马传染性贫血病毒驴白细胞弱毒疫苗株及其亲本驴强毒株长末端重复序列(LTR)的启动子活性比较

将马传染性贫血病毒驴强毒(donkey-adapted equine infectious anemia virus,D-A EIAV)、马传染性贫血病毒驴白细胞弱毒(donkey leukocyte attenuated EIAV,DLA EIAV)及EIAV Wyoming株的全长长末端重复序列(LTR)分别克隆到报告基因载体pCAT-Basic vector中,获得重组质粒p-D-A-LTR-CAT、p-DLA-LTR-CAT及p-Wyo-LTR-CAT.将这3个重组质粒分别体外转染EIAV阴性健康驴的白细胞,比较这三者LTR启动报告基因CAT表达的基础活性和在tat-pcDNA3共转染条件下激活活性的差异.结果表明:在驴白细胞中,DLA EIAV LTR启动CAT表达的活性略高于D-A EIAV LTR;而与EIAV Wyoming株LTR比较,DLA EIAV与D-A EIAV二者LTR启动CAT表达的活性都较低.在有共转染重组表达质粒tat-pcDNA3条件下,D-A EIAV、DLA EIAV及EIAV Wyoming株LTR起始CAT表达的活性都得到提高,分别提高了4.8倍、6.0倍和3.2倍.上述结果提示,LTR可能是体现DLA EIAV驴白细胞适应性的因素,不一定是影响其毒力减弱的根本原因.     

HCVIRES介导萤火虫荧光素酶翻译的双顺反子载体的构建与在小鼠体内的表达

将HCVIRES插入双报告基因海肾荧光素酶 (Rluc)基因和萤火虫荧光素酶 (Fluc)基因之间 ,建立了“依赖帽子的扫描机制”翻译表达Rluc ,HCVIRES调控Fluc翻译的双顺反子表达载体pCI Rluc HCVIRES Fluc ,通过酶切反应及转染HepG2细胞鉴定双荧光素酶瞬间表达活性等试验 ,证实获得了表达双荧光素酶的双顺反子载体 .并应用水压转染法将双顺反子表达质粒导入小鼠体内 ,在小鼠肝脏检测到高水平表达的Rluc和Fluc .该研究成功构建一种HCVIRES介导萤火虫荧光素酶基因表达的双顺反子载体 ,并在HepG2细胞及小鼠体内进行了瞬时表达 ,为进一步建立稳定评价靶向HCVIRES药物作用的细胞及小动物模型研究奠定了基础     

马传染性贫血强/弱毒嵌合病毒的体外构建

马传染性贫血病毒(equine infectious anemia virus,EIAV)引起马传染性贫血(简称马传贫),导致马持续性感染和反复病毒血症[1].EIAV与人免疫缺陷病毒Ⅰ型(HIV-1)同属反转录病毒科慢病毒属,二者有很多相似的特性[2].在反转录病毒前病毒基因组两端含有长末端重复序列(long terminal repeat,LTR).LTR含有真核启动子,其中含有病毒转录调控顺式作用位点,病毒编码的反式作用因子与其结合后可以反式激活基因的表达,对病毒基因的表达和其它生命活动起重要调控作用[3,4].因此,LTR序列的变异可能会引起病毒转录和复制方式的改变,进而引起其细胞嗜性和致病性的改变[5,6].为了探讨LTR在EIAV病毒复制和转录过程中的作用,并进一步研究EIAV的致病和免疫机制,用EIAV强毒L株LTR置换了以前构建的EIAV DLA(弱毒)感染性分子克隆中的LTR,构建了马传贫强/弱毒嵌合分子克隆,并获得了具有感染性的强/弱毒嵌合病毒.     

用于活体影像技术稳定表达荧光素酶的肺癌细胞系的建立

目的:建立稳定表达萤火虫荧光素酶的人肺癌细胞系并进行体外验证.方法:亚克隆萤火虫荧光素酶基因到真核表达载体pRc/CMV2上,构建重组质粒pRc/CMV2.1uc+,转染到肺癌细胞株spc-a-1,经过G418筛选获得单细胞抗性克隆.抗性克隆连续传代,并通过荧光素酶活性检测筛选出高水平表达荧光素酶的稳定细胞克隆,在体外检测细胞的生物发光能力与细胞数量的相关性.结果:构建的pRc/CMV2-1uc+真核表达质粒转染spe-a-1细胞后,经G418筛选出多个抗性克隆;传代至40代时确定有3株细胞的荧光素酶活性最高;活体影像系统体外检测证实阳性细胞株发光强度与数量呈正相关.结论:结果表明成功构建了稳定表达萤火虫荧光素酶的spc-a-1细胞系.     

