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双语推荐：型腺相关病毒

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[目的]包装并鉴定带有人白介素2(hIL-2)及ZsGreen的双基因共表达的重组5型腺相关病毒(recombinant adeno-associated virus 5,rAAV5),为今后利用5型重组腺相关病毒载体进行胰腺癌基因治疗提供研究基础。[方法]以5型腺相关病毒包装系统(helper-free system)为模版,PCR法扩增pLVX-IRES-ZsGreen和PES-IL-2模板上的IRES-ZsGreen和IL-2基因,利用酶切位点插入重组腺相关病毒骨架质粒,获得重组质粒pAAVhIL-2-IRES-ZsGreen。经三质粒共转染AAV293细胞包装重组腺相关病毒rAAV5-hIL-2-IRES-ZsGreen。荧光显微镜下检测病毒包装效率,超速离心纯化、浓缩重组病毒,qPCR测定病毒的滴度。重组病毒转导细胞经荧光显微镜检测、外源基因经PCR检测证实病毒包装结果。[结果]5型重组腺相关病毒骨架质粒pAAV-IL-2-IRES-ZsGreen经双酶切和测序鉴定,确定其序列正确。荧光显微镜下观察三质粒共转染AAV293细胞72h后,病毒包装效率达90%以上。5型重组病毒感染HEK 293细胞48h有荧光出现,重组病毒基因组成功扩增出外源基因IL-2,证明有感染力的重组病毒包装成功。[结论]成功包装带有hIL-2及ZsGreen标记的双基因共表达重组腺相关病毒,滴度达到2.62×1012。
Objective To pack and identify the recombinant adeno-associated virus type 5(rAAV5) vector co-expressing hIL2 and ZsGreen for offering the basement of gene therapy in pancreatic cancer. [Methods] The IRES-ZsGreen and hIL2 genes were amplified by PCR and inserted into pAAV-MCS by enzyme digestion sites respectively. The recombinant expression plasmid pAAV-hIL2-IRES-Zs Green was co-transfected into AAV293 cells with pHelper and pAAV-RC for packaging of recombinant AAV5. The efficiency of rAAV packaging was monitored under fluorescent microscope and re-combinant viral particles were harvested from infected host cells, and titer was measured through qPCR after concentration and purification. HEK293 cells were infected with the rAAV5 and the recombinant AAV5 was verified by PCR of the exogenous interest genes of hIL2. [Results] The recombinant plas-mid pAAV-hIL2-IRES-ZsGreen was verified by double digestion. Using the AAV helper-free system, ZsGreen expression could be observed under flu-or

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目的 构建1b型丙型肝炎病毒(HCV) NS34b基因重组腺相关病毒载体并了解其在HEK 293细胞中的表达,为进一步研究HCV重组腺相关病毒疫苗及其树突状细胞疫苗奠定前期基础.方法 收集基因1b型的丙型肝炎病人血清,用RT-PCR的方法扩增NS3-4b全长片段,与腺相关病毒的表达载体pAAV.CMV.eGFP重组,构建pAAV.CMV.HCV.NS3-4b重组表达载体,转染HEK293细胞,检测其蛋白表达情况.结果 PCR扩增获得的NS3-4b条带与预期大小(2838 bp)一致,重组质粒经双酶切和测序证实NS3-4b基因已重组成功,重组质粒转染HEK 293细胞后Western Blot图可见有目的蛋白表达.结论 成功构建腺相关病毒重组HCV NS3-4b载体并且其能在真核细胞中表达.
Objective To clone 1b type of HCV NS3-4b Gene and express in HEK 293 cells,lay the foundation for further study of the HCV NS3-4b recombinant adeno-associated virus vaccine and its dendritic cell vaccine.Methods HCV 1b patients'' serum was collected,and full length NS3-4b segment was amplified by RT-PCR and cloned into adeno-associated virus'' expression vector pAAV.CMV.EGFP in order to express in HEK 293 cells.At last,it was validated whether express or not by Western Blot.Results The 1b type gene NS3-4b were amplified and consistent to the expected size(2838 bp),the recombinant plasmid has been confirmed its successful restructured by double enzyme and sequencing,at last,Western Blot map can see objective protein expression after it transfect HEK 293 cells.Conclusion The adenoassocisted virus recombination HCV NS3-4b plasmid have successfully constructed and it can express in eukaryotic cells.

