C基因截短的HBV复制与包装

目的 探讨C基因截短型HBV变异体的复制与包装。方法 采用分子克隆、人工定点突变等技术构建C基因截短型HBV变异体质粒，用脂质体法转染HepG2细胞，提取细胞内及培养上清液中DNA分别进行Southem杂交，PCR及实时定量荧光PCR分析。结果 经DNA测序及酶切鉴定证实C基因截短型HBV质粒载体构建成功；C基因截短型HBV为复制缺损型，与辅助质粒共转染HepG2细胞，可在细胞内及培养上清液中检测到HBV各种DNA构型；DNA定量分析提示C基因截短型HBV的包装效率较野生型HBV提高3～40倍。结论 C基因截短型HBV变异体为复制缺损型，单独转染后不能在肝细胞内包装与复制，但在缺失包装信号ε的相应辅助病毒辅助下可有效复制并包装成子代病毒颗粒分泌到胞外，且包装效率大大提高。     

Construction and identification of a human liver specific microRNA eukaryotic expression vector

MiR-122 is one of the non-coding RNAs which showed its effects on the lipo-metablism, virus infection and HCC forming through regulation of liver gene expression. Its eukaryotic expression vector was constructed by using pSuper which was widely applied in the siRNA expression. The precursor of human miR-122 gene was amplified by polymerase chain reaction （PCR） from the human genomic DNA. The positive clones were screened by PCR and restriction enzyme digestion. The new expression vector of miR-122 was named pHsa-m122. PHsa-m122 and its controls were transfected to HepG2 cells. The miR-122 expression activity was evaluated by GFP122i sensor reporter plasmid through fluorescence detection and Western blot. It was shown that the fluorescence intensity of GFP122si and pHsa-m122 co-transfection group was weaker than that of the controls, so the functional activity of expressed miR-122 was detected. When HepG2 cells were co-transfected with HBV1.3 and pHsa-m122 plasmids, the results showed miR-122 may down-regulate the gene expression of HBV. The human liver specific microRNA eukaryotic expression vector of miR-122 was constructed successfully, which may facilitate further study of its function in the development of liver virus infection diseases and HCC. Cellular ＆ Molecular Immunology.     

HBx、MHBs~（t155）真核表达载体构建及在HepG2细胞中的表达

目的建立稳定、高效的HBx、羧基末端截短的中分子表面蛋白（MHBst）体外表达细胞株,以进一步研究HBx、MHBst蛋白在肝癌发生中的作用及其机理。方法设计特异性引物,采用PCR方法从adr亚型HBV质粒pHBV DNA中扩增HBx、羧基末端截短至155位氨基酸的中分子表面蛋白（MHBst155）编码基因,并定向插入绿色荧光蛋白（GFP）表达载体pEGFP-C1的BglⅡ、KpnⅠ和BglⅡ、BamHⅠ酶切位点,转化宿主菌DH5α,提取质粒,分别用上述内切酶酶切及DNA测序鉴定重组质粒。采用脂质体介导将空载体、重组质粒分别转染到人肝肿瘤细胞株HepG2中,G418筛选抗性细胞克隆,荧光显微镜下观察GFP表达,挑取表达GFP的抗性克隆扩大培养、传代。采用RT-PCR、蛋白印迹检测抗性细胞中HBx、MHBst155的mRNA及蛋白水平。结果经酶切及测序鉴定成功构建了pGFP-HBx、pGFP-MHBst155重组表达载体,将空载体及重组质粒转染HepG2,经G418筛选约20 d获得抗性细胞克隆。将带有绿色荧光的抗性克隆扩大培养并经传代40次,细胞仍表达强的绿色荧光。RT-PCR及蛋白印迹检测表明转染重组体的HepG2/GFP-HBx、HepG2/GFP-MHBst155细胞有相应的条带,而空载体、空白对照组未出现条带。结论成功构建了HBx、MHBst155真核重组表达载体pGFP-HBx、pGFP-MHBst155,获得了稳定表达融合蛋白GFP-HBx、GFP-MHBst155的HepG2细胞系,为进一步研究HBx及MHBst蛋白在肝癌发生中的作用及分子机制构建了良好的平台。     

