Stepwise prediction and statistical screening: B-cell epitopes on neuraminidase of human avian H5N1 virus

The B-cell epitopes of virus are associated with the antiviral drug and the vaccine screening. As the nucleotide sequences of neuraminidase （NA） of stain GD-01-06 were sequenced, we predicted the a-helix and β-fold structure and the indexes of the flexible regions of secondary structure of NA with methods of the Hydrophilicity plot by Kyte-Doolittle, the Surface probability plot by Emini and the An- tigenic index by Jameson-Wolf, and then screened statistically the parameters to predict B-cell epi- topes by the Hierarchical cluster and the Bivariate correlation and the quartiles with SPSS 13.0. The impact of variation of amino acids in NA on its epitopes was analyzed. The predictive results were evaluated by Wu＇s Antigenic Index and SWISS-MODEL. We found that the most possible epitopes on NA were located within or nearby its N-terminal Nos. 120--137, 81--84, 408--415, 273--282, 429--432, 356--368, 46--55, 146--155, 341 --350 and 198--209, which were the dominant regions of NA epitopes. Peptide 120--137 including the glycoprotein domain （NGT126 128） was first chosen as the B-cell epitopes on NA. NA in H5N1 strain isolated after 2003 lacked in No. 53 amino acid （I）, resulting in an increase in the surface flexible region of NA in GD-01-06 and an enlargement to their epitope regions （VEP48-48→ VEPISNTNFL46-55）. Conclusively, prediction of the B-cell epitopes on the NA based on multiple pa- rameters is useful for researches on the molecular immunology and drug screening and immuno-prophylaxis. A deletion of No. 53 amino acid （I） in NA in strain GD-01-06 might increase its anti- genicity.     

Delayed resistance to Cucumber mosaic virus mediated by 3’UTR-derived hairpin RNA

RNA silencing has been shown to function in the plant antivirus defense response, leading to viral RNA degradation induced by vsiRNA-containing RISC cleavage activity. Cucumber mosaic virus （CMV） 3′UTR sequences share a high conservation of nucleotide sequence and secondary structures that are important for CMV replication. Here, in an attempt to simultaneously target the multiple genomic and subgenomic RNAs of CMV for degradation, CMV 3′UTR were used to design hairpin RNA （hpRNA） to transform tobacco （Xanthi. nc） so as to constitutively produce viral siRNAs. Most of the transgenic plants expressing CMV Q strain （Q-CMV, subgroup Ⅱ strain） RNA3 3′UTR-derived hpRNA showed delayed resistance to Q-CMV infection and exhibited recovery phenotypes. Compared with Q-CMV-inoculated leaves, the upper leaves showed weak or no disease symptoms and a reduced accumulation level of viral RNAs. Together with transient assays, our results indicate that the 3′UTR-derived siRNAs were biologically active in targeting viral RNA for degradation. Recovery resistance in transgenic plants was also observed against subgroup IB strain SD-CMV infection, indicating a broad-spectrum anti-CMV effect of the 3′UTR-based antiviral silencing. Northern blot assays indicated that there was no strong correlation between the degree of resistance and the accumulation level of 3′UTR-derived siRNAs, suggesting that to target a highly structured RNA, such as the CMV 3′UTR, the quantity of siRNAs may not be the only determinant of silencing efficiency. Target RNA secondary structures may also affect target accessibility, siRNA-containing RISC-target recognition and the consequent antiviral effect.     

禽流感病毒型及亚型特异性免疫酶染色技术的研究

 在获得禽流感病毒型及亚型特异性单克隆抗体的基础上,研究建立型及亚型特异性免疫酶染色技术,用于检测或鉴定禽流感病毒．优化反应条件,确定单克隆抗体及辣根过氧化物酶标记二抗的最佳工作浓度,进行敏感性、特异性、重复性及稳定性分析,并与病毒分离鉴定比较．结果表明该方法敏感、特异,具有良好的重复性和稳定性,可用于检测临床组织样品、鸡胚及细胞培养物中的禽流感病毒．     

禽流感病毒H9亚型特异性抗原捕捉ELISA检测方法的研究

 采用禽流感病毒多克隆抗体及H9亚型特异性单克隆抗体,研究建立H9亚型特异性抗原捕捉ELISA检测方法,用于检测H9亚型禽流感病毒.优化了反应条件,确定了包被抗体、检测抗体及酶结合物的最佳工作浓度,对该方法的敏感性、特异性、重复性及稳定性分析,并与RT-PCR方法比较.通过使用该方法对野外样品进行检测.结果表明该方法敏感、特异,具有良好的重复性和稳定性,可用于检测临床样品、鸡胚培养物及细胞培养物中的H9亚型禽流感病毒.     

