对虾白斑综合症病毒一个基因的序列分析

对虾白斑综合症病毒)White Spot Syndrome Virus,WSSV)是一种无包含体、具囊膜、杆状的双链DNA病毒 ,其全基因组序列为 300kb左右 ,是目前已知的基因组最大的动物病毒之一1,2.该病毒的基因组结构非常特殊 ,与已知的病毒的基因组相差很远 ,拥有许多该病毒特有的基因 ,很可能为一种新的病毒科成员 ,其分类地位待于进一步研究。从已经登记到GenBank1的对虾白斑综合症病毒全序列知道 ,该病毒存在着至少两种不同的分离株 ,但基因组序列非常保守。本文报道该病毒的一个基因序列的分析结果以及三个不同的白斑综合症病毒分离株的同源基因片段序列的比较结果。       

对虾白斑综合征杆状病毒同源性比较的研究

比较我国沿海不同海域对虾白斑综合征杆状病毒三个分离株即唐海分离株（渤海湾）,宁波分离株（东海）,深圳分离株（南海）的同源性。三个WSSV分离株基因组的限制性内切酶(Sac I,Hind III,Pst I)酶切多态（RFLP）以及病毒结构蛋白图谱完全一致,证实造成我国从南至北对虾爆发性流行病的对虾白斑杆状病毒为同一种病毒。利用高保真Taq酶,分别以报道的日本对虾杆状病毒（RV-PJ＝PRDV）,斑节对虾白斑综合征杆状病毒（WSBV＝PmNOBIII）基因组核酸片段特异性引物进行PCR扩增,结果均能从中国对虾白斑杆状病毒（WSSV）基因组中扩增得到相应大小的PCR产物,扩增产物序列分析表明中国对虾白斑杆状病毒（WSSV）与斑节对虾白斑综合征杆状病毒（WSBV＝PmNOBIII）,日本对虾杆状病毒（RV－PJ＝PRDV）同源率分别为100％与97％,其结果为证实亚洲及太平洋地区对虾白斑综合征杆状病毒为同一种病毒或同一种病毒的不同株系提供了证据。     

对虾白斑综合征杆状病毒同源性比较的研究

比较我国沿海不同海域对虾白斑综合征杆状病毒三个分离株：即唐海分离株（渤海湾）、宁波分离株（东海）,深圳分离株（南海）的同源性。三个WSSV分离株基因组的限制笥内切酶（Sac Ⅰ,HindⅢ,PstⅠ）酶切多态（RFLP）以及病毒结构蛋白图谱完全一致,证实造成我国从南对北对虾爆发性流行病的对虾白斑杆状病毒为同一种病毒。利用高保真Taq酶,分别以报道的日本对虾杆状病毒（RV－PJ－PRDV）,斑节对虾白斑综合征杆状病毒（WSBV－PmNOBⅢ）基因组核酸片段特异性引物进行PCR扩增,结果均能从中国一杆状病毒（WSSV）基因组中扩增得到相应大小的PCR产物,扩增产物序列分析表明中国对虾白斑杆状病毒（WSSV）与斑节对虾白斑综合征杆状病毒（WSBV－PmNOBⅢ）,日本对虾相状RV-PJ=PRDV)同源率分别为100％与97％,其结果为证实亚洲及太平洋地区对虾白斑综合征杆状病毒为同一种病毒或同一种病毒的不同株系提供了依据。     

对虾白斑综合症病毒与虾鳃细胞膜特异性结合关系的确立

对虾白斑综合症病毒(White spot syndrome virus,WSSV)是养殖对虾的一个主要病原,也是目前发现的基因组最大的动物病毒(基因组约290kDa,双链环状)。WSSV病毒粒子为卵形杆状,外被囊膜,囊膜在尾部延伸成一长尾。它不仅能感染对虾,还能感染其它淡水及海水甲壳类。养殖对虾被感染后,3—10d内累积死亡率可达100％,给对虾养     