抗J亚群禽白血病病毒的鸡胚成纤维细胞系建立

将J亚群禽白血病病毒囊膜基因(ALV-Jenv)插入pcDNA3.1真核表达载体,构建成转移载体pcDNA-env,并转染鸡胚成纤维细胞系DF-1,通过Zeocin药物筛选获得转染阳性细胞。细胞经传30代后,冻存。13个月后复苏,置不含药物的培养基中传代培养。连续传40代后,分别用PCR、Southern blot、间接免疫荧光(IFA)及Western blot对该细胞中的env基因进行了检测,并进行了抗病毒感染分析。研究结果表明,通过Zeocin药物筛选获得了稳定表达ALV-Jenv基因的鸡胚成纤维细胞系,命名为pcDNA-env-DF-1细胞。该细胞在体外抗ALV-J感染试验中,能抵抗1×104个TCID50病毒感染量,提示所构建的细胞系具有良好的抗病毒作用。这一细胞系的构建为研究ALV-J Env蛋白与宿主细胞表面受体相互作用分子机理提供了很好的工具。     

拼接信号序列单碱基变异提高马传染性贫血病毒mRNA拼接效率

分别以马传染性贫血(马传贫)驴强毒(D—A EIAV)RNA和马传贫驴白细胞弱毒疫苗(DLA EIAV)RNA为模板,利用RT—PCR的方法,克隆到马传贫强、弱毒株基因组外显子2及其下游的核苷酸序列。然后将报告基因CAT插入到EIAV内含子2env阅读框架中,构成CAT拼接报告系统。同时在强毒株重组表达质粒的基础上,将其外显子-3上游拼接受体位点的核苷酸序列CAG突变为弱毒株相应位置的核苷酸序列TAG,得到强毒单核苷酸突变株重组表达质粒。用构建的3个重组表达质粒DNA转染驴血白细胞,ELISA检测转染细胞CAT浓度。结果表明：EIAV强毒株重组表达质粒中CAT蛋白表达量最高,EIAV强毒株重组表达质粒次之,EIAV强毒突变株重组表达质粒最低。由于CAT基因被插入于各重组质粒中的EIAV内含子-2里,EIAV外显子-2、3之间的拼接可导致该基因的删除,因而其拼接效率低于EIAVmRNA外显子-2、3之间的拼接效率。实验数据表明,EIAV SA2拼接信号序列单碱基变异提高了SD2-SA2拼接效率;D—AEIAV SA2-SD2拼接效率比DLA EIAV相应位点拼接效率高。     

RU5区对BFV3026基因表达的调控

以牛泡沫病毒(Bovine foamy virus,BFV)中国株BFV3026原病毒DNA为材料,构建R区系列缺失质粒,通过对其转染细胞中RT水平及对缺失质粒与luc报告质粒共转染细胞中萤火虫荧光素酶活性的测定,确立U5区对于BFV3026两类启动子LTR和IP均具有负调控作用;同时将带有不同R区的BFV3026结构基因片段克隆于异源启动子CMV之下,通过对其转染细胞293T中RT酶活性的测定,确立R区对于病毒结构基因pol的表达具有一定的调节作用,并将其功能区域初步界定在R区5′端100bp内。     

马传染性贫血病毒基因非编码区LTR嵌合克隆的构建

在已有全长感染性克隆pLGFD3 8 和pD70344 的基础上,根据马传贫弱毒疫苗致弱过程中不同代次毒株LTR序列的分析,在LTR U3区选取特定的酶切位点对EIAV非编码区LTR基因进行了部分替换。将替换的全基因克隆转染驴胎皮肤细胞(FDD)并以驴白细胞(DL)传代,用逆转录酶活性检测、RT PCR方法及Real time RT PCR验证其感染性。结果发现,其衍生病毒感染上述两种细胞均出现明显的细胞病变;细胞培养上清可检测到RT酶活性和RT PCR阳性。电镜下可见大量典型的EIAV颗粒。pLGFD9 12 嵌合克隆衍生病毒与其父本克隆衍生病毒pLGFD3 8具有相似的复制水平,pLGFD9 12嵌合克隆衍生病毒在DL细胞上复制水平略高于FDD细胞。此结果为进一步深入研究LTR对马传染性贫血病毒复制水平和毒力的影响奠定了基础。     