Gene transfer system derived from the caprine arthritis-encephalitis lentivirus

Lentiviruses are attractive candidates for therapeutic vectors, because of their ability to infect non-dividing target cells. Vectors based on HIV-1 efficiently transfer gene expression to a variety of dividing or quiescent cells, but are subject to reservations on safety grounds. Caprine arthritis encephalitis virus (CAEV) is a lentivirus inducing only minor pathology in its natural host and in related species after cross-species transmission. To test the CAEV potential as vector for gene transfer, a cassette expressing the green fluorescent protein (GFP) under control of a CMV promoter was inserted into the CAEV genome, producing the pK2EGFPH vector. When pseudotyped with vesicular stomatitis virus (VSV)-G envelope protein, this vector allowed efficient transfer of GFP expression in human cells (up to 86% of GFP-expressing cells into the TE671 cell line). Three vectors carrying different parts of the viral gag, pol and env genes were then developed, together with a CAEV packaging system. These vectors allowed delimitation of the minimal CAEV sequences necessary for an improvement of vector production compared to the previously described CAEV-based vectors [Mselli-Lakhal et al., 1998. Defect in RNA transport and packaging are responsible for low transduction efficiency of CAEV-based vectors. Arc. Virol. 143, 681-695]. While our previous vectors were produced in a helper/vector system, the present vectors are produced in a helper/free system. However, these vector titers remain lower than those obtained with other lentiviral vectors carrying equivalent packaging sequences. We discuss on possible reasons of such differences and possible improvements.     

Specific expression of human interferon-gamma controls hepatitis B virus replication in vitro in secreting hepatitis B surface antigen hepatocytes

Interferon-gamma (IFN-γ) has been reported to have antiviral activity against Hepatitis B virus (HBV) and to suppress HBV replication noncytolytically in vivo. Since systemic administration of IFN-γ may cause severe adverse effects, studies of the effects of liver-specific IFN-γ expression from adenoviral vectors in vivo have been investigated. In this study, a novel strategy has been described that drives specific expression of human IFN-γ in HBsAg-secreting hepatocytes. A bicistronic expression vector has been developed, pcDNA3.1-HBV antisense S gene-HCV core protein gene-HCV internal ribosome entry sites (IRES)-IFN-γ (pcDNA-SCIγ), by inserting four DNA fragments into pcDNA3.1. Tight modulation of HCV IRES-dependent translation by the HCV core protein was achieved using an antisense RNA technique with a bicistronic expression vector. HepG2 cells and HepG2.2.15 cells stably expressing HBV were transduced with pcDNA-SCIγ to test the responsiveness of IFN-γ to HBsAg expression. Gene transfer resulted in a low background and a 30-fold induction of IFN-γ expression from pcDNA-SCIγ in a cell-specific fashion. Hepatocyte-specific IFN-γ expression controlled effectively HBV replication in HBsAg-secreting HepG2.2.15 cells without cell toxicity.     

人NOD2基因启动子的绿色荧光蛋白表达载体的构建

人NOD2基因启动子的绿色荧光蛋白表达载体的构建。以人基因组DNA为模板,PCR扩增含有人NOD2基因启动子不同长度的4段序列,利用特定的限制性内切酶位点,以切除启动子的pEGFP-N3作为框架结构,将这4个序列片段进行酶切并插入表达载体pEGFP-N3中,用Vsp I和Pst I双酶切和PCR鉴定重组质粒,再将重组质粒进行DNA序列分析。构建的重组质粒经脂质体LipofectamineTM2000介导转染HTK293T细胞,转染24 h后加入TNF-α持续刺激细胞24 h或不加TNF-α刺激,在倒置荧光显微镜下观察其能否在NOD2基因启动子的调控下表达报告基因绿色荧光蛋白(green fluores-cent proteins,GFP)。结果:pEGFP-N3-NOD2(617 bp)、pEGFP-N3-NOD2(747 bp)、pEGFP-N3-NOD2(1 136 bp)、pEG-FP-N3-NOD2(1 387 bp)分别经酶切鉴定和序列测定证实目的基因已插入重组质粒;细胞转染结果表明,构建的4段重组质粒pEGFP-N3-NOD2(617 bp)、pEGFP-N3-NOD2(747 bp)、pEGFP-N3-NOD2(1 136 bp)、pEGFP-N3-NOD2(1 387 bp)转染的HTK293T均能表达绿色荧光,其中构建pEGFP-N3-NOD2(1 387 bp)重组质粒经TNF-α持续刺后激绿色荧光表达最强。4段不同长度的人NOD2基因启动子绿色荧光蛋白表达载体成功构建,这为进一步研究NOD2基因表达及调控机制奠定了良好的基础。     

Green fluorescent protein as a selectable marker of retrovirally transduced hematopoietic progenitors.

