真核表达NDV F基因重组质粒的构建及其在CEF细胞中的转染

通过PCR方法自含NDVZF基因的克隆质粒中扩增NDVF基因 ,将其与真核表达载体pcDNA3..1/V5_His_TOPO重组。经核酸内切酶酶切 ,阳性克隆鉴定准确无误 ,核酸序列测定结果与原始基因比较 ,同源性大于 99% ,启始密码和终止密码未出现变异 ,将该重组质粒命名为pcDNANDVZF。将pcDNANDVZF在脂质体作用下转染CEF细胞 ,用间接免疫荧光试验检测 ,结果证明pcDNANDVZF可在CEF细胞中大量表达F蛋白。     

真核表达载体构建表达ＰＲＶ闽Ａ株gE基因表位抗原编码区片段的重组质粒

根据伪狂犬病病毒闽Ａ株gE基因表位抗原编码区的序列与身份种真核表达载体pPICZaA、pAcGP67A序列与特性分别设计了两对ＰＣＲ引物。通过ＰＣＲ方法扩增到了两端具有不同酶切位点的gE基因表位抗原编码片段。将这２个片段分别克隆到pPICZaA与pAcGP67A载体，转化大肠杆菌ＴＯＰ１０菌档及ＸＬ１－Ｂlue菌株，获得了含伪狂犬病病毒闽Ａ株gE基因表位抗原编码区的重组质粒pICZaA-FS与pAcGP67A-FS。序测定结果显示两个重组质粒中插入片段的大小与方向均正确。     

Chromosomal and Plasmid Diversity of Agrobacterium Strains Isolated from Ficus benjamina Tumors

Agrobacteria were previously isolated from tumors developing on branches and aerial and hypogeous roots of weeping fig plants in Italy and in The Netherlands. A representative group of 48 strains was analyzed by PCR–RFLP of 16S and 16S + IGS ribosomal regions, PCR–RFLP of six Ti plasmid (pTi) regions and characterized for plasmid content. Two groups of agrobacteria were separated by cluster analysis of PCR–RFLP profiles of rrs gene: seventeen strains were similar to the new species Agrobacterium larrymoorei, while the remaining strains were included within the agrobacterium biovar 1 group. Sixteen different plasmid profiles from one to five plasmids were observed. In addition, 21 ribotypes and 20 pTi structures were arranged in many different combinations, showing that fig agrobacteria were characterized by a wide heterogeneity. A general lack of correlation between strain ribotypes and plasmid content was observed.     

基于CRISPR序列的肠道沙门氏菌分离株CSST分型

为验证一种新的CRISPR分型方法——CSST分型方法分型沙门氏菌的可行性,分别采用传统CRISPR与CSST两种分型方法,对20株临床肠道沙门氏菌分离株进行分型.结果显示:CSST分型方法同传统CRISPR分型方法分型沙门氏菌的结果完全一致,均将20株临床肠道沙门氏菌菌株分成了15种亚型,其中CR4/CT4和CR9/...     

Campylobacter spp. prevalence and fluoroquinolone resistance in chicken layer farms

Chicken is a major source of human campylobacteriosis. Chicken meat originates not only from broilers but also from spent layers; however, few reports have documented the prevalence and antimicrobial resistance of Campylobacter spp. in layers in Japan. Therefore, we investigated the prevalence and antimicrobial susceptibility of Campylobacter spp. in 47 layer farms in Japan. Fecal samples were collected from the youngest and oldest flocks on the farm, and Campylobacter spp. was isolated from 46/47 (97.9%) farms. Among the C. jejuni isolates, the resistance rates to ampicillin, tetracycline, and ciprofloxacin were 29.6%, 22.2%, and 19.8%, respectively. The ciprofloxacin resistance rate (7.3%) in C. jejuni isolated from old flocks was significantly (P<0.01) lower than that in young flocks (32.5%).     