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肠道腺病毒(enteric adenovirus,EAdv)是导致儿童腹泻的重要病原体,发病率仅次于轮状病毒、诺如病毒.研究表明在免疫缺陷患者中EAdv易引起并发症的发生,甚至导致死亡.国内外有EAdv暴发或流行的报道[1].虽然近年来的研究发现A、C、D、G等其他亚型的腺病毒均可引起婴幼儿急性腹泻[2],但EAdv仍以F亚群的40型和41型最为常见,其代表株分别为原型株Dugan-Hovix病毒和Tak病毒.自1995年程绪杰等在我国从儿童粪便中分离到EAdv以来,我国对EAdv的研究越来越重视.本文就肠道腺病毒及其相关研究进展进行综述.
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背景：重组9型腺相关病毒对心肌细胞具有良好的亲和力，是目前研究基因治疗心肌梗死的理想载体。目的：观察携带增强型绿色荧光蛋白基因的重组9型腺相关病毒(rAAV9-eGFP)对小鼠骨髓间充质干细胞的转染效率。 方法：将携带增强型绿色荧光蛋白基因的重组9型腺相关病毒以不同感染复数(1×105，1×106，1×107)转染体外培养的第4代小鼠骨髓间充质干细胞，并在转染后1-7 d连续用荧光倒置显微镜观察骨髓间充质干细胞中增强型绿色荧光蛋白阳性表达情况，寻找最佳感染条件；采用流式细胞仪检测最佳感染复数下携带增强型绿色荧光蛋白基因的重组9型腺相关病毒对小鼠骨髓间充质干细胞的转染效率。 结果与结论：转染后第1天，感染复数为1×107组可见增强型绿色荧光蛋白开始表达，转染后第2天，感染复数为1×105、1×106组开始表达；各组增强型绿色荧光蛋白表达强度随感染复数值的增高而增强，同时增强型绿色荧光蛋白的表达强度随着时间延长而逐渐增强；转染后第5天达到高峰，此时感染复数为1×107组转染效率约8%，结果表明携带增强型绿色荧光蛋白基因的重组9型腺相关病毒对小鼠骨髓间充质干细胞转染效率较低。
BACKGROUND:Recombinant adeno-associated virus type 9 (rAAV9) has a good affinity to myocardialcytes, and it is an ideal carrier for gene therapy of myocardial infarction. OBJECTIVE:To investigate the transfection efficiency of rAAV9-enhanced green fluorescence protein (eGFP) in transfection of mouse bone marrow mesenchymal stem cels. METHODS:rAAV9-eGFP was transfected into mouse bone marrow mesenchymal stem cels which were subcultured to the fourth generation at different multiplicities of infection (MOI=1×105, 1×106, 1×107 ). To find the best condition of transfection, we observed the eGFP expression under the inverted fluorescence microscope at 1-7 days after transfection. Then the transfection efficiency of rAAV9-eGFP under the optimal MOI was determined by flow cytometry. RESULTS AND CONCLUSION: The cels with rAAV9-eGFP transfection at MOl of 1×10 7 began to exhibit eGFP expression at the 1st day after transfection, and the cels transfected at MOI of 1×105 and 1×106

