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| 1 | IRES特异性IRNA对HCV IRES启动蛋白翻译细胞内抑制作用显示文摘目的:研究核糖体内部进入位点(internalribosomeentrysite,IRES)特异性抑制性RNA(inhibitorRNA,IRNA)细胞内对丙型肝炎病毒(HCV)IRES介导蛋白翻译的抑制作用.方法:体外应用脂质体细胞转染法,将IRNA及其突变体mIRNA真核表达载体pcRz-IRNA/pcRz-mIRNA转染人肝癌细胞株(HHCC),经G418筛选4wk后建立IRNA及mIRNA表达株;以相同的方法构建pcHCVcluc转染株;以脂质体介导细胞转染法将pCMVNCRLuc转染IRNA及mIRNA细胞株,于转染后48h检测荧光素酶表达量;将IRNA及mIRNA真核表达体转染pcHCVcLuc表达株,于转染后不同时间检测荧光素酶表达量.结果:HCVIRES介导蛋白翻译在IRNA表达株明显受到抑制,同样IRNA对HCV翻译复制子的蛋白翻译作用有明显抑制性;突变体mIRNA表达株和空载体对照株中未见相似的抑制性.结论:pcHCVcluc在HHCC细胞中获得有效表达;IRES特异性IRNA能有效的抑制HCVIRES介导细胞内蛋白翻译作用. | 梁雪松 连建奇 周永兴 聂青和 郝春秋 | 2003 | 世界华人消化杂志2003，11，2： | 4 |
| 2 | Expression of RNase H of human hepatitis B virus polymerase in Escherichia coli显示文摘AIM: To amplify HBV-RNase H gene fragment and expression of RNase H for further use in the studies of HBV associated liver diseases.METHODS: The encoding gene of HBV-RNase H was separately amplified for the first half and second half (H1 and H2) by PCR from full length HBV gene and cloned into pT7Blue-T vector. Clones were first screened by digestion with XbaI and Hind Ⅲ enzyme for the correct size, and analyzed further by DNA sequencing. The RNase H1 and H2fragments isolated from XbaI and HindⅢ digestion products of pT7 Blue-RNase H plasmid were ligated to the GSTag expressing vectors separately, and expressed in E.coli BL21.The expressed proteins were checked by PAGE gel and Western blot.RESULTS: Both H1 and H2 nucleotide seqences consisting of known genes and proteins, in correct size, were further confirmed by Western blot to be the GST and RNase H1 or H2 fusion proteins.CONCLUSION: The successful cloning and expression of HBV-RNase H will contribute to further research and application in HBV-associated diseases. | HongCheng Hui-ZhongZhang Wan-AnShen Yan-FangLiu Fu-ChengMa | 2003 | World Journal of Gastroenterology2003，9，3： | 3 |
| 3 | A small yeast RNA inhibits HCV IRES mediated translation and inhibits replication of poliovirus in vivo显示文摘AIM: To investigate the anti-virus infection activity of internal ribosome entry site (IRES) specific inhibitor RNA (IRNA).METHODS: IRNA eukaryotic vector pcRz-IRNA or mIRNA eukaryotic vector pcRz-mIRNA was tansfected into human hepatocarcinoma cells (HHCC), then selected with neomycin G418 for 4 to 8 weeks, and then infected with polio virus vaccinas line. The cytopethogenesis effect was investigated and the cell extract was collected. At last the polio virus titer of different cells was determined by plaque assay.RESULTS: Constitutive expression of IRNA was not detrimental to cell growth. HCV IRES-mediated capindependent