维生素A对视黄醇结合蛋白基因表达效果初探

采用半定量RT-PCR技术对视黄醇结合蛋白(RBP)基因的表达进行研究.在猪的成纤维细胞内添加浓度为0.5、5、10、30μg/μL维生素A,分别作用12、24、48、72 h后,比较分析RBP的表达情况.结果表明,添加10μg/μL的维生素A作用72 h后,RBP的表达量下降了92.6%;添加5μg/μL的维生素A作用72 h后,RBP的表达量最高,为对照组的1.7倍.适量的维生素A可以诱导RBP表达,但过量的维生素A则会抑制RBP表达.     

Purification and characterisation of proteins secreted by the entomopathogenic fungus Metarhizium anisopliae with insecticidal activity against adults of the Mediterranean fruit fly,Ceratitis capitata (Diptera: Tephritidae)

BACKGROUND: The control of the Mediterranean fruit fly (medfly) Ceratitis capitata (Wied) is usually performed with protein bait sprays incorporating chemical insecticides that may have adverse effects on humans, non‐target organisms and the environment. In recent years, scientists have sought more environmentally friendly insecticides for medfly control, such as plant‐ and microorganism‐derived compounds. Among these compounds, entomopathogenic fungi are an unexplored source of natural insecticides. RESULTS: The crude soluble protein extract (CSPE) of the entomopathogenic fungus Metarhizium anisopliae (Mestch.) (strain EAMa 01/58‐Su) shows chronic insecticidal activity when administered per os. Mortality in flies exhibits a dose response. The CSPE produces an antifeedant effect in adult flies, a result probably due to a progressive deterioration of the fly midgut after ingestion of the extract. Protease and temperature treatments show that insecticidal activity against C. capitata is due to proteinaceous compounds that are highly thermostable. Four monomeric proteins from this crude extract have been purified by liquid chromatography and gel electroelution. Although all four monomers seem to be involved in the insecticidal activity of the CSPE, the 15 kDa and the 11 kDa proteins appear to be mainly responsible for the observed insecticidal effect. CONCLUSIONS: Four new fungal proteins with insecticidal activity have been purified and identified. These proteins might be combined with insect baits for C. capitata biocontrol. Copyright © 2009 Society of Chemical Industry     

不同品种马血液蛋白(酶)遗传多态性及聚类分析

采用垂直平板不连续聚丙烯酰胺凝胶电泳技术分别对蒙古马、三河马及纯血马的8种血液蛋向进行多态性检测.计算了各位点的基因型频率、基因频率、遗传杂合度,并通过聚类分析探讨了群体间的亲缘关系和遗传距离.结果表明:种血液蛋白(酶)位点在不同程度上呈现出多态.平均杂合度分别为:蒙古马0.378 2,三河马0.395 6和纯血马0.257 1.聚类分析表明,蒙古马与三河马间的亲缘关系最近,与纯血马的亲缘关系相对较远.通过分析不同马品种的遗传关系,为中国马遗传资源的保护与育种提供依据.     

极端嗜热菌Pyrococcus furiosus热休克蛋白HSP60的克隆表达和纯化

[目的]研究极端嗜热菌Pyrococcus furiosus的热休克蛋白HSP60(Pfu-HSP60)的克隆表达与纯化方法。[方法]应用加拿大HSP60数据库及其BLAST和FASTA序列比较工具对Pfu-HSP60基因序列进行同源性分析,对HSP60基因进行了体外扩增并在大肠杆菌中成功克隆与表达,还进行了Pfu-HSP60蛋白的纯化。[结果]以Taq酶为DNA聚合酶,用PCR方法扩增出预期的Pfu-HSP60基因,插入质粒pET-21a,转化入大肠杆菌受体菌BL21-Codonplus(DE)3-RIL,0.1mmol/LIPTG诱导2 h,得到可溶性高表达,经超声、离心、上清85℃加热、再离心得到初步纯化的Pfu-HSP60蛋白,再经DEAE Sepharose Fast Flow阴离子交换层析纯化,收集峰超滤浓缩后经SephacrylS-200凝胶过滤柱,收集Pfu-HSP60蛋白峰,结果获得纯度为94.5%以上的重组Pfu-HSP60蛋白,总得率为28.0%。[结论]该研究为Pfu-HSP60蛋白的结构和功能及其他热休克蛋白相互作用机制等的研究奠定基础。     

水稻种子蛋白质含量及组分在品种间的变异与分布

采用近红外光谱技术测定分析了351份不同类型水稻品种(系)糙米中的蛋白质含量,结果显示粗蛋白含量在9.3%~17.7%之间,平均为12.4%;籼稻平均蛋白质含量为13.2%,比粳稻高约1个百分点。蛋白质含量低的粳稻品种明显偏多,表现出明显的遗传不平衡现象。现有生产上主栽品种稻米蛋白质含量大多处于中等水平,而高蛋白粳稻种质极少。但仍有部分蛋白质含量极高或低的种质,如饲料稻、早籼稻和一些籼粳交后代品系蛋白质含量较高,而部分粳稻和外来籼稻品种中的蛋白质含量较低。因此可以从一些地方品种、外来品种以及籼粳交后代中筛选到极端类型的种质,为遗传育种提供研究的原材料。SDS-PAGE分析结果显示不同类型水稻间各贮藏蛋白组分具一定差异。     