人雄激素受体的雄激素结合区cDNA在E.coli中的高效表达

将编码人雄激素受体（ｈＡＲ）的雄素结合区（ＬＢＤ）的ｃＤＮＡ片段（１００５ｂｐ）克隆到由Ｐ１启动子控制的硫氧还蛋白表达载体ｐＴｒｘＦｕｓ上,构建了表达质粒ｐＴｒｘＡＲ,并转化到大肠杆菌Ｇ１７２４中,经色氨酸诱导表达后,ＳＤＳ－ＰＡＧＥ分析,可观察到一高效表达的融合蛋白产物,此融合蛋白 分子了量与理论值相吻合,氨基酸组分分析证明了ＬＢＤ目的基因的表达。     

Comparison of the Value of Pulsed-field Gel Electrophoresis,Random Amplified Polymorphic DNA and Amplified rDNA Restriction Analysis for Subtyping Taylorella equigenitalis

Eight strains of Taylorella equigenitalis were identified by a polymerase chain reaction using a primer pair specific to the 16S rDNA of T equigenitalis. These eight strains were chosen because they had previously been shown to represent eight distinct genotypes by pulsed-field gel electrophoresis analysis after separate digestion of the genomic DNA with ApaI or NotI. The eight strains could be classified into six or seven types by random amplified polymorphic DNA analysis using different kinds of primers. Amplified rDNA restriction analysis after separate digestion with five restriction enzymes, including AluI and MboI, of the 1,500 bp fragments of rDNA amplified by polymerase chain reaction did not discriminate the genomic variations among the eight strains of T equigenitalis. Thus, pulsed-field gel electrophoresis was shown to discriminate these eight organisms better than random amplified polymorphic DNA analysis, while amplified rDNA restriction analysis was found to be unsuitable for subtyping T equigenitalis.     

Identification and molecular characterization of border disease virus (BDV) from sheep in India

Pestiviruses isolated from sheep and goats in India thus far have been bovine viral diarrhoea virus 1 (BVDV-1) or BVDV-2. During routine genetic typing of pestiviruses in the years 2009-10, border disease virus (BDV) was detected in eight Indian sheep of a flock showing clinical signs of BD by real time RT-PCR. All the samples yielded positive virus isolates in cell culture but were found negative by a BVDV antigen ELISA. A representative BDV isolate was characterized at genetic and antigenic level. Phylogenetic analysis carried out in 5′-UTR, N^pro and E2 regions of genome typed the Indian BDV isolate as BDV-3. A more detailed analysis in N^pro and entire region coding structural proteins showed that the N^pro (168), C (100 aa), E^rns (227 aa), E1 (195 aa) and E2 (373 aa) proteins were of size characteristic for BDV reference strain X818. Antigenic differences were evident between the BDV-3 isolate and previously reported BDV-1, BDV-5 and BDV-7 strains. Although origin of BDV-3 in India is not clear, the results reflect probable introduction through trade in sheep between India and other countries or BDV-3 may be more widely distributed. Additionally, this study suggests that for diagnosis of BDV infection, the commercial BVDV Ag-ELISA should be used with caution. This is the first identification of BDV in sheep in India which highlights the need for continued pestivirus surveillance and assessing its impact on sheep and goat production.     

基于SNP标记的爆裂玉米农家品种遗传多样性

为了评价广西爆裂玉米农家品种的遗传多样性,利用56K SNP芯片技术对中国广西45个爆裂玉米农家品种、中国2个爆裂玉米杂交种和6个南美爆裂玉米种质进行全基因组扫描,获得多态性标记14 338个,基因分型为6 种类型。其中A/G类型最多,为5 805个;A/T类型最少,为149个。1号染色体的多态性SNP位点最多,为2 302个;10号染色体最少,为848个。45个农家品种间平均遗传相似系数为0.62,变幅为0.41~0.99,总体遗传相似度高。聚类分析将参试品种划分为三大类群,相同来源的农家品种大多聚在一起;主成分分析显示大部分农家品种聚在一起,与杂交品种和南美品种之间没有交集,表明农家品种遗传相似性较高,与杂交品种和南美品种遗传差异较大。