Recombinant retroviruses are most commonly used in hematopoietic stem cell gene therapy trials, but gene transfer efficiency is still inadequate with the present vectors. One approach for overcoming this problem is to develop methods of selecting and enriching the successfully transduced cells. We investigated the feasibility of using the green fluorescent protein (GFP) gene as a selectable marker of hematopoietic cells. When M1 murine leukemia cells were electroporated with GFP expression vectors, a red-shifted mutant (S65T) GFP showed several-fold greater fluorescence than the wild-type GFP and generated readily detectable green light under control of SRalpha or CAG promoter. We then inserted an SRalpha-S65T GFP cassette into the MSCV retrovirus vector and established virus producer cells. Infection of primary murine bone marrow cells resulted in a distinct population with green fluorescence, which was separated by fluorescence-activated cell sorting. The fractionated bright cells gave rise to fluorescent spleen colonies in lethally irradiated mice, while the fluorescence-negative cells yielded only dark colonies. These results indicated that GFP is a faithful marker in gene transfer into hematopoietic progenitor/stem cells, facilitating selection of the transduced cells and tracking of their progeny in vivo.     

A method of alphavirus replicon particle titration based on expression of functional replicase/transcriptase

Alphavirus replicon particles are being exploited for a variety of purposes both in vitro as gene expression vectors, and in vivo as vaccines or gene therapy vectors. There is a need for a simple and universal method of titration of replicon particles that is independent of expression of the foreign protein. We devised a method that uses modified vaccinia virus Ankara (MVA) as an indicator virus, to deliver a Venezuelan equine encephalitis virus (VEE) defective helper RNA encoding green fluorescent protein (GFP). Co-infection of cells with the MVA-based indicator and Venezuelan equine encephalitis virus replicon particles (VRP) results in expression of the GFP gene. VRP titer is readily determined by counting fluorescent cells.     

GFP／HBVX融合蛋白重组载体的构建及稳定表达细胞系的建立

目的构建绿色荧光蛋白（GFP）与人乙型肝炎病毒（HBV）X基因的重组表达载体，建立稳定表达HBVX蛋白（HBx）与GFP融合蛋白的HepG2细胞系，以进一步研究HBx的生物学功能及其在肝癌发生中的作用。方法应用PCR法从adr亚型HBV质粒pHBVDNA中扩增HBVX基因片段，PCR产物经HindⅢ和KpnⅠ双酶切后定向插入绿色荧光蛋白真核表达载体pEGFP-C1的相应酶切位点，转化宿主菌DH5α，采用上述双酶切及DNA测序鉴定重组质粒pGFP-HBx；采用脂质体转染法将pEGFP-C1质粒、pGFP-HBx重组质粒DNA转染人肝母细胞瘤细胞系HepG2细胞，G418选择抗性细胞克隆，荧光显微镜下观察GFP表达，挑取表达GFP的抗性克隆扩大培养、传代。采用RT-PCR检测转染细胞HBVX基因的表达。结果经酶切及测序鉴定成功构建了GFP-HBVX重组表达载体pGFP-HBx；将pEGFP-C1、pGFP-HBx重组质粒转染HepG2．，经G418筛选15d获得抗性细胞克隆。将带绿色荧光的抗性克隆细胞扩大培养并经传代70次，细胞仍表达强的荧光蛋白。RT-PCR检测表明转染pGFP-HBx重组体的HepG2／GFP-HBx细胞有HBVX转录、表达。结论成功构建了GFP．HBVX真核重组表达载体pGrP-HBx；获得了稳定表达GFP-HBx融合蛋白的HepG2细胞系，这为进一步研究FIBx的生物学功能以及HBx在肝癌发生中的作用与机制打下了基础。     

