转鲑鱼降钙素基因酵母及RANKL对大鼠血管平滑肌细胞体外钙化的影响

观察核因子kB受体激活物配体(RANKL)对大鼠血管平滑肌细胞体外钙化的调节作用,以及转鲑鱼降钙素基因酵母对RANKL调节作用的影响.体外培养大鼠血管平滑肌细胞,建立体外血管平滑肌细胞钙化模型,在培养介质中分别加入RANKL及转鲑鱼降钙素基因酵母蛋白上清液,观察细胞钙沉积、细胞碱性磷酸酶活性及骨钙素的变化情况.结果表明,RANKL促进钙化培养的大鼠血管平滑肌细胞的钙沉积、细胞碱性磷酸酶活性及骨钙素的表达,而转鲑鱼降钙素基因酵母能够抑制RANKL的促钙化作用.RANKL可能通过增加细胞碱性磷酸酶活性及骨钙素的表达从而促进血管平滑肌细胞向成骨细胞转化;转鲑鱼降钙素基因酵母可能通过OPG/RANKL/RANK途径抑制血管平滑肌细胞钙化.     

Occurrence and Diversity of Pichia spp. in Marine Environments

A total of 328 yeast strains from seawater, sediments, mud of saltems, the guts of marine fish and marine algae were obtained. The results of routine identification and molecular methods show that five yeast strains obtained in this study belonged to Pichia spp., including Pichia guilliermondii luv-small, Pichia ohmeri YF04d, Pichiafermentans YF12b, Pichia burtonii YF11A and Pichia anomala YF07b. Further studies revealed that Pichia anomala YF07b could produce killer toxin against pathogenic yeasts in crabs while Pichia guilliermondii luv-small could produce high activity of extracellular inulinase. It is advisable to test if Pichia ohmeri YF04d obtained in this study is related to central-venous-catheter-associated infection.     

凡纳滨对虾翻译控制肿瘤蛋白Fortilin的毕赤酵母表达及其对血细胞免疫反应的影响

翻译控制肿瘤蛋白Fortilin是1种多功能蛋白,参与重要的细胞活动.并且,对虾fortilin通过抑制病毒复制干扰病毒感染.参照Genbank中fortilin基因序列设计引物,PCR扩增得到凡纳滨对虾fortilin目的基因片段,EcoR I/XbaI双酶切插入表达载体pGAPZαA,转化大肠杆菌,经博来霉素(Zeocin)抗性筛选及测序分析,获得分泌型重组酵母表达载体pGAPZαA-F.酶切线性化后,经电穿孔法转入毕赤酵母细胞X-33,经Zeocin抗性筛选,得到阳性转化子.表达产物的上清液经SDS- PAGE电泳和质谱分析,表明在酵母中成功表达fortilin.以体外原代培养的对虾血细胞检测重组蛋白的免疫活性,结果显示重组蛋白显著提高了血细胞酚氧化酶和超氧化物歧化酶活性.该结果为下一步研究重组fortilin 在对虾养殖中的应用奠定了基础.     

鉴定病毒受体的研究方法

研究病毒受体对了解病毒感染机制、病毒病的预防与治疗、病毒分类等都有重要意义。本文重点就酵母双杂交系统以及病毒蛋白铺覆技术、噬菌体展示、生物芯片、荧光共振能量转移等可用来进行病毒受体鉴定研究的几种方法做了介绍。     

Isolation and identification of a marine killer yeast strain YF07b and cloning of the gene encoding killer toxin from the yeast

It was found that the marine yeast strain YF07b could secrete a large amount of killer toxin against a pathogenic yeast strain WCY which could cause milky disease in Portunus trituberculatus.The marine yeast strain YF07b was identified to be Pichia anomala according to the results of routine yeast identification and 18S rDNA and ITS sequences.The gene encoding killer toxin in the marine yeast strain YF07b was amplified by PCR technology.After sequencing,the results show that an open reading frame,consisting of 1 281 bp,encoded a presumed protein of 427 amino acids.The sequence of the cloned gene was found to have 99% match with that of the gene encoding killer toxin in Pichia anomalas strain K.A signal peptide including 17 amino acids appeared in the N-terminal domain of the killer toxin.Therefore,the mature protein consisted of 410 amino acids,its molecular mass was estimated to be 47.4 ku and its isoelctronic point was 4.5.     

海洋红酵母不同添加剂量对凡纳滨对虾生长、免疫酶活性及抗病力的影响

研究了海洋红酵母(菌株编号：RM)不同添加浓度对凡纳滨对虾幼虾的生长性能、免疫酶活性及对副溶血弧菌抵抗力的影响。研究分别设置了4个海洋红酵母添加浓度(1.0×10⁸、1.0×10⁹、1.0×10¹⁰和1.0×10¹¹ cfu/kg)和1个无海洋红酵母添加的对照组,每个处理分别设置了5个重复,进行了42 d的养殖实验。养殖实验结束后,以副溶血弧菌进行病原攻毒实验,周期为14 d。结果表明：各实验组对虾的养殖总体成活率均大于90%,与对照组相比,未见显著性差异(P>0.05);除海洋红酵母1.0×10⁸ cfu/kg处理组外,其它各处理均显著提高了凡纳滨对虾的末体重(P<0.05),其中,1.0×10⁹和1.0×10¹⁰ cfu/kg添加组的对虾特定生长率及饲料转化效率均显著高于对照组。海洋红酵母不同浓度添加量对对虾血清非特异性免疫酶活性影响差异较大,1.0×10⁹ cfu/kg添加水平下,对虾血清中超氧...     

