Identification of antiviral- relevant genes in the cultured fish cells induced by UV-inactivated virus

~~Identification of antiviral- relevant genes in the cultured fish cells induced by UV-inactivated virus~~     

Detection of Grass Carp Hemorrhage Virus （GCHV） from Vietnam and Comparison with GCHV Strain from China

Grass carp plays an important role in small-scale aquaculture in Vietnam. However, a severe disease, known in Vietnam as “Red Spot Disease“, is causing significant economic loss in grass carp aquaculture. In this study, the tissue samples isolated from the grass carp with Red Spot Disease in Vietnam are investigated and eomparied with the control GCHV isolated in China by experimental infection, culture cell infection, serological cross reactivity, and RT-PCR amplifica-tion. Infected grass carp exhibits hemorrhage symptoms about 5 days after experimental injection with GCHV-V (Vietnam) strain. The symptoms and lethality induced by the GCHV-V strain are identical to that induced by the Chinese GCHV-9014 strain. The Chinese GCHV-873 strain in-duces typical cytopathogenic effects in 4 cell lines, such as CIK, CAB, FHM and GCO, from the6 fish cell lines examined. No cytopathogenic effects are observed in all the 6 examined cell lines,including CAB, FHM, CIK, EPC, CCO and G(X), infected by the GCHV-V strain and GCHV-9014 strain. Immunodiffusion assays demonstrate an obvious cross-reactivity among three GCHVstrains. Precipitin lines are clearly observed not only between the anti-GCHV-873 serum and thetwo strains GCHV-873 and GCHV-9014, but also between the anti-GCHV-873 serum and the GCHV-V strain. GCHV can be detected by immunodiffusion assays after three generations of blind propagations in the cell lines inoculated by GCHV-V strain. This implicates that GCHV-Vviruses have been replicated and amplified despite there being no cytopathogenic eifects observed in these examined cell lines. Three genome segments of GCHV, including S8, S9 and S10, are amplified by three sets of PCR primers designed according to the segment sequences published re-cently. The Q8fp and Q8rp primer set specific for genome segment S8 amplifies a 955 bp frag-ment from the extracted sample of diseased fish with Red Spot Disease, and the fragment size is i-dentical to that amplified by the same primer set from control GCHV-873 strain. Simultaneously,the Q9fp and Q9rp primer set specific for genome segment S9 generates a same 635 bp product,and the Q10fp and Q10rp primer set specific for genome segment S10 produces a same 697 bp fragment from both template samples of diseased fish with Red Spot Disease and control GCHV-873 strain. The RT-PCR amplification and corresponding size comparison data indicate that the three GCHV-V genome segments extracted from the diseased grass carp with Red Spot Disease in Vietnam should be identical to that in control GCHV-873 strain from China. The data confirm that the causative agent of grass carp Red Spot Disease in Vietnam is a virus, and the virus is closely similar to GCHV strain in China.     

Identification of genes associated with cotyledon senescence in upland cotton

Leaf senescence in plants is an essential develop- mental phase, and an understanding of senescence is important not only for pure scientific reasons, but also for practical purposes. During the last decade, a number of senescence-associated genes (SAGs) …     

Identification of differentially expressed genes in esophageal cancer through SSH in combination with high throughput reverse Northern screening

To understand the molecular mechanisms of carcinogenesis of esophagus and to isolate genes with different expression levels in esophageal cancer, suppression subtractive hybridization (SSH) was combined with PCR-based cDNA synthesis and reverse Northern on the cancer tissues and matched almost normal mucosa using 5 microgram of total RNA as starting marterial. Eight genes were found expressed differentially in esophageal cancer, in which 5 were known genes and 3 were novel ones; and 6 were down-regulated in cancer tissues, while 2 were up-regulated; 6 were of mid-high abundance and 2 were of low abundance in esophagus. The results revealed that alteration in expression level of multiple genes underlied the initiation and development of esophageal cancer. The differentially expressed genes identified in this study such as liporcotinⅠ, cystatin A, cystatin B, cytokeratin 13 may play roles in dedifferentiation, transformation and malignant proliferation of esophageal cancer. The combination of SSH with PCR-based double- strand cDNA synthesis and high throughput reverse Northern screening is an efficient way to isolate differentially expressed genes from microgram of total RNA.     

Identification of the candidate genes associated with cellular rejection in pig-to-human xenotransplantation

To identify the genes associated with cellular rejection in pig-to-human xenotransplantation, the suppression subtractive hybridization (SSH) was used in screening the up-regulated genes from a co-culture of human peripheral blood mononuclear cells (PBMCs) and porcine vascular endothelial cell line PIEC. The up-regulated cDNAs were cloned into pGEM-T Easy vector and then sequenced. Nucleic acid homology searches were performed using the BLAST program. A subtracted cDNA library including about 300 clones with the expected up-regulated genes was obtained. Twenty-four of these clones were analyzed by sequencing and homology comparison was made. These clones represent the genes of human perforin (PRF1), proteasome, lymphocyte specific interferon regulatory factor/interferon regulatory factor 4 (LSIRF/IRF 4), muscleblind-like (MBNL) protein and a porcine expressed sequence tag (EST) which has 81% homology with human oxidative-stress responsive 1 (OSR 1). These genes might be the candidate genes which are associated with cellular rejection in pig-to-human xenotransplantation.     