translation was markedly inhibited in cells constitutively expressing IRNA compared to control hepatoma cells. However, cap-dependent translation was not significantly affected in these cell line. Additionally, HHCC cells constitutively expressing IRNA became refractory to infection of polio virus.CONCLUSION: IRES specific IRNA can inhibit HCV IRES mediated translation and poliovirus replication. | Xue-Song Liang Jian-Qi Lian Yong-Xing Zhou Qing-He Nie Chun-Qiu Hao The center of diagnosis and treatment for infectious diseases of Tangdu Hospital of Military Medical University of PLA,Xi’an 710038,Shaanxi Province,China | 2003 | World Journal of Gastroenterology2003，9，5： | 2 |
| 4 | 前S2抗原基因对乙型肝炎DNA疫苗免疫应答的影响显示文摘目的:探讨前S2抗原基因对乙型肝炎DNA疫苗免疫应答的影响. 方法:采用常规PCR法从adr亚型全基因DNA序列中分别扩增HBV S和前S2+S基因片段,重组到VR1012载体中, 转染COS-7细胞并免疫BALB/C小鼠.采用蛋白印迹、ELISA,ELISPOT等方法检测其在COS-7细胞内的表达及小鼠的体液及细胞免疫. 结果:转染的COS-7细胞表达相应的目的蛋白,且前S2+ s转染的细胞表达明显高于S转染的细胞的表达;免疫接种小鼠后2wk产生HBsAb及HBsAg特异性CTL,前S2+ S抗原免疫产生的HBsAb及HBsAg特异性CTL均强于S 抗原免疫. 结论:前S2抗原基因可增强乙型肝炎DNA疫苗刺激的免疫应答. | 赫兢 辛绍杰 毛远丽 貌盼勇 沈宏辉 杨健洋 徐军 孔维 | 2004 | 世界华人消化杂志2004，12，9： | 2 |
| 5 | 人工构建含丙型肝炎病毒核糖体插入位点的双顺反子表达载体显示文摘目的:研究丙型肝炎病毒核糖体插入位点启动翻译蛋白质功能,构建双顺反子真核表达载体.方法:逆转录PCR(RT-PCR)扩增丙型肝炎基因组中核糖体插入位点序列(IRES),定向克隆到pcDNA3-S质粒多克隆酶切位点中。在IRES下游克隆乙型肝炎病毒核心基因,测序鉴定后获得的质粒经脂质体转染hepG2细胞,间接免疫荧光染色和Western-blot检测.结果:在间接免疫荧光染色后,荧光显微镜下可见已型肝炎病毒S基因和核心基因表达。转染细胞破碎后免疫沉淀检测和图像分析表明,两种基因有相似的表达量。结论:人为截断HCV 5’NTR区17个碱基,并不影响IRES对下游基因蛋白质翻译,为成功构建了含丙型肝炎病毒IRES的双顺反子表达载体打下基础。 | 阚全程 余祖江 雷延昌 杨东亮 郝连杰 | 2003 | 世界华人消化杂志2003，11，10： | 2 |
| 6 | 增强子Ⅰ对乙型肝炎病毒DNA疫苗免疫应答的影响显示文摘目的研究乙型肝炎病毒(HBV)自身增强子Ⅰ(enhancerⅠ,ENHⅠ)对HBVDNA疫苗免疫应答的影响。方法采用PCR法以HBVadr亚型全基因DNA序列为模板分别扩增表面抗原(HBsAg)和HBsAg-ENHⅠ基因片段,重组到载体VR1012中,构建两种HBVDNA疫苗,转染COS-7细胞及HepG2细胞并免疫BALB/c小鼠。采用蛋白印迹、ELISA、ELISPOT等方法检测其在COS-7和HepG2细胞内的表达及小鼠的体液及细胞免疫应答效果。结果转染的HepG2和COS-7细胞均表达HBsAg;连接ENHⅠ的HBVDNA疫苗转染HepG2细胞后HBsAg表达量明显升高,两种疫苗转染COS-7细胞表达HBsAg无明显差异;免疫小鼠后第2周产生HBsAb及HBsAg特异性细胞毒T淋巴细胞(CTL),两种疫苗免疫产生的HBsAb及HBsAg特异性CTL无明显差异。结论ENHⅠ可使HBVDNA疫苗转染HepG2细胞表达HBsAg明显增加,对转染COS-7细胞表达HBsAg及接种BALB/c小鼠引起的免疫应答无明显影响。 | 赫兢 辛绍杰 侯俊 胡燕 杨健洋 貌盼勇 | 2006 | 解放军医学杂志2006，31，2： | 2 |
| 7 | HCV-Fc融合基因疫苗真核表达载体的构建及表达显示文摘目的:构建能表达HCV C-Fc融合基因的真核表达载体,为进一步修饰和转染树突状细胞,制备能高效表达HCV C和Fc基因的树突状细胞疫苗做准备。方法:用分别含有Kpn Ⅰ和Xhol Ⅰ酶切位点的HCV C区基因上、下游引物,以含有HCV H株基因序列的质粒pBRTM/HCV1-3011为模板,通过PCR扩增获得HCV C区基因片段回收后,以Kpn Ⅰ和Xhol Ⅰ双酶切,定向插入到含IgG1 Fc基因的质粒pIgC3双粘端位点之间,获得重组表达质粒pHCVFc。通过Kpn Ⅰ双位点酶切、PCR及插入片段序列测定对质粒进行了鉴定,以抗HCV C单克隆抗体为一抗,利用间接免疫荧光法检测了pHCV-IgFc在人肝癌细胞7721中的瞬时表达。结果:酶切、PCR及测序鉴定证实,pHCV-IgFc插入片段为HCV C区基因片段,免疫荧光法检测表明其可以在7721细胞中瞬时表达。结论:构建的质粒pHCV-IgFc可以在7721细胞中瞬时表达HCV C区基因,为研究HCV C-Fc融合基因修饰的树突状细胞的功能奠定了基础。 | 冯志华 王全楚 周永兴 郝春秋 聂青和 | 2003 | 世界华人消化杂志2003，11，6： | 2 |