重组慢病毒对不同哺乳动物细胞基因转移及表达效率的研究

慢病毒是一种具有独特优点和巨大应用潜力的哺乳动物细胞基因转移载体,我们对慢病毒载体对不同哺乳动物细胞的基因转移及表达效率进行了平行比较研究.应用第三代重组慢病毒系统构建了携带CMV启动子-EGFP报告基因表达元件的重组慢病毒Lenti-EGFP,分别对多种不同哺乳动物细胞进行转导实验,在转导48 h后应用流式细胞仪检测报告基因在不同细胞株中的转移及表达效率.我们共使用了29种哺乳动物细胞株,包括14种人类组织细胞,5种猴组织细胞,9种鼠组织细胞,1种兔组织细胞.结果显示,重组慢病毒具有良好的基因转移能力,可有效进入多数哺乳动物细胞,对不同种属来源的细胞没有表现出特别的偏嗜性,但对贴壁培养细胞的基因转移效率明显高于对悬浮培养细胞.本研究为重组慢病毒系统的合理使用提供了基础.     

植物病毒的反RNA干扰机制

RNA干扰(RNAi)是个古老而保守的机制,其作用如同“基因组的免疫系统”,广泛存在于真核生物真菌、植物、动物中,是天然的抵御病毒侵袭的系统.植物病毒在和植物长期而复杂的协同进化中,为了生存繁殖,提高自身复制、运动、感染寄主的能力,也演化出了对付宿主沉默反应的机制,即反干扰机制.目前的研究表明,病毒编码沉默抑制子是反干扰机制的最主要方式.本文主要就病毒编码的沉默抑制子作用特点和功能进行了概述.并介绍了缺损干扰RNA及卫星RNA的特殊复杂二级结构、快速复制与运动躲避沉默信号等反干扰机制,以及探讨了病毒反干扰的研究应用前景.     

HIV-1病毒样颗粒真核表达系统的建立

目的 利用哺乳动物细胞表达HIV-1病毒样颗粒,为HIV-1病毒的生物学研究和进一步设计HIV-1中和优势表位预防性疫苗奠定基础.方法 以293T细胞作为宿主细胞,利用磷酸钙沉淀法将带有HIV-1病毒被膜蛋白gp160基因的pcDNA3.1/BaL gp160真核表达载体和带有HIV-1核心蛋白(gag)的pVRC3900真核表达载体共转染至293T细胞中,收集培养上清,经蔗糖不连续梯度离心,收集病毒样颗粒,经磷钨酸负染,透射电镜观察结果 .结果 以磷酸钙沉淀DNA转移技术,EGFP真核表达质粒为靶基因,293T细胞为宿主细胞,最佳转染效率达30%以上;将peDt NA3.1/BaL gp160(env)和pVRC3900(gag)真核表达质粒共转染,宿主细胞成功表达目的 蛋白,转染细胞培养上清经蔗糖密度梯度离心,电镜观察可见自行装配的HIV病毒样颗粒.结论 在真核细胞中表达HIV-1病毒样颗粒.     

慢病毒及其载体系统研究进展

基于慢病毒的病毒载体的研究已经发展到一定水平,因此用慢病毒作为基因转移媒介的临床研究已经开始,在实验室和临床应用中,慢病毒具有特别的优点,尤其是能整合未分裂细胞的独特能力.现在基于HIV的慢病毒载体的临床研究已经开始应用于人体.本文介绍基于HIV-1的慢病毒和慢病毒载体的结构,慢病毒载体的优点以及慢病毒载体应用的展望.     

Characterization of codon usage bias in the dUTPase gene of duck enteritis virus

A comparative analysis of the codon usage bias in the newly discovered dUTPase gene （Assigned Accession No.： DQ486149） of the duck enteritis virus （DEV） and the dUTPase gene of 32 reference herpesviruses was performed. The results indicated that the DEV dUTPase gene encodes a protein of 477 amino acids, which includes five conserved motifs with a 3-1-2-4-5 arrangement. The codon adaptation index （CAI）, effective number of codons （ENC）, and GC3s values indicated synonymous codon usage bias in the dUTPase gene of herpesviruses, and this synonymous bias was correlated with host evolution. The codon usage patterns of the DEV dUTPase gene were phylogenetically conserved and similar to that of the dUTPase genes of the avian alphaherpesvirus. Although codon usage in each microorganism was different, there were no strain-specific differences among them. Sixty-one codons in the predicted polypeptide, with a strong bias towards A and T at the third codon position, were used. Comparison of the codon usage in the dUTPase gene of different organisms revealed that there were 19 codons showing distinct codon usage differences between the DEV and Escherichia coli dUTPase genes; 16 between the DEV and yeast dUTPase genes; and 15 between the DEV and human dUTPase genes. Analysis of variance （ANOVA） showed significant differences between the DEV and yeast dUTPase genes （r = 0.536, P 〈 0.01）. The extent of codon usage bias in the DEV dUTPase gene was highly correlated with the gene expression level, therefore the results may provide useful information for gene classification and functional studies.     

苹果树腐烂病防治状况及植物性防治剂

苹果树腐烂的防治主要依靠福美胂等高毒农药,长期使用会造成对环境和果品的砷污染.苹果树腐烂病植物性防治剂为一种新型的高效、低毒、低残留、与环境相协调的新型植物性农药.具有显著的治疗和预防效果.该制剂加工简单,使用方便,为生产绿色食品及有机食品提供了保障.