乙型肝炎病毒X基因重组腺病毒的构建及在HepG2细胞中的表达

从携带有双拷贝乙肝病毒adw亚型全基因组的质粒pecob6获得X基因片段,将其亚克隆到AdEasy腺病毒系统的穿梭质粒pAdTrack-CMV上,和骨架质粒pAdEasy-1在BJ5183细菌内同源重组,获得重组腺病毒质粒pAd-X,PacI酶切线性化后脂质体法转染293细胞进行包装、扩增,获得重组腺病毒Ad-X,Ad-X可感染HepG2细胞,Western-blot法检测到X蛋白的表达,重组腺病毒Ad-X构建成功,为进一步研究X蛋白的生物学功能奠定了基础。     

Thyroid hormone (T3)-modification of polyethyleneglycol (PEG)-polyethyleneimine (PEI) graft copolymers for improved gene delivery to hepatocytes

Targeting of gene vectors to liver hepatocytes could offer the opportunity to cure various acquired and inherited diseases. Efficient gene delivery to the liver parenchyma has been obscured from efficient targeting of hepatocytes. Here we show that the thyroid hormone, triiodothyronine (T3), can be used to improve the gene transfer efficiency of nonviral gene vectors to hepatocytes in vitro and to the liver of mice in vivo. T3 conjugated to the distal ends of fluorescent labeled PEG-g-dextran resulted in T3-specific cellular endosomal uptake into the hepatocellular cell line HepG2. PEG-g-PEI graft copolymers with increasing molar PEG-ratios were synthesized, complexed with plasmid DNA, and transfected into HepG2 or HeLa cells. Gene transfer efficiency decreased as the number of PEG blocks increased. T3 conjugation to PEI and the distal ends of PEG blocks resulted in T3 specific gene transfer in HepG2 cells as evidenced by reduction of gene transfer efficiency after pre-incubation of cells with excess of T3. In vivo application of T3-PEG-g-PEI based gene vectors in mice after tail vein injection resulted in a significantly 7-fold increase of gene expression in the liver compared with PEG-g-PEI based gene vectors.     

靶向腺病毒载体介导的EGFP基因在甲胎蛋白阳性肝癌细胞的特异表达

目的：建立一种在甲胎蛋白(AFP)阳性的肝癌细胞中靶向表达目的基因的重组腺病毒载体。方法： 基于腺病毒载体Adeno-XTM Expression system,以300 bp AFP特异启动子替换穿梭质粒Pshuttle中CMV启动子，将增强型绿色荧光蛋白(EGFP)基因作为报告基因亚克隆至Pshuttle，HEK293细胞包装腺病毒，收集病毒后分别转染人正常肝脏LO2细胞，人肝癌HepG2细胞及HeLa细胞；通过Northern杂交检测EGFP基因在3种细胞中的转录水平，荧光显微镜下观察3种细胞中绿色荧光蛋白的表达。结果：Northern杂交显示，HepG2细胞中有大量EGFP基因的转录，而正常肝细胞LO2和HeLa细胞中仅能检测到微量基因的转录；荧光显微镜检测发现HepG2细胞内有强绿色荧光表达，而在LO2以及HeLa细胞内见极弱绿色荧光。 结论： 在AFP特异启动子作用下，腺病毒携带的目的基因在AFP阳性的肝癌细胞中得到显著转录和表达，而在非AFP阳性细胞仅微量转录，蛋白表达极弱。该腺病毒载体可作为AFP阳性的肝癌基因靶向治疗的适宜载体。     

重组人肝细胞生长因子-NK4腺病毒的制备及初步鉴定

目的制备表达人肝细胞生长因子（HGF）-NK4蛋白的复制缺陷型重组腺病毒。方法酶切pcDNA3-hNK4质粒获得NK4基因编码区序列并克隆至穿梭载体构建重组pAdTrack—CMV—NK4载体，线性化后与pAdEasy-1共转化BJ5183，通过同源重组得到重组pAd—NK4病毒载体。将重组腺病毒载体转染HEK293包装细胞制备重组腺病毒，用病毒悬液感染人肝癌细胞株HepG2。RT—PCR法检测感染肿瘤细胞中NK4mRNA表达。结果酶切鉴定得到阳性pAd—NK4重组腺病毒载体，该载体能有效转染HEK293细胞并在细胞内成功包装。在转染2d后能观察到绿色荧光蛋白（GFP）表达。制备的Ad—NK4在体外能有效感染HepG2细胞并获得NK4基因高水平表达。结论成功构建了NK4基因的重组腺病毒载体并制备重组腺病毒颗粒，为进一步研究NK4基因的功能及应用NK4进行基因治疗提供实验依据。     