| 8 | 热休克蛋白70与IL-2对小鼠肝癌移植模型的治疗比较显示文摘目的:比较分析肿瘤热休克蛋白70(HSP70)与IL-2对小鼠肝癌移植模型的治疗作用,评价HSP70的疗效,为应用HSP70治疗人类恶性肿瘤提供有重要参考价值的实验依据。方法:细胞培养、蛋白质纯化技术、电泳技术、Westernblot法、动物实验等。结果:IL-2及HSP70对小鼠肝癌移植模型均有治疗作用,其中IL-210万U、HSP7010μg效果最佳,IL-25万U及HSP705μg有部分治疗作用,10万U的IL-2与5μgHSP70治疗效果相当,HSP7010μg治疗后有40%小鼠移植肿瘤全部消退,长期生存790d,与对照组及其他各组相比,差异具有显著性(P<0.01,P<0.05)。结论:HSP70具有良好的抗肿瘤免疫活性,其疗效明显优于IL-2,本研究对于应用HSP70治疗人类的恶性肿瘤有十分重要的借鉴意义。 | 傅庆国 沈晓东 孟凡东 郭仁宣 | 2003 | 世界华人消化杂志2003，11，4： | 2 |
| 9 | Hepatitis B virus S gene enhances immune responses of a multiple-epitope DNA construct of hepatitis C virus in mice显示文摘The purpose of this study was to construct an eukaryotic DNA vector encoding a multiple-epitope antigen (MFC) of hepatitis C virus (HCV) and a hepatitis B surface antigen (HBsAg) , and explore the effect of HBsAg gene on the immunity of HCV multiple-epitope DNA construct in vitro and in vivo in mice. An HCV DNA vector (pVAX1-HBs-MFC) was constructed by fusing HBsAg gene to the N-terminal of an HCV multiple-epitope antigen gene. The pVAX1-HBs-MFC was transfected into HEK 293T cells and its expression was measured by ELISA and Western blotting. BALB/c mice were intramuscularly immunized with the pVAX1-HBs-MFC, and an ELISA approach was applied to determine the specific antibody titers and subtypes in the mouse serum. The cross-reactivity of the antibodies was also checked with two synthesized HCV hypervariable region 1 (HVR1) peptides. The IFN-γ production and cell proliferation of the mouse spleen cells were evaluated by ELISA and MTS (3-[4, 5-dimethylthiazol-2-yl]-5-[3-carboxymethoxyphenyl]-2-[4-sulfophenyl] -2H-tetrazolium, inner salt) assays, respectively. The expression of pVAX1-HBs-MFC was detectable in the transfected HEK 293T cells. The serum antibody response was effectively elicited in BALB/c mice injected with pVAX1-HBs-MFC. The highest titer of antibody against HCV (MFC) was 1∶1280, and the ratio of IgG2a/IgG1 was 1.50±0.12 at the fifth week after first immunization. Moreover, the collected mouse serum antibody had the ability to cross-react with the two synthesized HCV HVR1 peptides. The stimulation index of the mouse splenocytes to MFC was 1.79 ±0.07, and the IFN-γ level was 287±6?pg/ml at week 21 after first immunization. The highest titer of the antibody in control BALB/c mice immunized with pVAX1-MFC was 1∶320, and the ratio of IgG2a/IgG1 was 1.33±0.11 at week 5 post-immunization. Furthermore, the stimulation index of the mouse splenocytes cells to MFC was 1.52±0.06, and the IFN-γ level was 225±9.3?pg/ml at week 21 post-immunization. The HBsAg gene can enhance the effects of an HCV multiple-epitope DNA construct on its humoral and cellular immune responses. This HBsAg enhanced HCV multiple-epitope DNA vector may be of potential use in the development of HCV vaccines. | YUPINGGONG JUNGAO PINGZHAO XIAOPINGYANG ZHONGTIANQI | 2005 | Journal of Microbiology and Immunology2005，3，1： | 1 |
