Nucleolin/Nsr1p binds to the 3′ noncoding region of the tombusvirus RNA and inhibits replication

Previous genome-wide screens identified > 100 host genes affecting tombusvirus replication using yeast model host. One of those factors was Nsr1p (nucleolin), which is an abundant RNA-binding shuttle protein involved in rRNA maturation and ribosome assembly. We find that overexpression of Nsr1p in yeast or in Nicotiana benthamiana inhibited the accumulation of tombusvirus RNA by ∼10-fold. Regulated overexpression of Nsr1p revealed that Nsr1p should be present at the beginning of viral replication for efficient inhibition, suggesting that Nsr1p inhibits an early step in the replication process. In vitro experiments revealed that Nsr1p binds preferably to the 3′ UTR in the viral RNA. The purified recombinant Nsr1p inhibited the in vitro replication of the viral RNA in a yeast cell-free assay when preincubated with the viral RNA before the assay. These data support the model that Nsr1p/nucleolin inhibits tombusvirus replication by interfering with the recruitment of the viral RNA for replication.     

单纯疱疹病毒I型糖蛋白D酵母表达载体的构建

目的:构建含有单纯疱疹病毒I型包膜糖蛋白D全基因片段的克隆载体及酵母表达载体,为进一步真核表达重组gD蛋白奠定基础。方法:应用PCR方法从HSV-I基因组中扩增糖蛋白D的全长基因,产物回收后与pMD-18T载体连接,并转化,经氨苄筛选及测序,建立克隆载体pMD18T-gD;克隆载体与表达载体双酶切后,产物连接、转化大肠杆菌JM109,筛选、测序后确定构建了酵母表达载体pGAPZαA-gD。结果:重组质粒pMD18T-gD和pGAPZαA-gD经PCR和酶切均出现相应长度的片段,测序结果证实为gD基因,序列分析同源性为99.66%。结论:成功构建了含有单纯疱疹病毒I型糖蛋白D全基因的克隆载体及酵母表达质粒。     

基因工程杂交酵母艾滋病疫苗研究

目的 对以酵母为载体所制得的艾滋病候选疫苗进行研究。方法 构建表达Gag及IL-2的质粒载体，将表达载体分别转化入酵母细胞，通过酵母杂交获得同时表达两种基因的酵母杂交体，放大培养后经灭活、分装、冻干，制得细胞干粉作为试验的疫苗。用小鼠及食蟹猴对试验疫苗进行有效性及安全性研究。结果 杂交酵母可以稳定表达Gag及IL-2；经皮下注射可以分别在小鼠及食蟹猴体内诱导一定水平的细胞免疫反应；对小鼠肿瘤模型的研究表明，疫苗可以诱导有效的免疫保护反应；疫苗的安全性良好，没有明显的毒副作用，只在注射部位引起轻微的红肿，个别动物有低热反应，但均在一周左右恢复正常。结论杂交酵母艾滋病疫苗具有诱导细胞免疫反应的能力；杂交酵母载体疫苗在相关疾病的治疗方面有良好的应用前景。     

雄激素受体相关蛋白267-α酵母双杂交诱饵重组质粒的构建及转化

目的 构建和转化雄激素受体相关蛋白267—α(ARA267—α)的酵母诱饵重组质粒，为通过酵母双杂交研究ARA267—α的功能奠定基础。方法 用PCR扩增ARA267—α全长cDNA中编码PWWP和PHD-SET蛋白结构域的两个基因片段；将基因片段与pGBKT7载体定向重组；用酶切和测序鉴定重组质粒；将核苷酸序列正确的重组质粒转化入AH109酵母菌株。结果 成功构建pGBKT7-PWWP和pGBKT7—PHDSET重组质粒。分别转化有2种重组质粒和pGBKT7空载体的3种AH109酵母都能在SD／-Trp／X—α—gal培养基中长成白色菌落，但都不能在SD／-HiS／-Trp／2．5mmol／L 3-AT／X—α—gal和SD／-Ade／-Trp／X—α—gal培养基中生长，在SD／-Trp液中培养16h后，A600均值分别为0．8、0．9、0．9。这表明重组质粒表达的融合蛋白没有激活HIS3、ADE2和MEL1酵母报告基因表达的活性，也没有酵母毒性作用。结论 构建的诱饵重组质粒可以用于下一阶段的cDNA文库筛选。     

The VP1 structural protein of enterovirus 71 interacts with human ornithine decarboxylase and gene trap ankyrin repeat