shRNA随机文库结合TK自杀基因筛选靶向HIV-1LTR相关宿主因子

构建shRNA随机文库与HIV-1 LTR启动胸苷激酶基因(TK基因)稳定表达的稳定细胞系HEK293/TK,将两者结合起来,筛选靶向HIV-1 LTR相关宿主因子.方法:通过化学合成含有19个随机脱氧核苷酸的发夹结构,将其退火补平后与合成的接头Linker连接进行PCR反应,将PCR产物酶切后置于慢病毒载体pLenti-U6启动子下游由此构建shRNA随机文库;利用重叠PCR将HIV-1 LTR片段和TK基因连接起来,连接产物经酶切后与pcDNA3.1载体连接;将连接正确的质粒转染HEK293细胞同时用G418加压筛选获得稳定细胞系HEK293/TK;将所获得的文库质粒包装成慢病毒后侵染所构建的HEK293/TK细胞系,通过加入药物GCV进行加压筛选获得存活细胞.结果:成功筛选到加药后存活下来的细胞,抽提细胞基因组,采用巢式PCR扩增目的干扰序列并用Western blot对干扰序列进行验证,鉴定获得一个克隆所表达的shRNA能对TK基因的表达起到抑制作用,通过测序分析获得其干扰序列,该序列很有可能针对HIV-1 LTR某宿主相关因子.结论:成功构建了一种筛选HIV-1 LTR相关宿主因子的方法,筛选所得序列可以定位到具体相关宿主因子,为靶向筛选抗HIV-1药物提供了重要手段.     

以HIV-1 5＇LTR为靶点的药物筛选细胞模型的构建

构建以人类免疫缺陷病毒（HIV-1）5＇LTR为靶点的药物筛选细胞模型,用于体外筛选潜在的对HIV-1启动子具有抑制作用的药物,或用于筛选与HIV-1复制相关的宿主因子。通过PCR扩增HIV-1 5＇LTR片段和胸苷激酶基因（thymidine kinase gene,TK基因）,以这2片段为模板进行重叠PCR将两者连接起来,连接产物经酶切后与pcDNA3.1载体连接;将连接正确的质粒转染HEK293细胞同时用G418加压筛选获得稳定细胞系;加入药物更昔洛韦（GCV）检测TK基因的表达。成功构建了HIV-1 5＇LTR调控TK基因表达的稳定细胞系,在其培养过程中加入药物更昔洛韦时,细胞逐渐死亡。构建了以HIV-1 5＇LTR为靶点的药物筛选细胞模型,该模型利用TK基因作为报告基因方便灵敏,可用于筛选针对HIV-1 5＇LTR的潜在抗HIV药物。     

Mapping of equine lentivirus receptor 1 residues critical for equine infectious anemia virus envelope binding

The equine lentivirus receptor 1 (ELR1), a member of the tumor necrosis factor receptor (TNFR) protein family, has been identified as a functional receptor for equine infectious anemia virus (EIAV). Toward defining the functional interactions between the EIAV SU protein (gp90) and its ELR1 receptor, we mapped the gp90 binding domain of ELR1 by a combination of binding and functional assays using the EIAV SU gp90 protein and various chimeric receptor proteins derived from exchanges between the functional ELR1 and the nonbinding homolog, mouse herpesvirus entry mediator (murine HveA). Complementary exchanges of the respective cysteine-rich domains (CRD) between the ELR1 and murine HveA proteins revealed CRD1 as the predominant determinant of functional gp90 binding to ELR1 and also to a chimeric murine HveA protein expressed on the surface of transfected Cf2Th cells. Mutations of individual amino acids in the CRD1 segment of ELR1 and murine HveA indicated the Leu70 in CRD1 as essential for functional binding of EIAV gp90 and for virus infection of transduced Cf2Th cells. The specificity of the EIAV SU binding domain identified for the ELR1 receptor is fundamentally identical to that reported previously for functional binding of feline immunodeficiency virus SU to its coreceptor CD134, another TNFR protein. These results indicate unexpected common features of the specific mechanisms by which diverse lentiviruses can employ TNFR proteins as functional receptors.     