| 10 | Construction of eukaryotic expression plasmids of hepatitis B surface antigen and helper T lymphocyte epitope显示文摘 | | 2004 | Hepatobiliary & Pancreatic Diseases International2004，3，2： | 1 |
| 11 | 病毒性肝炎基因治疗的研究和面临的挑战显示文摘病毒性肝炎是目前严重危害人类健康的最常见的世界性感染性疾病之一,迄今仍无有效的抗病毒药物,抗病毒基因疗法作为一种新的治疗手段,可望从病因上解决病毒性肝炎的治疗问题,本文结合我们自己的研究综述抗肝炎病毒基因疗法的研究现状和面临的挑战,重点介绍抗肝炎病毒核酶和DNA免疫的研究。 | 贾战生 冯志华 周永兴 | 2003 | 世界华人消化杂志2003，11，6： | 1 |
| 12 | Elicitation of strong immune responses by a DNA vaccine expressing a secreted form of hepatitis C virus envelope protein E2 in murine and porcine animal models显示文摘AIM: To characterize the immunogenicity of a hepatitis C virus (HCV) E2 DNA vaccine alone or with a protein vaccine boost in murine and porcine animal models. METHODS: A DNA vaccine expressing a secreted form of HCV E2 protein was constructed and used to vaccinate mice and piglets with or without boosting with a recombinant E2 protein vaccine formulated with CpG ODN and 10% Emulsigen. The immunogenicity of HCV E2 vaccines was analyzed by ELISA for antibody responses, MTT assay for lymphocyte proliferation, ELISPOT for the number of interferon-γ secreting cells, and cytotoxic T lymphocyte assays. RESULTS: Intradermal injection of E2 DNA vaccine induced strong Th1-like immune responses in mice. In piglets, E2 DNA vaccine elicited moderate and more balanced immune responses. A DNA vaccine prime and protein boost vaccination strategy induced significantly higher E2-specific antibody levels and shifted the immune response towards Th2-like ones in piglets. CONCLUSION: A DNA vaccine expressing a secreted form of HCV E2 protein elicited E2-specific immune responses in mice and piglets. Recombinant E2 protein vaccination following DNA immunization significantly increased the antibody response in piglets. These HCV E2 vaccines may represent promising hepatitis C vaccine candidates for further investigations. | Yi-Ping Li Hye Na Kang Lorne A Babiuk Qiang Liu | 2006 | World Journal of Gastroenterology2006，12，44： | 1 |
| 13 | 科研实例在生物化学与分子生物学教学中的应用显示文摘生物化学与分子生物学是生命科学中的研究热点。近些年来生命科学领域的实验技术手段日新月异、各类研究成果层出不穷,但都要遵循生物化学与分子生物学的理论知识基础。以各类生物化学与分子生物学科研成果作为科研实例,应用于生物化学与分子生物学理论课教学,激发学生学习兴趣,从而提高教学质量。 | 董博翰 戴广丽 徐蕾 孙玲玲 | 2013 | 基础医学教育2013，15，11： | 1 |