Enterovirus 71 (EV71) is a major etiological agent of hand, foot and mouth disease (HFMD). Several outbreaks in East Asia were associated with neurological complications and numerous deaths. EV71 possesses four structural proteins VP1-VP4 that are necessary in the formation of the pentameric icosahedral capsid. The viral capsid contributes to virulence, and VP1 is a prime target for EV71 vaccine development. Using yeast two-hybrid analysis, we demonstrated binding affinity between VP1 and three human proteins, i.e. ornithine decarboxylase (ODC1), gene trap ankyrin repeat (GTAR), and KIAA0697 expressed in brain tissue. These interactions were authenticated by co-immunoprecipitation experiments, and by indirect immunofluorescent confocal microscopy of transfected and EV71-infected Vero cells. The significant interaction between VP1 and ODC1 may compromise the latter's activity, and interfere with polyamine biosynthesis, growth and proliferation of EV71-infected cells. The interaction between VP1 and GTAR is noteworthy, since ankyrin proteins are associated with certain neural cell adhesion molecules and with the CRASH neurological syndrome. Given that VP1 is synthesized in large amounts during productive infection, these viral-host protein interactions may provide insights into the role of VP1 in the pathogenesis of EV71 disease and its neurological complications such as acute flaccid paralysis and encephalitis.     

Antigenic characterisation of yeast-expressed lyssavirus nucleoproteins

In Europe, three genotypes of the genus Lyssavirus, family Rhabdoviridae, are present, classical rabies virus (RABV, genotype 1), European bat lyssavirus type 1 (EBLV-1, genotype 5) and European bat lyssavirus type 2 (EBLV-2, genotype 6). The entire authentic nucleoprotein (N protein) encoding sequences of RABV (challenge virus standard, CVS, strain), EBLV-1 and EBLV-2 were expressed in yeast Saccharomyces cerevisiae at high level. Purification of recombinant N proteins by caesium chloride gradient centrifugation resulted in yields between 14-17, 25-29 and 18-20 mg/l of induced yeast culture for RABV-CVS, EBLV-1 and EBLV-2, respectively. The purified N proteins were evaluated by negative staining electron microscopy, which revealed the formation of nucleocapsid-like structures. The antigenic conformation of the N proteins was investigated for their reactivity with monoclonal antibodies (mAbs) directed against different lyssaviruses. The reactivity pattern of each mAb was virtually identical between immunofluorescence assay with virus-infected cells, and ELISA and dot blot assay using the corresponding recombinant N proteins. These observations lead us to conclude that yeast-expressed lyssavirus N proteins share antigenic properties with naturally expressed virus protein. These recombinant proteins have the potential for use as components of serological assays for lyssaviruses.     

重组人胱蛋白酶抑制剂C在毕氏酵母菌中的高效表达

目的：构建人胱蛋白酶抑制剂C（Cystatin C)的表达载体并进行蛋白的初步纯化。方法：用逆转录聚合酶链反应从人胎盘组织中扩增含有380bp的Cystatin C cDNA基因片段，并将其插入到pPIC9质粒中，携带有Cystatin C片段的pPIC9质粒转化毕氏酵母菌菌株GS115，醇氧化脱氢酶1（AOX1）启动子被甲醇诱导而产生可溶性Cystatin C.重组Cystatin C采用Hitrap-SP阳离子交换柱进行初步纯化。结果：核酸序列测定与预期一致。Cystatin C 表达水平达到16mg/L，约占菌体总可溶性蛋白的60％，蛋白纯化回收率为40．5％，Cystatin C在表达体系中至少可稳定3d，结论：Cystatin C在毕氏酵母力胞外有较高水平的分泌，既可作为抗原免疫动物获取抗体，也可作为测定的标准品，为今后进行国产Cystatin C免疫学测定剂研制奠定了基础。     

乙型肝炎病毒前S2基因酵母表达载体的构建及表达

目的：探讨乙型肝炎病毒（HBV）前S2蛋白的功能。方法：以质粒pCP10 (含有HBV ayw亚型全长序列)为模板，多聚酶链反应（PCR）扩增HBV前S2基因，克隆到pGEM-T载休整 ，测序鉴定、酶切后回收，与酵母表达质粒pGBKT7连接。将重组载体转化酵母细胞AH109，提取酵母蛋白质，进行十二烷基磺酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）和Western免疫印迹分析。结果：成功构建HBV前S2基因酵母表达载体，Western免疫印迹显示HBV前S2蛋白在酵母细胞中表达，表达产物在胞内存在，分子量24kD左右。结论：HBV前S2蛋白在酵母细胞中表达成功。