海洋源芽孢杆菌的分离鉴定及其消化酶代谢产物的测定

【目的】从海洋生物和环境中分离鉴定出产消化酶代谢产物的芽孢杆菌,为微生态制剂的研制提供益生菌种。【方法】从雷州半岛近岸海域采集样品,用高温处理、富集培养和分离培养的方法获得分离菌株;通过菌落形态、细胞结构、染色特征、生化试验和16S rRNA基因测序对分离菌株进行鉴定;采用福林-酚比色法、淀粉-碘比色法和橄榄油乳化法测定分离菌株代谢产物中的蛋白酶、淀粉酶和脂肪酶活性。【结果】从22份样品中初步鉴定出疑似芽孢杆菌43株,筛选3种消化酶综合活性最强的8株,经生化试验和16S rRNA基因测序鉴定,有蜡样芽孢杆菌3株、暹罗芽孢杆菌3株、特基拉芽孢杆菌1株、解木糖赖氨酸芽孢杆菌1株。其中,解木糖赖氨酸芽孢杆菌BC1产脂肪酶活力最高,为52.13 U/mL;蜡样芽孢杆菌BC3产蛋白酶活力最高,为4.78U/mL;暹罗芽孢杆菌BC6产淀粉酶活力最高,为2.35 U/mL。【结论】成功筛选出产消化酶代谢产物的海洋源芽孢杆菌。     

cDNA cloning and characterization of the warm-temperature-acclimation-associated protein Wap65 from carp, Cyprinus carpio

We determined full-length cDNA of carp warm-temperature-acclimation-associated 65-kDa protein (Wap65). It encoded 439 amino acid residues with a signal peptide of 22 residues and showed an amino acid sequence identity of 88% to that of goldfish reported before (J. Biol. Chem. 1995. 270: 17087–17092). The number of potential N-linked glycosylation sites of carp Wap65 was two in contrast to three for goldfish. In addition, molecular mass determined by SDS-PAGE was apparently different from that of goldfish. These results suggest that the amount of oligosaccharide is different between the carp and goldfish protein. As in goldfish, carp Wap65 mRNA showed marked accumulation in hepatopancreas of the 30 °C- acclimated fish, which was 8-fold higher than that of the 10 °C-acclimated fish. Carp Wap65 showed 30% amino acid identity to mammalian hemopexins, which appeared to be considerably low in comparison with those among mammalian hemopexins (72 to 80%), or among carp Wap65 and rainbow trout hemopexin-like protein (70%). However, although mammalian hemopexins contain residues comprising the heme binding pocket, carp Wap65 lacked one of the two histidine residues to serve as heme axial ligands in hemopexins. Our data on carp protein substantiates the previous observation for goldfish and indicates that Wap65 might have some important functions in warm-temperature-acclimation of fish.     

2株猪流行性腹泻病毒的分离鉴定及ORF3基因的克隆与序列分析

为研究、防控广东地区猪流行性腹泻,首先通过RT-PCR法确定某猪场粪便样品中存在猪流行性腹泻病毒,随后利用Vero细胞进行盲传,最后经核酸类型鉴定、RT-PCR、病毒TCID50的测定、目的基因测序和序列分析等方法,确认分离到2株猪流行性腹泻病毒毒株,命名为PEDV GDKP-2013和PEDV GDKP2-2013,通过ORF3全基因核苷酸序列分析可知,GDKP-2013株与GDKP2-2013株与经典参考毒株的氨基酸序列一致性为 JX261936-CHGD-01,98.2%,98%;JQ039903-CH-GD-2011,98.6%,98.4%;AF353511-CV777,95.4%,95.1%;KC344845-STPED0810,95.1%,94.8%;EU054929-DR13,95.5%,95.2%;KF272920-USA-Colorado-2013,95.1%,94.8%,表明此2毒株与国内分离株的氨基酸同源性高,与韩国、泰国和美国分离株的氨基酸同源性要低一些,同时,与疫苗参考株的氨基酸同源性也相对较低;基因系统进化分析可知,GDKP-2013株与GDKP2-2013株主要处在Ⅰa分支,均与弱毒疫苗株（Ⅰb分支）相互之间遗传进化关系较远。     

Isolation,in vitro propagation,genetic analysis,and immunogenic characterization of an Ehrlichia canis strain from southeastern Brazil

Amplification of the 16S rRNA gene from a blood sample obtained from a dog in southeastern Brazil was used to confirm a naturally acquired Ehrlichia (E.) canis infection. Following isolation and culturing of the new bacterial strain called Uberlândia, partial sequences of the dsb and p28 genes were obtained. The dsb partial sequence of the novel strain was 100% similar to dsb gene sequences of E. canis obtained from different geographic areas around the world. Conversely, the p28 partial sequence for the E. canis Uberlândia strain differed at several nucleotides from other sequences available in GenBank. To confirm the antigenic profile of the Uberlândia strain, an indirect immunofluorescence assay against E. canis antigens was performed using dog sera collected from two different areas in Brazil (Uberlândia and São Paulo). The results suggest that both antigens were able to identify animals seropositive for E. canis in Brazil since these Brazilian strains appear to be highly conserved.     

