供帮助。     

针对马铃薯卷叶病毒复制酶基因负链的突变核酶的克隆及体外转录

根据特异性切割马铃薯卷叶病毒中国分离株 ( PLRV-Ch)复制酶基因负链 RNA的锤头状核酶 ,设计、合成了编码与其相应的突变核酶的 c DNA,并克隆到质粒 p GEM-4 Z中 ,经序列分析及体外转录表明得到完整的突变核酶基因重组质粒 ,从而为突变核酶的转基因及进一步研究核酶在转基因马铃薯中表达引起的抗性及机理创造了条件     

尤文肉瘤细胞总RNA转染的DC疫苗体外诱导尤文肉瘤患者特异性抗肿瘤免疫的实验研究

为了探讨尤文肉瘤细胞总RNA转染的DC疫苗体外诱导特异性抗肿瘤免疫的能力,采用分离尤文肉瘤患者外周血单核细胞体外诱导DC细胞,Trizol法提取患者尤文肉瘤细胞总RNA,用总RNA转染DC并诱导特异性CTL的扩增,用RT-PCR方法检测肿瘤总RNA加载的DC细胞的表达,用MTT法检测淋巴细胞的增殖和CTL的杀伤活性.结果RT-PCR测定显示尤文肉瘤细胞总RNA转染的DC细胞可以呈递肿瘤特异性抗原,并特异性地表达其特有序列EWS-FLI1.经尤文肉瘤细胞总RNA转染的DC特异性表面标志及功能相关分子表达均上调,转染后的DC可显著刺激自体T淋巴细胞增殖.诱导的特异性CTL对携带EWS-FLI1抗原的靶细胞的杀伤率显著高于LAK细胞和未经转染的DC.说明尤文肉瘤细胞总RNA转染的DC疫苗可在体外诱导出特异性抗肿瘤免疫.     

HCV非结构区NS3-5B蛋白的真核表达载体构建及其在BHK-21细胞中的表达

通过PCR方法扩增出HCV NS3-5b全长基因序列,克隆入真核表达载体pIRES2-EGFP中,构建重组质粒pIRES2-EGFP-NS3-5b。利用脂质体将该质粒转染至BHK-21细胞,通过荧光成像和Western Blot检测NS3-5b基因的表达。结果显示成功构建了真核表达质粒pIRES2-EGFP-NS3-5b,并且NS3/4A蛋白和NS5B蛋白得到特异性表达,为下一步建立HCV RdRp活性的细胞评价系统和动物模型评价系统奠定了实验和理论基础。     

体外诱导骨肉瘤特异性抗肿瘤的免疫实验

采用分离骨肉瘤患者外周血单核细胞体外诱导DC细胞,Trizol法提取患者骨肉瘤细胞总RNA,用总RNA转染DC并诱导特异性CTL的扩增,用MTT法检测淋巴细胞的增殖和CTL的杀伤活性。探讨骨肉瘤细胞总RNA转染的DC疫苗体外诱导特异性抗肿瘤免疫的能力。经骨肉瘤细胞总RNA转染的DC特异性表面标志及功能相关分子表达均上调,转染后的DC可显著刺激自体T淋巴细胞增殖,诱导的特异性CTL对靶细胞的杀伤率显著高于单纯淋巴细胞和未经转染的DC。说明骨肉瘤细胞总RNA转染的DC疫苗可在体外诱导出特异性抗肿瘤免疫。     

丙型肝炎病毒NS5A分子及其干扰素敏感决定区的研究进展

丙型肝炎病毒(HCV)是慢性肚炎的主要病因之一，其基因组为单股正链RNA，NSA是划HCV编码的一种非结构蛋白。位于HCV NS5A区的干扰素敏感决定区(ISDR)，与肝细胞中RNA依赖性蛋白激酶(PKR)之间的相互作用，可能决定着HCV对干扰素（IFN-α）的敏感性。     

丙型肝炎病毒感染与ras,p53基因表达的相关性

为了探讨丙型肝炎病毒（ＨＣＶ）感染与肝细胞癌（ＨＣＣ）的关系以及ＨＣＶ可能的致癌机理，采用免疫组织化学方法及巢式ＰＣＲ法检测了１３６例肝细胞癌等肝病组织中的ＨＣＶＮＳ３抗原、ＨＣＶＲＮＡ及Ｐ２１、Ｐ５３蛋白。结果表明，肝细胞癌及癌周肝组织中有ＨＣＶＮＳ３抗原及ＨＣＶＲＮＡ检出，支持ＨＣＶ与ＨＣＣ的关联。Ｐ２１在ＨＣＣ、肝炎后肝硬化、慢性肝炎、体质性黄疸各组中的检出率随病变的加重而逐渐增高，在ＨＣＣ的癌及癌周组织中Ｐ２１呈致密的过量表达，提示ｒａｓ癌基因的激活在ＨＣＣ的发生过程中起一定作用。Ｐ５３的阳性率较Ｐ２１低，但ｐ５３的突变似乎也是肝癌发生的协同因素之一。组织中Ｐ２１的过量表达与ＨＣＶＮＳ３抗原阳性检出呈正相关，ＨＣＶＮＳ３抗原与Ｐ２１的这种关联提示，ＨＣＶ感染作为ＨＣＣ的密切相关因素之一，可能通过激活某些癌基因或使某些抑癌基因突变而致肝细胞癌变     