乙型肝炎病毒上调HepG2细胞表面HLA-I表达的机制

目的 :探讨HBV野生株 (WT)及核壳蛋白变异株L97、V6 0上调HepG2细胞表面HLA I表达的机制。方法 :将已构建的重组表达载体EBO WT、EBO L97及EBO V6 0 ,分别经脂质体介导转染HepG2细胞 ,以空载体EBO作为对照。用RT PCR半定量法 ,检测细胞内HLA A基因及抗原提呈相关基因LMP2、TAP1和tapasinmRNA的表达 ;用Westernblot测定细胞内HLA I蛋白的表达。结果 :3株HBV重组表达载体转染的细胞 ,均呈现明显的HLA AcDNA的PCR扩增条带及HLA I蛋白条带 ,但其条带的强度有差异 ,EBO L97、EBO WT和EBO V6 0依次减弱。TAP1cDNA扩增带清晰 ,3株HBV间无明显差别。对照细胞未呈现HLA A的条带和仅呈现微弱的TAP1扩增带。各转染细胞均未检出LMP2及tapasinmRNA的表达。 结论 :HBV能诱导HepG2细胞内HLA I分子的合成量 ,使TAP1基因转录增强 ,HLA I的表达上调。核壳蛋白变异株L97和V6 0 ,可使宿主细胞内HLA ImRNA和其蛋白的表达水平发生变化     

Generation of replication-defective helper-free vectors based on simian immunodeficiency virus

A systematic study on generating simian immunodeficiency virus (SIV)-based vectors was carried out. The goal was to generate helper-free, replication-defective SIVmac-based vectors at high titers. The general approach was to cotransfect into human 293T cells a plasmid carrying the vector construct along with two helper plasmids that together expressed the SIVmac virion proteins. Initial vectors carried the bacterial beta-galactosidase gene (beta-gal). These vectors had a technical difficulty: "pseudotransduction" of beta-gal protein produced during the 293T cell transfections. As a result, infection of cultures with these vector stocks also resulted in passive transfer into, and X-gal staining of, cells that had not actually been infected by the vector. A second generation of vectors expressing the enhanced jellyfish green fluorescence protein (EGFP) was not subject to this artifact. A systematic study of the SIVmac-based EGFP vectors was carried out. Helper-free vector stocks were obtained when helper plasmids lacking the SIVmac packaging signals were used. By employing envelope helper plasmids derived from different SIVmac isolates, it was possible to generate SIVmac-based vectors pseudotyped with envelope proteins of different cell tropism. Optimization of vector and helper plasmid structures, transfection conditions, and infection procedures ultimately yielded vector titers in excess of 10(6)/ml.     

Stable expression of foreign proteins in herbaceous and apple plants using Apple latent spherical virus RNA2 vectors

Summary. Infectious cDNA clones of Apple latent spherical virus (ALSV)-RNA1 (pEALSR1) and -RNA2 (pEALSR2) were constructed using an enhanced 35S promoter. A viral vector was constructed from pEALSR2 by creating artificial protease processing sites by duplicating the Q/G protease cleavage site between 42KP and Vp25. Eight RNA2-derived vectors expressing GFP with varied sizes of duplications around the 42KP/Vp25 junction were constructed and tested for infectivity in Chenopodium quinoa. The results indicated that greater than five aa from the C-terminus of 42KP and N-terminus of Vp25 in duplication are necessary for systemic infection. In infected C. quinoa plants, GFP fluorescence was observed in both inoculated and upper leaves. Serial passages of the viruses derived from the above vectors in C. quinoa showed that the size of duplications affected the stability of the GFP gene. The version of the RNA2-vector (pER2L5R5GFP) with the shortest duplications and its silent mutant version could stably express GFP in leaves even after at least nine serial passages. ALSV-RNA2 vector has a capacity to maintain a DNA insert as long as 1300bp because Apple chlorotic leaf spot virus movement protein (50KP) gene could be expressed in C. quinoa. Inoculation of a virus derived from pER2L5R5GFP to apple seedlings resulted in the expression of GFP fluorescence in uninoculated upper leaves, indicating that the vector is available for the expression of foreign genes in apple trees.     