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目的构建一种由腺病毒AdMax包装系统表达目的基因——黑色素瘤分化相关基因-7(MDA-7)的复制缺陷型腺病毒,并初步探索其抗肿瘤活性。方法通过基因操作技术将目的基因(MDA-7)插入到5型腺病毒AdMax包装系统的E1A区域,构建复制缺陷型腺病毒Ad.hMDA-7-EGFP;同时构建对照病毒Ad.EGFP。病毒滴度计算按寇化法(Karber法)进行腺病毒感染性滴度(TCID50)测定。应用免疫组化染色法检测MDA-7在感染2种病毒的肺腺癌A549细胞中的表达状态;通过二苯甲亚胺-碘化丙啶(Hoechst33342-PI)双染色法及流式细胞仪检测2种病毒对肿瘤细胞杀伤的差异。结果成功构建携带目的基因的腺病毒载体,经聚合酶链反应(PCR)鉴定正确;MDA-7在感染Ad.hMDA-7-EGFP的肺腺癌A549细胞中表达,以相同感染复数(MOI)值感染Ad.EGFP时肺腺癌A549细胞中未检测到MDA-7表达;Ad.hMDA-7-EGFP对肿瘤细胞株杀伤能力明显高于Ad.EGFP。结论成功构建复制缺陷型腺病毒Ad.hMDA-7-EGFP,并证实MDA-7蛋白在肿瘤细胞中过表达,诱导肺腺癌A549细胞株发生凋亡。
Objective To construct replication-deficient recombinant adenovirus of melanoma differentiation associated gene(MDA)-7,which is expressed by adenovirus vector AdMax packing system,and to initially explore its antineoplastic activity. Methods Ad.hMDA-7-EGFP of replication-deficient recombinant adenovirus was constructed by inserting the targeted gene (MDA-7)into E1A area of type 5 adenovirus vector AdMax packing system with gene manipulation,meanwhile the contrast virus of Ad. EGFP was constructed. Karber′s method was used to calculate virus titer,the tissue culture infective dose 50 (TCID50) was adopted to determine infective titer of adenovirus vector ,and the immunohistochemical staining was employed to detect the expression status of MDA-7 in A 549 cell lines infected with 2 viruses. The difference of the two viruses on tumor cell was assessed by Hoechst33342-PI staining method and flow cytometry(FCM). Results Adenovirus vector carrying targeted gene was constructed succe

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背景：动物研究显示，自体髓核细胞移植能有效修复椎间盘退变。然而髓核细胞体外增殖能力差，这就限制了其作为种子细胞在椎间盘退变性疾病治疗中的研究及应用。 目的：构建包含外源性人端粒酶反转录酶基因的腺相关病毒2载体，观察其转染人髓核细胞后人端粒酶反转录酶基因的表达。 方法：构建pSNAV2.0-pRSV-hTERT质粒并鉴定，采用AAVMaxTM包装系统进行重组腺相关病毒2-人端粒酶反转录酶载体的构建，以 PCR 及酶切方法验证构建的质粒，构建成功后扩增，并纯化。利用腺相关病毒2-增强型绿色荧光蛋白载体转染第1代人髓核细胞，测定最佳感染复数。参照此感染复数值，确定腺相关病毒2-人端粒酶反转录酶对人髓核细胞转染的相关感染复数；对照组采用不含外源性人端粒酶反转录酶基因的腺相关病毒2进行转染。转染后1，2，4周分别采用RT-PCR对人端粒酶反转录酶基因mRNA水平进行半定量检测。 结果与结论：实验成功构建了腺相关病毒2-人端粒酶反转录酶载体；并获得了滴度达2×1011 v·g/mL的腺相关病毒2-人端粒酶反转录酶载体。测得腺相关病毒2-人端粒酶反转录酶载体对人髓核细胞的最佳感染复数为5×104 v·g/cel。在以1×104，5×104，1×105 v·g/cel 转染人髓核后，均可检测到人端粒酶反转录酶基因mRNA的高量表达。采用RT-PCR半定量检测方法，发现以转染后2周时人端粒酶反转录酶 mRNA表达量相对最高(P <0.05)，4周时仍可见人端粒酶反转录酶基因mRNA的稳定表达。而对照组无论在何时间点均未能检测到人端粒酶反转录酶mRNA的表达。提示利用腺相关病毒2可以成功构建包含外源性人端粒酶反转录酶基因的病毒载体，腺相关病毒2-人端粒酶反转录酶能有效转染人髓核细胞并稳定表达人端粒酶反转录酶基因mRNA，此结果可能为增强髓核细胞性能提供新的策略。
BACKGROUND:In animal experiments, transplantation of autologous nucleus pulposus cellscan effectively repair the intervertebral disk degeneration. However, nucleus pulposus cells have a poor ability of proliferation in vitro, which limits its application as seed cells in treatment of intervertebral disk disease. OBJECTIVE:To construct recombinant adeno-associated virus type-2 vector carrying human telomerase reverse transcriptase and observe the human telomerase reverse transcriptase mRNA expression in human nucleus pulposus cells in vitro. METHODS:After the plasmid pSNAV2.0-pRSV-hTERT was constructed and identified, recombinant adeno-associated virus type-2 vector carrying human telomerase reverse transcriptase were constructed, amplified and purified by AAVMaxTM package and purification system. The optimal multiplicity of infection for human nucleus pulposus cells was detected by recombinant adeno-associated virus type-2 vector carrying enhanced green fluorescent protein. According t