An equine infectious anemia virus variant superinfects cells through novel receptor interactions

Wild-type strains of equine infectious anemia virus (EIAV) prevent superinfection of previously infected cells. A variant strain of virus that spontaneously arose during passage, EIAV_vMA-1c, can circumvent this mechanism in some cells, such as equine dermis (ED) cells, but not in others, such as equine endothelial cells. EIAV_vMA-1c superinfection of ED cells results in a buildup of unintegrated viral DNA and rapid killing of the cell monolayer. Here, we examined the mechanism of resistance that is used by EIAV to prevent superinfection and explored the means by which EIAV_vMA-1c overcomes this restriction. We found that the cellular receptor used by EIAV, equine lentivirus receptor 1 (ELR1), remains on the surface of cells chronically infected with EIAV, suggesting that wild-type EIAV interferes with superinfection by masking ELR1. The addition of soluble wild-type SU protein to the medium during infection blocked infection by wild-type strains of virus, implicating SU as the viral protein responsible for interfering with virion entry into previously infected cells. Additionally, interference of wild-type EIAV binding to ELR1 by the addition of either anti-ELR1 antibodies or the ELR1 ectodomain prevented entry of the wild-type strains of EIAV into two permissive cell populations. Many of these same interference treatments prevented EIAV_vMA-1c infection of endothelial cells but only modestly affected the ability of EIAV_vMA-1c to enter and kill previously infected ED cells. These findings indicate that EIAV_vMA-1c retains the ability to use ELR1 for entry and suggest that this virus can interact with an additional, unidentified receptor to superinfect ED cells.     

Replication of Equine Infectious Anemia Virus in Engineered Mouse NIH 3T3 Cells

We employed the equine lentivirus equine infectious anemia virus (EIAV) to investigate the cellular restrictions for lentivirus replication in murine NIH 3T3 cells. The results of these studies demonstrate that NIH 3T3 cells expressing the EIAV receptor ELR1 and equine cyclin T1 supported productive replication of EIAV and produced infectious virions at levels similar to those found in a reference permissive equine cell line. The studies presented here demonstrate, for the first time, differential levels of restriction for EIAV and human immunodeficiency virus type 1 (HIV-1) replication in murine cells and suggest that these differences can be exploited to reveal critical virus-cell interactions required for HIV-1 assembly and budding of lentivirus particles.     

Engineering Viral Promoters for Gene Transfer to Human Neuroblasts

1. The strength and activity of several viral promoters in human neuroblasts were evaluated in vitro. 2. Several luciferase reporter gene contructs under the control of different viral promoters (HIV-1 LTR, HTLV-I LTR, MMTV LTR, RSV LTR, CMV, SV40), in the presence or in the absence of the viral SV40 enhancer, were transfected into two well-established human neural cell lines, including one derived from human embryonic olfactory cells (B4) and one derived from an adrenal neuroblastoma (SH-SY-5Y). The epithelial cell line HeLa was used as a control.3. The enzymatic activity of luciferase was evaluated after normalization with an internal control. The results indicated that in the context of the reporter gene constructs, the CMV promoter alone was, overall, the most active in any tested cell line. However, addition of the SV40 enhancer to the CMV promoter abolished luciferase activity in SH-SY-5Y cells while significantly increasing luciferase expression in the CNS derived B4 fetal neuroblasts.4. The results suggest that gene therapeutic vectors aimed to promote enzymatic activity through gene transfer into undifferentiated human neural cells are feasible. However, since differences in promoter activity in neuroectodermal-derived cells are very relevant, gene construct variants should be considered to optimize the system.     