沛县鸭传染性浆膜炎的病原分离鉴定及药敏试验

收集沛县具有典型鸭传染性浆膜炎症状的34份病死鸭进行细菌的分离培养。总共得到18株疑似鸭疫里默氏杆菌（RA）,从中随机选取10株RA进行生化鉴定及动物回归试验,经确认后使用本地区常见的抗菌药进行了纸片法药敏试验,结果发现RA分离菌株对头孢噻肟、氧氟沙星、阿莫西林＋棒酸等药物敏感度较好,对氟苯尼考、丁胺卡那、阿奇霉素等药物敏感度很低。研究结果为该地区鸭传染性浆膜炎的药物防治提供了很好的理论依据。     

Tanjungides A and B: New Antitumoral Bromoindole Derived Compounds from Diazona cf formosa. Isolation and Total Synthesis

Tanjungides A (1) (Z isomer) and B (2) (E isomer), two novel dibrominated indole enamides, have been isolated from the tunicate Diazona cf formosa. Their structures were determined by spectroscopic methods including HRMS, and extensive 1D and 2D NMR. The stereochemistry of the cyclised cystine present in both compounds was determined by Marfey’s analysis after chemical degradation and hydrolysis. We also report the first total synthesis of these compounds using methyl 1H-indole-3-carboxylate as starting material and a linear sequence of 11 chemical steps. Tanjungides A and B exhibit significant cytotoxicity against human tumor cell lines.     

Coupled magnetic nanoparticle-mediated isolation and single-cell image recognition to detect Bacillus' cell size in soil

Microbial morphology fundamentally constrains how species interact with their environment, and hence ultimately affects their niche. However, the methodology of functional microbes in the soil ecosystem is still poorly studied since it is difficult to capture and identify the active monospecific community from the complicated environment and enormous number of microbial species in soils. To comprehensively reveal the morphology of active microbes in soil ecosystem, magnetic nanoparticle-mediated isolation (MMI) and single-cell image recognition (SCIR) were employed to study soil active Bacillus community, which functionally boosted the soil fertility in organic fertilisation compared to mineral fertilisation and unfertilised control treatments in our previous study. The results showed that MMI and SCIR can efficiently isolate active Bacillus from soil particles and other microorganisms. High throughput sequencing showed that the captured Bacillus showed similar community structure in different long-term fertilisation soils, while SCIR revealed that the active Bacillus was greater in number and larger in size in organic fertilisation treatment compared to mineral fertilisation and unfertilised control treatments. Our study demonstrates that the combination of MMI and SCIR is a potentially powerful tool to capture and identify the morphology of active and functional microbes in the soil ecosystem.     

一株鸭瘟病毒的分离与鉴定

从湖北省武汉市某种鸭场送检的死亡鸭肝脏中分离到1株病毒,结合流行病学调查,剖检病理变化特征初步诊断为鸭瘟病毒感染。根据Gen Bank上公布的鸭瘟病毒UL6基因序列设计一对引物,提取病毒核酸DNA进行PCR扩增,得到大小约为416 bp的目的片段。测序结果表明与Gen Bank上公布的鸭瘟病毒UL6基因的序列相似性达到99%。鸭胚感染试验表明,感染的鸭胚在第四天开始死亡,并出现典型的鸭瘟病毒感染症状。上述结果表明,该种鸭场种鸭死亡是由鸭瘟病毒所引起的。     

洗涤厂排污口可培养中度嗜热菌的多样性

为考查洗涤厂热废水排放口土壤在常年人为热环境下的可培养嗜热菌的多样性,采集洗涤厂热废水排放口附近的湿热泥土,应用不同培养基分离纯化泥土中的可培养嗜热菌,并对获得的纯培养细菌进行16S rDNA鉴定和系统发育相关分析。结果表明：分离纯化得到的29株不同表型的纯培养菌株均属于坚壁菌门芽孢杆菌纲,归属于5个属11个种,分别是地芽孢杆菌属 Geobacillus 3个种,芽孢杆菌属 Bacillus 3个种,无氧芽孢杆菌属 Anoxybacillus 3个种,解脲芽孢杆菌属Ureibacillus 1个种和高温放线菌属 Thermoactinomyces 1个种,属种多样性丰富,菌株资源具有潜在开发应用价值。     

百色市动物饲料中沙门氏菌分离鉴定检测报告

随机抽查百色市2009—2012年468份饲料进行沙门氏菌的检测,通过预增菌、选择性增菌和分离培养鉴定,检出阳性样品7份,阳性率1.5%。其中猪浓缩饲料样品检出5份,鸡配合饲料样品检出1份,自配大鸡混合饲料检出1份。检测发现猪浓缩饲料检出的几率较高,鸭、鱼、兔配合料未检出阳性样品,饲料中沙门氏菌污染主要来源于添加的动物蛋白质原料,添加动物蛋白质原料比例越高,检出的几率越大。