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目的 监测北京市丰台区环境污水中的肠道病毒的血清型分布及流行情况,为北京市丰台区肠道病毒相关的传染病预防和控制提供实验室支持.方法 选择吴家村污水处理厂为监测点,2012年每月采集污水(共12份),用阴离子膜-碱洗脱法进行病毒浓缩,然后进行病毒分离,并对阳性分离物进行肠道病毒血清型鉴定.结果 北京市丰台区环境监测污水标本中共分离到60株肠道病毒、6株腺病毒;病毒分离以B组肠道病毒为主;夏季(5-9月)的病毒分离率较高;部分环境中分离的肠道病毒与手足口患者体内标本有同源性.结论 环境监测可以为北京市丰台区肠道病毒病例监测系统提供重要的补充信息.本研究可以为丰台区乃至北京市的EV研究提供背景信息.
Objective To provide laboratory data for the prevention and control of the human enterovirus infection diseases in Fengtai district of Beijing,human enterovirus in enviromental sewage was monitored.Methods The Wu-jia-cun sewage treatment plant was chose as surveillance site,the sewage samples were collected monthly from in Fengtai district,from January 2012 to December 2012.Viral isolation was performed for the sewage samples.Results 60 enteroviruses and 6 adenoviruses were isolated from sewage water samples,HEV-B were the mainly enteroviruses during the environmental surveillance.Virus separation rate is higher in May,Jun,Jul,Aug and Sep.Part of the enterovirus isolated from environment specimens have homology with hand-foot-mouth of patients specimens.Conclusions Environmental surveillance can provide important supplementary information for enteroviruses case surveillance system which could provides background information on the EV study for the Fengtai district.

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探讨柯萨奇病毒-腺病毒受体(CAR)在肺腺癌部分亚型中的表达及其与临床病理和患者预后的关系。方法:采用EnVision免疫组化二步法检测CAR在137例具有细支气管肺泡癌特征的肺癌(PWBF)组织中的表达,分析其与临床因素的相关性;并收集患者的生存资料,采用Kaplan-Meier曲线描述生存率,行Log-rank检验。结果:CAR在PWBF、其他类型肺癌及正常组织中的阳性率分别为71.5%、50.0%、13.3%,其差异具有统计学意义;CAR蛋白的表达与病理分型和组织学分级相关,与性别、年龄、临床分期等无关。CAR阳性表达患者的生存时间较阴性者长,但无统计学差异。结论:CAR的表达与肺癌的发生发展相关,并同肺腺癌部分亚型(PWBF)的关系密切。CAR在PWBF中的较高表达为以腺病毒(Ad)载体的基因治疗开辟了更为广阔的空间;同时CAR的高度可调节性也为肺癌其他类型的基因治疗提供了可靠的依据和美好的前景。
Objective:This study aims to investigate the protein expression of coxsackie-virus and adenovirus receptor (CAR) in partial subtypes of pulmonary adeno-carcinoma, as well as its expression with clinico-pathological factors and prognosis. Methods:CAR expression was immunohistochemically assessed in 137 cases of lung cancer with bronchiole-alveolar carcinoma (PWBF) fea-tures, and analyzed in relation to various clinico-pathological parameters. All data were analyzed using SPSS statistics software, and Ka-plan-Meier survival curves were constructed. A Log-rank test was also conducted. Results: The CAR positive rates in PWBF, other types of lung cancer, and normal lung tissue were 71.5%, 50.0%, and 13.3%, respectively. The difference was statistically significant (P<0.05). In addition, a statistically significant difference was observed between the positive expression of CAR and other clini-co-pathologic parameters, such as pathological type and histological differentiation. No statistical