对虾白斑综合征病毒基因组快速定性定量方法的建立

对于对虾白斑综合征病毒(white spot syndrome virus,WSSV)全基因组样品,使用限制性内切酶酶切以及荧光绝对定量的方法进行分析,建立了WSSV全基因组快速定性定量的方法。定性实验通过对GenBank中WSSV基因组序列深度分析,选择确定合适的限制性内切酶BamHI对基因组进行酶切,通过比对实际酶切结果和软件模拟酶切结果,以定性待测样品。定量实验使用荧光定量试剂盒,通过建立标准曲线的方法对未知浓度的WSSV基因组样品进行绝对定量。实验结果表明,结合使用酶切分析和荧光定量的方法可以准确、快速、方便、经济地对WSSV基因组样品进行定性定量分析,为进一步深入研究WSSV基因组奠定坚实基础。     

从噬菌体展示抗体文库筛选对虾白斑综合征病毒的单链抗体

对虾白斑综合征是一种严重危害对虾养殖业的病毒性疾病.由于目前对其病原体对虾白斑综合征病毒（WSSV）的研究不够深入,所以对WSSV的有效防治仍然是一大难题.为此,用完整的对虾白斑综合征病毒粒子作为靶抗原固相包被,淘选噬菌体展示单链抗体文库,得到两个能够与WSSV结合的单链抗体：E2和H4.单链抗体H4能够结合病毒并抑制病毒对原代培养的对虾淋巴细胞的感染,这些结果表明此单链抗体具有开发为诊断试剂盒和抗病毒药物的潜力.     

WSSV囊膜蛋白VP37和VP39基因酵母双杂交诱饵载体的构建及其激活作用检测

对虾暴发性流行病是近十年来危害对虾养殖业发展的重要病害之一,其主要病原为对虾白斑综合症病毒（WSSV）^[1]。近年来对WSSV的研究主要集中在其囊膜蛋白、黏附蛋白等结构蛋白方面^[2]。本实验室经病毒结合分析^[3]和病毒铺覆蛋白印迹技术（Virus overlay protein blot assay,VOPBA）初步研究,已证实WSSV存在4种病毒黏附蛋白（VAP）,其中VAP1已确定为WSSV囊膜蛋白VP37^[4],该蛋白存在有特征性的细胞结合域（RGD）。编码的蛋白包含281个碱基,与Huang C,et al.^[5]报道的VP37一致,     

一种改良的对虾白斑综合征病毒的提纯技术

对虾白斑综合征病毒(White spot syndrome virus,WSSV)是对虾养殖业的主要病原,自1992年以来一直严重影响对虾的产量和质量,造成巨大的经济损失.     

对虾白斑综合症病毒与虾鳃细胞膜特异性结合关系的确立

对虾白斑综合症病素养(White spot syndrome virus,WSSV)是养殖对虾的一个主要病原,也是目前发现的基因组最大的动物病素养(基因组约290kDa,双链环状).     

注意杆状病毒科分类的变化

杆状病毒由于被广泛用作目的基因的表达载体倍受重视.近年来证实某些杆状病毒对虾具有致病性,从而成为研究热点之一.目前公认的最重要的对虾致病病毒是对虾白斑综合征病毒(white spot syndromviruS,WSSV),在我国养殖对虾中广为流行,引致重大经济损失.不少文献将WSSV称作对虾白斑病杆状病毒,其实并不准确.     

Conversion of the Salmonella phase 1 flagellin gene fliC to the phase 2 gene fljB on the Escherichia coli K-12 chromosome.

The Escherichia coli-Salmonella typhimurium-Salmonella abortus-equi hybrid strain EJ1420 has the two Salmonella flagellin genes fliC (antigenic determinant i) and fljB (determinant e,n,x) at the same loci as in the Salmonella strains and constitutively expresses the fliC gene because of mutations in the genes mediating phase variation. Selection for motility in semisolid medium containing anti-i flagellum serum yielded 11 motile mutants, which had the active fliC(e,n,x) and silent fljB(e,n,x) genes. Genetic analysis and Southern hybridization indicated that they had mutations only in the fliC gene, not in the fljB gene or the control elements for phase variation. Nucleotide sequence analysis of the fliC(e,n,x) genes from four representative mutants showed that the minimum 38% (565 bp) and maximum 68% (1,013 bp) sequences of the fliC(i) gene are replaced with the corresponding sequences of the fljB(e,n,x) gene. One of the conversion endpoints between the two genes lies somewhere in the 204-bp homologous sequence in the 5' constant region, and the other lies in the short homologous sequence of 6, 8, or 38 bp in the 3' constant region. The conversions include the whole central variable region of the fljB gene, resulting in fliC(e,n,x) genes with the same number of nucleotides (1,503 bp) as the fljB gene. We discuss the mechanisms for gene conversion between the two genes and also some intriguing aspects of flagellar antigenic specificities in various Salmonella serovars from the viewpoint of gene conversion.     