Screen and effect analysis of immunostimulants for sea cucumber,Apostichopus japonicus

Immunostimulants may improve disease resistance of aquaculture animals by promoting the nonspecific immunity response of the organisms. Five types of saccharides, including chitosan, yeast polysaccharide, burdock oligosaccharide, seaweed polysaccharide and lentinus edodes polysaccharide, were screened for potential use as immunostimulants by using spectrophotometry. The saccharides were injected into Apostichopus japonicus, a sea cucumber, and the lysozyme and superoxide dismutase (SOD) activities of the coelomic fluid and epidermal slime were monitored in six consecutive days. The results show that the lysozyme activity of the animal's coelomic fluid was significantly stimulated on day 2, day 4 and day 6 after the injection of the saccharides (P<0.05). The effects of chitosan and yeast polysaccharide were the most notable. The lysozyme activity of the epidermal slime was significantly increased by chitosana, yeast polysaccharide, seaweed polysaccharide, and burdock oligosaccharide on day 1 and day 2 after the injection (P<0.05). The SOD activity of the coelomic fluid was significantly promoted by the saccharides on day 2 and day 4 post-injection (P<0.05), while the SOD activity of the epidermal slime increased on day 2. These findings indicate that chitosan and yeast polysaccharide are the most effective immunostimulants and potential healthy anti-disease feedstuff for A. japonicus.     

南极酵母Rhodotorula sp.NJ298的鉴定及其产虾青素的研究

本研究对产虾青素的南极菌株NJ298进行了鉴定,同时对影响虾青素产量的主要环境因子进行了测试。形态特征、生理生化特征及ITS序列的系统发育分析表明,南极菌株NJ298属于红酵母属(Rhodotorula);虾青素积累主要在NJ298生长的对数期和稳定期;乙酸钠、蛋白胨是合成虾青素的最佳碳、氮源;在0-15℃之间,南极酵母NJ298均可以生长,但是低温更有利于虾青素的积累;pH值在6-8范围内NJ298均可生长,最适pH值为7.5;该菌对盐度要求不严格。     

谷胱甘肽酵母水解物对干露胁迫下凡纳滨对虾肝胰腺糖代谢相关基因的影响

【目的】探究富含谷胱甘肽的酵母水解物对干露胁迫下的凡纳滨对虾（Litopenaeus vannamei）肝胰腺糖代谢相关基因的影响,为谷胱甘肽酵母水解物在水产养殖中的应用提供参考。【方法】在基础饲料（鱼粉质量分数17.7%）中分别按质量分数0%、0.3%、0.5%、0.7%、0.9%、1.1%的比例添加谷胱甘肽酵母水解物,配制等氮等脂饲料6组（分别记为G0、G0.3、G0.5、G0.7、G0.9、G1.1）,饲喂初始体质量（0.24±0.01）g凡纳滨对虾4周,测定对虾生长性能指标;对对虾进行干露胁迫30 min,测定对虾肝胰腺相关基因的相对表达。【结果】各处理组对虾增重率、特定生长率和饲料系数无显著差异（P> 0.05）。干露胁迫后,G0组凡纳滨对虾肝胰腺缺氧诱导因子（HIF-1α）、果糖磷酸激酶（PFK）和乳酸脱氢酶（LDH）基因的表达显著上调（P <0.05）,琥珀酸脱氢酶（SDH）、苹果酸脱氢酶（MDH）和细胞色素氧化酶（CCO）基因显著下调（P <0.05）,葡萄糖转运蛋白1(GLUT1)、己糖激酶（HK）和丙酮酸激酶（PK）基因的表达无显著变化（P>...     

草鱼呼肠孤病毒vp5基因诱饵重组载体的构建、转化与自激活作用检测

根据草鱼呼肠孤病毒(Grass Carp Reovirus,GCRV)的vp5基因设计特异性引物,并扩增其开放阅读框序列(open reading frame,ORF),然后连接至pGBKT7载体上构建酵母双杂交诱饵载体pGBKT7-vp5,转化至酵母菌Y2HGold,并进行PCR鉴定以及自激活性和毒性检测。PCR、酶切以及测序表明,pGBKT7-vp5重组诱饵载体构建成功。自激活性和毒性检测显示,阳性重组菌株pGBKT7-vp5/Y2HGold和阴性对照菌株pGBKT7/Y2HGold在SD/-Trp平板上出现大小相一致的乳白色菌落,且在SD/-Trp/X-α-Gel、SD/-Trp/AbA+/X-α-Gel平板上生长出蓝色菌落,而在SD/-Trp/-Ade/-His无菌落出现。表明重组菌株pGBKT7-vp5/Y2HGold表达的融合蛋白激活了报告基因AUR1-C和MEL1,没有激活报告基因ADE2和HIS3并且对宿主菌无毒性。该重组诱饵载体可用于酵母双杂交系统进一步筛选cDNA文库中与其相互作用的蛋白。