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目的 探讨病毒感染在VAP中的发生情况,总结病毒感染与VAP的相关性.方法 将35例应用呼吸机治疗未发生肺炎患者作为对照组及35例发生肺炎患者作为观察组,对比两组呼吸道合胞病毒(RSV)、腺病毒(ADV)、单纯疱疹病毒Ⅰ型(HSV-I)、巨细胞病毒(CMV)Ig-M的阳性率,并分析RSV、ADV、HSV-I、CMV与VAP发生的相关性.结果 VAP组RSV、ADV、HSV-I、CMV阳性率分别为5.57％、8.57％、22.85％及11.43％,总阳性率为48.57％,而对照组阳性率分别为2.85％、2.85％、5.57％及2.85％,总阳性率为14.19％,VAP组各病毒阳性率均明显高于对照组(P＜0.05).同时HSV-I阳性与VAP均存在显著的正向直线相关性(P＜0.05),而RSV、CMV及ADV阳性与VAP无相关关系(P＞0.05).结论 VAP患者病毒感染阳性率明显升高,且HSV-I感染与VAP发生存在显著的相关性,提示HSV-I感染可能为VAP发生的危险因素.
Objective To investigate the correlation between virus infection and VAP summary.Methods 35 patients using respirator treatment without pneumonia were collected as control group,and 35 patients using respirator treatment with pneumonia were collected as research group.Ig-M of respiratory syncytial virus (RSV),adenovirus (ADV),herpes simplex virus type Ⅰ (HSV-I),cytomegalovirus (CMV) were detected and the data was analyzed.Results Ig-M of RSV,ADV,HSV-I and CMV were 5.57％,8.57％,22.85％ and 11.43％ for the research group,2.85％,2.85％,5.57％ and 2.85％ for the control group.At the same time,there was linear correlation between HSV-I positive and VAP positive (P ＜ 0.05).Conclusions There was significant correlation between VAP patients with virus infection.

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干扰素基因刺激蛋白(stimulator of interferon genes,STING)是天然免疫信号通路中重要接头蛋白,环鸟苷酸-腺苷酸合成酶(cyclic GMP-AMP synthase,cGAS)是其上游DNA识别受体,两者共同参与的cGAS-STING通路在识别外源性DNA、介导产生Ⅰ型干扰素的过程中发挥重要。最近研究结果表明,人类免疫缺陷病毒(human immunodeficiency virus,HIV)入侵宿主细胞后,能激活cGAS-STING通路,诱导天然免疫反应,抑制HIV病毒活性。本文将对该信号通路的作用机制及相关内容加以综述,以期为开拓新的抗HIV药物靶点和分子设计提供新思路。
Stimulator of interferon genes(STING)are important adaptor proteins in innate immune-related signal pathway. The upstream protein,cyclic guanosine monophosphate-adenosine monophosphate synthase(cGAS),is cytosolic DNA sensor. Detection of cytosolic DNA,cGAS-STING pathway can be activated and subsequently activate the type Ⅰ interferon (IFN)antiviral response. Recent studies indicated that retroviruses especially HIV can activate the host innate immune system through cGAS-STING pathway and trigger robust immune response. In this review,we briefly describe the mechanism of innate immune activated by the newly discovered cGAS-STING pathway and highlight recent progress in STING agonist.

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