Detection of HIV-1 Neutralizing Antibodies in a Human CD4+/CXCR4+/CCR5+ T-Lymphoblastoid Cell Assay System

Sensitive assays are needed to meaningfully assess low levels of neutralizing antibodies (NAbs) that may be important for protection against the acquisition of HIV-1 infection in vaccine recipients. The current assay of choice uses a non-lymphoid cell line (TZM-bl) that may lack sensitivity owing to over expression of CD4 and CCR5. We used transfection of a human CD4+/CXCR4+/α₄β₇+ T-lymphoblastoid cell line (A3.01) with a CMV IE promoter-driven CCR5neo vector to stably express CCR5. The resulting line, designated A3R5, is permissive to a wide range of CCR5-tropic circulating strains of HIV-1, including HIV-1 molecular clones containing a Tat-inducible Renilla luciferase reporter gene and expressing multiple Env subtypes. Flow cytometric analysis found CCR5 surface expression on A3R5 cells to be markedly less than TZM-bl but similar to CD3.8 stimulated PBMC. More importantly, neutralization mediated by a diverse panel of monoclonal antibodies, HIV-1 positive polyclonal sera and sCD4 was consistently greater in A3R5 compared to TZM-bl cells. The A3R5 cell line provides a novel approach to guide the development and qualification of promising new HIV-1 vaccine immunogens.     

三种丙型肝炎包膜感染性假病毒颗粒的制备及初步应用研究

In this study, three expression vectors encoding unmodified glycoproteins E1 and E2 from H77 (1a), Hebei (1b) and JFH1 (2a) strains were constructed to form pVRC-H77-E1E2, pVRC-HeBei-E1E2 and pVRC-JFH1-E1E2 expressing constructs. The protein expression was confirmed by immunofluorescene assay(IFA) and Western blot. The Lentiviral vector has the ability to package the cellular membrane into pseudo-particles. The plasmid expressing HCV E1-E2 glycoproteins in native form was co-transfected into 293FT cells with a lentiviral packaging plasmid (pHR'CMV delta R8.2)and a self-inactivated (SIN) transfer plasmid (pCS-CG) containing a reporter EGFP gene to produce infectious HCV pseudo-particles(pp). Flow cytometry assays showed that the HCVpp could infect Huh7 and Huh7-CD81, and the infectivity in Huh7-CD81 was about 2-3 times higher than that in Huh7 cells. Meanwhile, HCVpp could neither infect non-liver cells, for example, the 293 cells, nor HepG2 cell . Titration of HCVpp by p24 ELISA assay or infection assay showed that this HCVpp may contain 5-25 ng/mL p24 or 10(4)-10(5) TU (transducing unit)/ ml. An in vitro HCV neutralizing assays based on HCVpp (1a, 1b, 2a) were then established using AP33, a monoclone antibody with cross-neutralizing ability to different HCV strains. The neutralizing ability of the antibodies from HCV infected patients was further studied with this HCVpp system. In summary, three kinds of HCVpp (1a, 1b, 2a subtype) were successfully developed; In vitro HCV neutralizing assays based on HCVpp and SIN lentiviral system were established. This system paves a way for characterization of early steps of HCV infection (host tropisms, receptor binding, membrane fusion, et al. ) or screening anti-HCV drugs (such as inhibitor to virus entry). This system can be further applied to assess the human immune responses in HCV patients or evaluate HCV vaccine candidates.     

汉坦病毒囊膜糖蛋白G1基因重组腺病毒载体的构建及其在Vero E6细胞中的表达

拟构建汉坦病毒Gl基因重组腺病毒载体并在VeroE6细胞中表达,为汉坦病毒基因疫苗的研究提供实验基础。PCR法从含汉坦病毒-76118株M基因的M56质粒扩增糖蛋白G1基因片段,利用穿梭质粒pShuttle,将其克隆入Adeno—X病毒DNA,获得重组腺病毒DNA,转染HEK293细胞,包装、扩增后得到汉坦病毒Gl基因重组腺病毒原种,感染VetoE6细胞,用IFA法和ELISA法检测表达产物。得到了含汉坦病毒G1基因的重组腺病毒,其滴度约为10^11pfu／ml,感染VeroE6细胞后检测到汉坦病毒糖蛋白G1的表达。     

A New Indicator Cell Line Established to Monitor Bovine Foamy Virus Infection

In order to improve the accuracy for quantitating the bovine foamy virus (BFV) in vitro,we developed a baby hamster kidney cell (BHK)-21-derived indicator cell line containing a plasmid that encodes the firefly luciferase driven by the BFV long terminal repeat promoter (LTR,from-7 to 1012).The BFV titer could be determined by detecting the luciferase expression since the viral trans-activator BTas protein activates the promoter activity of the LTR.One clone,designated BFVL,was selected from ten neomycin-res...