Analysis of the regions flanking the human insulin gene and sequence of an Alu family member.

The regions around the human insulin gene have been studied by heteroduplex, hybridization and sequence analysis. These studies indicated that there is a region of heterogeneous length located approximately 700 bp before the 5' end of the gene; and that the 19 kb of cloned DNA which includes the 1430 bp insulin gene as well as 5650 bp before and 11,500 bp after the gene is single copy sequence except for 500 bp located 6000 bp from the 3' end of the gene. This 500 bp segment contains a member of the Alu family of dispersed middle repetitive sequences as well as another less highly repeated homopolymeric segment. The sequence of this region was determined. This Alu repeat is bordered by 19 bp direct repeats and also contains an 83 bp sequence which is present twice. The regions flanking the human and rat I insulin genes were compared by heteroduplex analysis to localize homologous sequences in the flanking regions which could be involved in the regulation of insulin biosynthesis. The homology between the two genes is restricted to the region encoding preproinsulin and a short region of approximately 60 bp flanking the 5' side of the genes.     

Indel-II region deletion sizes in the white spot syndrome virus genome correlate with shrimp disease outbreaks in southern Vietnam

Sequence comparisons of the genomes of white spot syndrome virus (WSSV) strains have identified regions containing variable-length insertions/deletions (i.e. indels). Indel-I and Indel-II, positioned between open reading frames (ORFs) 14/15 and 23/24, respectively, are the largest and the most variable. Here we examined the nature of these 2 indel regions in 313 WSSV-infected Penaeus monodon shrimp collected between 2006 and 2009 from 76 aquaculture ponds in the Mekong Delta region of Vietnam. In the Indel-I region, 2 WSSV genotypes with deletions of either 5950 or 6031 bp in length compared with that of a reference strain from Thailand (WSSV-TH-96-II) were detected. In the Indel-II region, 4 WSSV genotypes with deletions of 8539, 10970, 11049 or 11866 bp in length compared with that of a reference strain from Taiwan (WSSV-TW) were detected, and the 8539 and 10970 bp genotypes predominated. Indel-II variants with longer deletions were found to correlate statistically with WSSV-diseased shrimp originating from more intensive farming systems. Like Indel-I lengths, Indel-II lengths also varied based on the Mekong Delta province from which farmed shrimp were collected.     

A molecular basis for discrete size variation in human ribosomal DNA.

The tandemly repeated human ribosomal RNA (rRNA) genes contain a region of size heterogeneity that is present in the nontranscribed spacer of every individual examined. This heterogeneity has been previously examined by Southern analysis of BamHI-digested human DNA. Using a ribosomal DNA (rDNA) probe specific for the 3' end of the 28S rRNA gene, at least four discrete sizes of BamHI fragments were seen in human populations. Molecular analysis of the cloned DNA from this region reveals tandem duplication of a segment of spacer rDNA located 388 base pairs (bp) 3' to the end of the 28S ribosomal RNA gene. Five hundred fifty bp of DNA, flanked on either side by a 150-bp repeated element, is either duplicated or deleted to produce a series of spacers that differ in size by 850 bp. These duplications/deletions appear to be the product of unequal homologous exchange, mediated by the small repeated element. Thus, human rDNA fragments cloned in lambda vectors and propagated in E. coli generate the same apparent size variation seen in genomic DNA. This study suggests that unequal homologous exchange is the molecular basis for the observed length heterogeneity in the spacer rDNA and may be a common mechanism for the generation of human genetic diversity.     