| 14 | Inhibitor RNA blocks the protein translation mediated by hepatitis C virus internal ribosome entry site in vivo显示文摘AIM:To investigate the inhibitory effect of hepatitis C virus internal ribosome entry site (HCV IRES) specific inhibitor RNA(IRNA) on gene expression mediated by HCV IRES in vivo.METHODS:By using G418 screening system, hepatoma cells constitutively expressing IRNA or mutant IRNA (mIRNA) were established and characterized, and HCV replicons containing the 5′ untranslated region (5′UTR) were constructed by using the same method. Cotransfection of pCMVNCRluc containing HCV 5′UTR-Iuc fusion genes and eukaryotic vector of IRNA into human hepatic carcinoma cells (HepG2) was performed and the eukaryotic expression plasmid of IRNA was transfected transiently into HCV replicons, pCMVNCRluc or pCDNA-luc was cotransfected with pSV40-β Gal into IRNA expressing hepatoma cells by using lipofectamine 2000 in vitro. Then the reporting gene expression level was examined at 48h after transfection by using a luminometer and the expressing level of HCV C antigen was analysed with a confocal microscope.RESULTS: Transient expression of IRES specific IRNA could significantly inhibit the expression of reporter gene and viral antigen mediated by HCV IRES by 50% to 90% in vivo, but mIRNA lost its inhibitory activity completely. The luciferase gene expression mediated by HCV IRES was blocked in the HHCC constitutively expressing IRNA. At 48h after transfection, the expression level of reportor gene descreased by 20%, but cap-dependent luciferase gene expression was not affected. IRNA could inhibit the HCV replicon expression 24h after transfection and the highest inhibitory activity was 80% by 72h, and the inhibitory activity was not increased until 7d after transfection.CONCLUSION:IRNA can inhibit HCV IRES mediated gene expression in vivo. | Xue-SongLiang Jian-QiLian Yong-XingZhou Mo-BinWan | 2004 | World Journal of Gastroenterology2004，10，5： | 0 |
| 15 | HBV RNA复制子疫苗、DNA疫苗在BHK-21细胞中表达情况的比较显示文摘为建立更有效的乙肝疫苗,通过将所构建的HBVRNA疫苗和DNA疫苗分别转染BHK-21细胞,并比较其在细胞中表达情况及细胞凋亡现象。以pSFV为基础构建的疫苗载体的表达效率高于以pcDNA3.1为基础构建的疫苗载体,pSFV为基础构建的疫苗载体出现细胞凋亡的现象。HBVRNA疫苗比DNA疫苗表达效果更好,安全性更高,更具有应用前景。 | 李莉 任林柱 张英 杨东 闫森 李小平 高飞 肖雪斌 欧阳红生 逄大欣 | 2010 | 中国农学通报2010，26，10： | 0 |
| 16 | 乙肝疫苗研究进展显示文摘乙肝病毒(HBV)可通过多种途径感染人体,引起肝病的传播,对乙型肝炎预防和治疗的研究具有重大的经济效益和社会效益。目前使用的乙肝疫苗存在诸多问题,作者就乙肝疫苗存在的问题及解决方法进行综述。 | 郝玉兰 钱淑凤 郑翠玲 | 2008 | 中国畜牧兽医2008，35，10： | 0 |
| 17 | 乙肝DNA疫苗显示文摘DNA疫苗可以诱导长期的体液及细胞免疫 ,打破免疫耐受 ,作为未来治疗病毒、胞内菌、寄生虫等病原体感染及肿瘤的工具已进行了大量研究。本文就慢性乙型肝炎DNA疫苗的研究进行了总结 ,主要描述了二十一世纪以后乙肝DNA疫苗的发展。 | 赫兢 辛绍杰 | 2004 | 国外医学(病毒学分册)2004，11，5： | 0 |
| 18 | 增强核酸疫苗免疫应答的策略及方法显示文摘 | 何煦 雷秉钧 | 2005 | 华西医学2005，20，4： | 0 |
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