水稻矮化突变体差异表达cDNA片段的克隆与分析

利用抑制性消减杂交（Suppressive subraction hybridization,SSH）分离本实验室获得的一株水稻矮化突变体dwarf69与其野生型对照品系特光矮-2（Rryza sativa,TGA-2 indica）间表达有差异的cDNA片段,分别以dwarf69作为驱赶子,TGA-2为检测子,以及以dwarf69作为检测子,TGA-2为驱赶子建立了两个差异表达cNDA文库,分别代表在TGA-2和dwarf69中特异表达的cDNA,经反式Northern检测这两个文库共得到10个阳性克隆。     

Dynamic changes of apoptosis in duck embryo fibroblasts induced by new type Gosling viral enteritis virus

The monolayer duck embryo fibroblast （DEF） cells were experimentally infected with new type Gosling viral enteritis virus （NGVEV） and the dynamic changes of apoptosis were detected at different time points after NGVEV infection by transmission electron microscopy （TEM）, DNA agarose gel electrophoresis and Annexin V-FITC/PI stained fluorescence-activated cell sorter （FACS）. The result shows that NGVEV can induce infected cells undergoing apoptosis and changing regularly. A series of characteristic apoptotic morphological changes including shrinkage of the cells, chromatin condensation and margination, as well as formation of apoptotic bodies, were observed by TEM. The typical ladder pattern of DNA fragmentation was demonstrated by agarose gel electrophoresis. And using flow cytometry analysis of Annexin V-FITC/PI staining, the dead, viable, apoptotic and necrotic cells could be analyzed quantitatively.     

草鱼日粮中虾蛋白肽对幼龄草鱼生长性能的影响

用300尾(3.74±0.23)g的草鱼Ctenopharyngodon idella作日粮中不同添加量虾蛋白肽(平均肽链长为3.61)对幼龄草鱼生长性能影响研究.实验分为4组,每组75尾鱼,每25尾鱼饲于0.25m3的水族箱中,每组设3个平行水族箱.试验1、2、3组日粮中分别添加w为0.25%虾蛋白肽 w为0.75%鱼粉、w为0.5%虾蛋白肽 w为0.5%鱼粉、w为1%虾蛋白肽,对照组添加w为1%鱼粉.试验期56 d.结果表明:试验各组草鱼其特定生长率、蛋白保留效率、饲料系数和血浆中磷、镁含量与小肽总量均高于对照组,特别是试验2、3组与对照组有显著性差异(P＜O.05);草鱼日粮中适宜的虾蛋白肽添加量为w为0.5%.草鱼日粮中添加一定比例的虾蛋白肽均可提高饲料表观消化率和蛋白消化率,增加血液循环中生物活性肽的含量,提高机体对日粮中氨基酸的利用率,从而增加体内氮沉积,减少肝胰脏和肠系膜脂肪储积.     

Identification of auxin responsive genes in Arabidopsis by cDNA array

The plant hormone auxin influences a variety of developmental and physiological processes. But the mechanism of its action is quite unclear. In order to identify and analyze the expression of auxin responsive genes, a cDNA array approach was used to screen for genes with altered expression from Arabidopsis suspension culture after IAA treatment and was identified 50 differentially expressed genes from 13824 cDNA clones. These genes were related to signal transduction, stress responses, senescence, photosynthesis, protein biosynthesis and transportation. The results provide the molecular evidence that auxin influences a variety of physiological processes and pave a way for further investigation of the mechanism of auxin action. Furthermore,we found that the expression of a ClpC (regulation subunit of Clp protease) was repressed by exogenous auxin, but increased in dark-induced senescing leaves. This suggests that ClpC may be a senescence-associated gene and can be regulated by auxin.     

Host cell apoptosis induced by infection with duck swollen head hemorrhagic disease virus

This study aimed to examine the host cell apoptosis in the tissues of Peking ducks infected with duck swollen head hemorrhagic disease virus (DSHDV). The dynamic changes associated with apoptosis occurring in the internal tissues were evaluated at different time points postinoculation (PI) by performing hematoxylin and eosin (HE) staining, followed by light microscopy, terminal deoxynucleotidyl transfe- rase dUTP nick-end labeling (TUNEL) assay, and transmission electron microscopy (TEM). The results showed that DSHDV infection could induce apoptosis in host cells, including those of the bursa of Fabricius (BF), thymus, spleen, liver, intestinal tract, kidney, and esophagus. The apoptotic index (AI) values increased with time from 2 h to 72 h PI, and the highest values were recorded at 72 h PI. Further, cell death due to classic necrosis was observed in the dying or deceased ducks after 72 h PI. In conclusion, host cell apoptosis can be induced by DSHDV and may play an important role in the pathogenesis of duck viral swollen head hemorrhagic disease (DVSHD).     