Nucleotide sequences of heat shock activated genes in Drosophila melanogaster. I. Sequences in the regions of the 5' and 3' ends of the hsp 70 gene in the hybrid plasmid 56H8.

We present the sequences at the 5' and 3' ends of one hsp 70 gene variant which is derived from the chromosomal locus 87A7. The 5' end of the hsp 70 mRNA has also been determined. 550 bp upstream from the 5' end of the hsp 70 mRNA, there is a very A+T rich region shown by heteroduplex analysis to be also present at the same position in other hsp 70 genes9. The 5' end of the hsp 70 mRNA was found 26 bp after a characteristic "Hogness box". The first ATG codon was found 250 bp downstream from the 5' end of the hsp 70 mRNA. We also determined the termination codon at the 3' end of the hsp 70 gene. Comparisons with other genes are discussed.     

The histidine tRNA genes of yeast

Yeast has at least seven nuclear histidine tRNA genes although there is a single tRNAHis. We have sequenced three of the histidine tRNA genes. The genes have identical coding sequences and the DNA anti-codon sequence GTG corresponds to the GUG anti-codon in tRNAHis. None of the three yeast histidine tRNA genes has an intervening sequence. Two of the three genes contain repeated DNA elements in the region adjacent to the 5' end of the histidine tRNA gene. One of the elements, sigma, is 18 base pairs (bp) from the 5' end of each of these genes, sigma elements are highly conserved and flanked by 5-bp repeats. The other element, delta, is at variable distances from the tRNA gene; one is 439 bp from a histidine tRNA gene and the other is 52 bp from a histidine tRNA gene. These solo delta elements are quite divergent when compared with delta s associated with transposon yeast elements and are not flanked by 5-bp repeats.     

Direct sequencing of deleted mitochondrial DNA in myopathic patients

To investigate the mechanism of mitochondrial DNA deletion in human diseases, we amplified the deleted mitochondrial DNA of five patients with mitochondrial myopathy by using the polymerase chain reaction, and directly sequenced the crossover regions of the deleted mitochondrial DNA without cloning. In Patient 1, a 7-bp directly repeated sequence of 5'-ATCCCCA-3' was found at the boundaries of deleted segment spanning 7,039 bp between the ATPase 6 and the cytochrome b genes. In Patients 2, 3, and 4, a 13-bp sequence of 5'-ACCTCCCTCACCA-3' was found in the boundaries of deleted segment spanning 4,977 bp between the ATPase 8 and the ND5 genes. In Patient 5, a 3-bp sequence of 5'-CCT-3' was found in the boundaries of deleted segment spanning 3,717 bp between the ATPase 6 and the ND5 genes. Similar directly repeated sequences may contribute to mitochondrial DNA deletions in human degenerative diseases.     

Intergenic spacer (IGS) polymorphism: a new genetic marker for differentiation of Toxoplasma gondii strains and Neospora caninum

The region between the 28S and 18S rRNA genes, including the intergenic spacer (IGS) region and the 5S rRNA gene, from 32 strains of Toxoplasma gondii and the NC1 strain of Neospora caninum was amplified and used for DNA sequencing and/or restriction fragment length polymorphism (RFLP) analysis. The 5S rDNA sequences from 20 strains of T. gondii were identical. The IGS region between the 5S and 18S rRNA genes (nontranscribed spacer 2 or NTS 2) showed 10 nucleotide variations. Six of the 10 variant positions correlated with the murine virulence of the strains. Intraspecific polymorphisms distinguished the virulent strains of zymodemes 5, 6, and 8 from other virulent strains (in zymodeme 1). RFLP methods (IGS-RFLP) were developed and used to characterize the virulent and avirulent patterns among 29 T. gondii strains. Sequence diversity of 19.8% was found between T. gondii and N. caninum when comparing a region of 919 bp at the 3' end of NTS 2. The sequence variation in ribosomal IGS could therefore be a useful marker for Toxoplasma strain identification and for distinguishing N. caninum from T. gondii.