Microfluidic platform for hepatitis B viral replication study

Hepatocytes, the cells responsible for the metabolic and detoxification processes in the liver, are the predominant target of hepatitis B virus (HBV) infections, a major cause of liver cancer. The limited availability of normal human hepatocytes for cell-culture based studies is a significant challenge in HBV-associated liver cancer research. Therefore, there is a need for miniaturized cell-culture systems that can serve as a platform for studying the effect of HBV infections on hepatocyte physiology. Here, we present a microfluidic platform that can be used to study HBV replication in both rat and human hepatocytes. Polydimethylsiloxane (PDMS) microchannels fabricated using soft lithography techniques served as a culture vessel for both primary rat hepatocytes (PRH) and a human hepatoblastoma cell line, HepG2. The micro cell-culture chamber was then used as a model for HBV replication studies. Cells were grown in static culture conditions and either transfected with an HBV-genome cDNA or infected with the viral genome expressed from a recombinant adenovirus. Supernatants collected from the microchannels were assayed for secreted HBV using polymerase chain reaction (PCR). We achieved approximately 40 and 10% transfection efficiencies in HepG2 cells and PRH respectively, and 80–100% adenoviral infection efficiency in PRH comparable to standard tissue culture methods. Moreover, we successfully detected replicated HBV using our novel platform. This platform can be easily extended to studies involving DNA transfection or HBV infection of primary human hepatocytes since only a small number of cells are required for studies in microfluidic chambers.     

Induction of intercellular adhesion molecule-1 (CD54) on human hepatoma cell line HepG2: influence of cytokines and hepatitis B virus-DNA transfection.

Human hepatocyte expression of intercellular adhesion molecule-1 (ICAM-1) (CD54) was studied in vitro by exposing the well differentiated human hepatoblastoma cell line HepG2 to various cytokines. In addition, hepatitis B virus (HBV)-DNA transfected HepG2 cells were also analysed. Expression of ICAM-1 on HepG2 cells was then revealed with an immunohistochemical procedure. Untreated HepG2 cells were unreactive, but showed strong cytoplasmic ICAM-1 immunoreactivity after treatment with interferon-gamma (IFN-gamma). This induction was completely inhibited by addition of a neutralizing antibody directed to IFN-gamma. IL-1, IL-6, tumour necrosis factor-alpha (TNF-alpha) and IFN-alpha, used alone or in combination, did not induce ICAM-1 expression, neither did they inhibit the IFN-gamma-induced expression of this adhesion molecule on HepG2 cells. Untreated hepatitis B virus-DNA transfected HepG2 cells expressed membranous ICAM-1. These results indicate that IFN-gamma is the main cytokine trigger for ICAM-1 expression on HepG2 cells, suggesting that in areas of liver inflammation this adhesion molecule is up-regulated on hepatocytes by locally released IFN-gamma. In addition, expression of ICAM-1 by hepatitis B virus-DNA transfected HepG2 cells suggests other, still unknown, triggering mechanisms in the induction of such adhesion molecules, for instance gene activation by viral genome, or autocrine virus-induced hepatocellular cytokine production.     

The C-terminal region of the hepatitis B virus X protein is required for its stimulation of HBV replication in primary mouse hepatocytes

Hepatitis B virus (HBV) infection is an important risk factor for hepatocellular carcinoma (HCC). The hepatitis B virus X protein (HBx), a multifunctional regulatory protein encoded by HBV, is known to be involved in stimulation of viral replication by modulating cell cycle status. HBx is required for maximal virus replication in plasmid-based replication assays in immortalized human liver HepG2 cells and in primary rat hepatocytes. Moreover, the C-terminal region of HBx is important for HBV replication in HepG2 cells. However, in normal hepatocytes, the region of HBx that is responsible for its effect on cell cycle regulation and HBV replication is unclear. We have demonstrated that HBx is similarly required for maximal HBV replication in primary mouse hepatocytes and that the C-terminus of HBx is essential for its ability to stimulate HBV replication by inducing quiescent hepatocytes to exit G0 phase of the cell cycle but stall in G1 phase. Our studies establish that primary mouse hepatocytes support HBx-dependent HBV replication, and provide further evidence for the effect of the C-terminal region of HBx on HBV infection and replication.