草鱼对葡萄糖和淀粉作为能源的利用研究

通过10周生长实验和同位素示踪实验探讨草鱼对不同结构糖的利用。生长实验结果显示,草鱼摄食以葡萄糖作为糖源的饲料后其相对生长率、饲料效率、蛋白质效率均显著高于摄食以淀粉作为糖源的饲料组。同位素示踪实验结果显示,草鱼灌喂^14C－标记葡萄糖饲料后24h内每2小时间隔内呼出的^14CO2放射活度比均高于灌喂^14C－标记淀粉饲料组。说明草鱼对于葡萄糖作为能源的利用优于对淀粉作为能源的利用。     

Characterization of variation induced by low-energy N+ and cloning of differentially expressed cDNA of a mutant in Arabidopsis thaliana

Using Arabidopsis thaliana as experimental materials, the variations induced by low-energy N+ have been investigated. Germination rate of the treated seeds is lower than that of the control, and it decreases with the intensification of the radiation. The phenotypic variations have been observed in M2 plants irradiated with higher doses, such as chlorisis, semilethality, plant morphology, and changes of blooming habit and fertility. In random amplified polymorphic DNA (RAPD) analysis on M2 seedlings, some differences including band deletions or additions are found in treated plants compared to the control and the differences are associated with the radiation doses. One of the M1 plants from the seeds irradiated with the dose of 80×1015 N+/cm2 is a dwarf variant. Its stable M6 generation, mutant T80II, is used to construct subtractive cDNA library and to clone differentially expressed cDNA. A 721 bp cDNA fragment is partly homologous with GRF7 gene.     

全息图的转印及效率研究

本文叙述了全息图转印的原理和转印方式，同时介绍了转印所用材料和怎样提高转印效率．     

HCV不同基因型对慢性丙型肝炎抗病毒治疗疗效的影响

目的探讨干扰素联合利巴韦林治疗慢性丙型肝炎患者临床疗效的相关影响因素.方法对110例慢性丙型肝炎患者进行PEG-IFNα-2a 180μg/周联合RBV 800~1200 mg/d的标准治疗,并完成治疗后24周随访,同时分析HCV基因型、HCV-RNA定量对持续病毒学应答(SVR)的影响.应用罗氏试剂检测丙肝病毒定量,应用反转录-套式聚合酶链反应(PT-PCR)法检测HCV基因型.结果在110例进行标准联合治疗、并完成治疗后24周随访的慢性丙型肝炎患者中,68例患者获得SVR.其中,HCV基因1b型感染者SVR率为51.4%(37/72),HCV基因N-1b型感染者SVR率为81.5%(31/38);基线HCV-RNA水平1.0×10~5IU/m L的患者SVR率为71.2%(42/59),基线HCV-RNA水平≥1.0×10~5IU/m L的患者SVR率为50.9%(26/51).结论非1b基因型SVR率高于1b基因型;基线低病毒RNA水平(血清HCV-RNA水平1.0×10~5IU/m L)SVR率高于基线高病毒RNA水平(血清HCV-RNA水平≥1.0×10~5IU/m L).     

Specific immune responses induced by a multi-epitope antigen of hepatitis C virus in mice and rabbits

Five highly conserved and immunogenic epitopes of hepatitis C virus (HCV) have been chosen to form a multi-epitope antigen gene and fused with β-galactosidase gene to express a hybrid GZ-PCX antigen, which could be specifically recognized by human HCV sera. High level of anti-GZ-PCX IgG has been induced when mice or rabbits were immunized with GZ-PCX antigen emulsificated with complete Freund’s adjuvant or mixed with killed attenuatedSalmonella typhimurium SL3261. The specific anti-GZ-PCX IgG reached a high titer of 10^-6, which remained for several months. Specific cytotoxic T lymphocyte (CTL) effects, delayed type hypersensitivity reaction (DTH) and proliferation of peripheral lymphocytes have been induced by GZ-PCX antigen or synthetic peptides. High level of anti-GZ-PCX slgG has been detected in mice’s intestinal washing fluids, which indicates that the antigen induced mucosal immunity as well as systematic immunity. The studies show that the HCV multi-epitope antigen induces high level of specific immune responses without obvious toxicity, which might be able to provide protectivity to any HCV genotypes and isolates.     

中华绒鳌蟹卵巢发育相关的新基因EJO3的克隆和特征分析

运用了抑制性差减杂交和cDNA芯片技术鉴定中华绒鳌蟹卵巢发育相关基因,并用5'和3'RACE的方法克隆了一个与Ⅱ期相比在卵巢发育的Ⅲ期高表达的新基因EJO3(GenBank登录号:AY185919).EJO3基因的开放阅读框(ORF)长1 368 bp,编码455个氨基酸.从氨基酸序列推算的EJO3的等电点和相对分子质量分别为5.79和50 000.生物信息学分析表明,EJO3可能是一个含有VWD结构域的分泌蛋白.EJO3在Ⅱ期和Ⅲ期卵巢的差异表达用Northern blot进行了进一步验证.组织表达谱分析表明EJO3在卵巢高表达,在肠、心脏、肌肉和肝胰腺中弱表达或不表达.本研究结果为进一步深入研究中华绒鳌蟹卵巢发育的分子机制奠定了重要的前期工作基础.