Innate immunity induced by composition-dependent RIG-I recognition of hepatitis C virus RNA

Innate immune defences are essential for the control of virus infection and are triggered through host recognition of viral macromolecular motifs known as pathogen-associated molecular patterns (PAMPs). Hepatitis C virus (HCV) is an RNA virus that replicates in the liver, and infects 200 million people worldwide. Infection is regulated by hepatic immune defences triggered by the cellular RIG-I helicase. RIG-I binds PAMP RNA and signals interferon regulatory factor 3 activation to induce the expression of interferon-alpha/beta and antiviral/interferon-stimulated genes (ISGs) that limit infection. Here we identify the polyuridine motif of the HCV genome 3' non-translated region and its replication intermediate as the PAMP substrate of RIG-I, and show that this and similar homopolyuridine or homopolyriboadenine motifs present in the genomes of RNA viruses are the chief feature of RIG-I recognition and immune triggering in human and murine cells. 5' terminal triphosphate on the PAMP RNA was necessary but not sufficient for RIG-I binding, which was primarily dependent on homopolymeric ribonucleotide composition, linear structure and length. The HCV PAMP RNA stimulated RIG-I-dependent signalling to induce a hepatic innate immune response in vivo, and triggered interferon and ISG expression to suppress HCV infection in vitro. These results provide a conceptual advance by defining specific homopolymeric RNA motifs within the genome of HCV and other RNA viruses as the PAMP substrate of RIG-I, and demonstrate immunogenic features of the PAMP-RIG-I interaction that could be used as an immune adjuvant for vaccine and immunotherapy approaches.     

人源性14-3-3σ真核表达载体的构建及鉴定

目的：构建人源性14-3-3σ真核表达载体pCMV-Myc-SFN,并观察其在真核细胞中的表达。方法：通过反转录-聚合酶链反应从HeLa细胞（人宫颈癌细胞系）中获得编码14-3-3σ的cDNA,定向克隆至真核表达载体pCMV-Myc中,经酶切和测序鉴定正确后,应用脂质体法转染HeLa细胞,并通过蛋白免疫印迹法检测细胞内14-3-3σ的表达。结果：构建了真核表达载体pCMV-Myc-SFN,将其转染HeLa细胞48h后,蛋白免疫印迹法检测到细胞内14-3-3σ的表达显著增高。结论：成功构建了真核表达载体pCMV-Myc-SFN,为研究14-3-3σ在上皮细胞源性肿瘤中的作用奠定了基础。     

Cloning of murine BRI3 gene and study on its function for inducing cell death

To understand the molecular mechanism of TNFα effects, the cDNA of murine BRI3 gene was cloned from the total RNA of murine brain endothelial cells (bEnd.3)treated with hTNFα by using the suppression subtractive hybridization (SSH) and the RT-PCR method. The fusion expression vector harbouring BRI3 gene and enhanced green fluorescence protein (EGFP) thus obtained were designated as pEGFP/I3. Then pEGFP/I3 was transiently transfected into L929 cells and the fusion protein EGFP/I3 was localized in cytoplasm. It is found that the expression of EGFP/I3 could induce cell death in L929 cells detected by TUNEL method and flow cytometry. And the overexpression of Bci-2 in L929 cells can block cell death induced by EGFP/I3, indicating that murine BRI3 gene might related to the TNFα mediated cytotoxicity.     

Antiviral compounds and one new iridoid glycoside from Cornus officinalis

From Cornus officinalis Sieb. et Zucc., bioassay-guided fractionation led to the isolation of four active tannin compounds with high effectiveness of inhibiting Hepatitis C virus NS3 serine protease in vitro. The compounds are: 1,2,3,6-tetragalloyl-β-D-glucopyranose (1), 1,2,3,4,6- pentagalloyl-β-D-glucopyranose (2), Tellimagrandin Ⅰ (3) and Tellimagrandin Ⅱ (4). The four compounds could inhibit HCV NS3 protease in vitro with IC50 values of 6.98, 5.11, 7.0 and 4.8 μmol/L respectively. In addition, a new iridoid glycoside (5) was also isolated from Cornus officinalis Sieb. et Zucc., which was assigned to be 7-O-butyl morroniside by spectroscopic analysis.     

Cloning of murine BRI3 gene and study on its function for inducing cell death

To understand the molecular mechanism of TNFα effects, the cDNA of murine BRI3 gene was cloned from the total RNA of murine brain endothelial cells (bEnd.3) treated with hTNFα by using the suppression subtractive hybridization (SSH) and the RT-PCR method. The fusion expression vector harbouring BRI3 gene and enhanced green fluorescence protein (EGFP) thus obtained were designated as pEGFP/I3. Then pEGFP/I3 was transiently transfected into L929 cells and the fusion protein EGFP/I3 was localized in cytoplasm. It is found that the expression of EGFP/I3 could induce cell death in L929 cells detected by TUNEL method and flow cytometry. And the overexpression of Bcl-2 in L929 cells can block cell death induced by EGFP/I3, indicating that murine BRI3 gene might related to the TNFa mediated cytotoxicity.