小麦品种“陕253”低分子量谷蛋白亚基基因的克隆及原核表达

利用LMW-GS特异引物,从强筋小麦品种陕253中克隆了1个1 498 bp的片段(GenBank登录号为FJ172533),该片段包含全长为912 bp的低分子量谷蛋白亚基的完整编码序列.经比较推导氨基酸序列的同源性,发现该基因属于Glu-D3位点编码低分子量谷蛋白亚基的基因,编码产物N-端具有LMW-m型低分子量谷蛋白亚基的典型特征,系统演化分析也支持这一结果.构建了该基因的表达载体pET32a-GluD3-S253,在宿主菌E. coll Rosetta-gami B(DE3)中经IPTG诱导表达融合蛋白.SDS-PAGE和Western blot检测表达产物,证实融合蛋白表达成功.     

3株水产动物病原弧菌主要分泌性蛋白的鉴定及其分泌性序列的分析

从3株水产动物病原菌鳗弧菌MN、鳗弧菌3101、费氏弧菌培养液中分别获取了分泌性蛋白,进行SDS-PAGE,对表达量较高的5条蛋白区带进行MALDI-TOF/TOF质谱鉴定表明,它们分别是鳗弧菌MN金属蛋白酶(empA-MN)、鳗弧菌3101金属蛋白酶(empA-3101)、锌金属蛋白酶(zincempA)、费氏弧菌VFMJ11_1094蛋白(VFMJ11_1094)和费氏弧菌ES114的外膜蛋白(OMP).根据所鉴定的序列设计引物,分别从鳗弧菌MN、鳗弧菌3101和费氏弧菌中扩增出相应基因,克隆后测定emp-MN、empA-3101、zinc empA、VFMJ11-1094和OMP相应基因的序列,经在线软件SignaIP 3.0分析,确定emp-MN、empA-3101、zinc empA、VFMJ11-1094和OMP均存在不同的分泌性信号肽序列angMN-35、ang3101-35、ang3101-25、vf-38和vf-23,通过在线软件PSORT分析表明,所有信号肽均定位在细胞的周质空间或细胞外膜上.     

暗纹东方鲀饲料中豆粕替代鱼粉的营养生理效应及其与大豆抗原蛋白的相关性

选择体质量为(39.84±3.09)g暗纹东方鲀,分为8组,分别饲喂等氮等能的高鱼粉组、低鱼粉组、低鱼粉发酵豆粕组、低鱼粉豆粕组4组实用饲料和添加大豆抗原蛋白的半纯化饲料(大豆抗原蛋白含量分别为0%、5%、8%和12.5%),进行为期两个月的生长实验,探讨大豆抗原蛋白对暗纹东方鲀生长和生理生化的影响。结果显示,用发酵豆粕替代部分鱼粉的低鱼粉发酵豆粕组生长性能显著优于低鱼粉组,用豆粕替代部分鱼粉的低鱼粉豆粕组与低鱼粉组无显著差异。半纯化饲料中随着大豆抗原蛋白含量增加,生长率呈现先增后降趋势;实用饲料和半纯化饲料对肌肉组成有不同程度的影响;在肝脏丙二醛、总抗氧化能力和血清丙二醛指标中,低鱼粉豆粕组显著高于低鱼粉组,低鱼粉发酵豆粕组则与低鱼粉组无显著性差异,半纯化饲料系列在8%组出现峰值,显著高于其他组。低鱼粉豆粕组血清谷草转氨酶活性显著高于低鱼粉组,低鱼粉发酵豆粕组与低鱼粉组相比无显著性差异,半纯化饲料组谷草转氨酶和谷丙转氨酶呈现先增后减趋势。研究表明,与豆粕替代鱼粉相比,用发酵豆粕替代鱼粉更能促进鱼体生长;大豆抗原蛋白是影响豆粕替代鱼粉效果的重要因素,但对生长、抗氧化性能和生理生化指标的影响则因大豆抗原蛋白的含量与单独存在与否而异。     

对虾白斑综合征病毒（WSSV）囊膜蛋白VP110部分重组表达及其与对虾鳃细胞膜蛋白结合分析

将VP110基因的部分序列克隆到pET-28a载体中构建pET28a-vp110b重组质粒并进行原核表达,获得重组表达的蛋白rVP110-B;用rVP110-B注射凡纳滨对虾Litopenaeus vannamei后,经WSSV感染,实验表明,该蛋白注射使凡纳滨对虾感染WSSV的半数死亡时间比对照组延长了20%。用表达纯化的该重组蛋白制备了兔抗rVP110-B多克隆抗体,该抗体用于凡纳滨对虾鳃细胞膜蛋白与rVP110-B的Far-western分析显示,凡纳滨对虾鳃细胞膜蛋白中除90 kDa左右的血蓝蛋白外,在41.7 kDa存在结合条带,经质谱分析表明这条鳃细胞膜蛋白是肌动蛋白。     

Effects of DL‐methionine supplementation on the success of fish meal replacement by plant proteins in practical diets for juvenile gibel carp (Carassius auratus gibelio)

A ten‐week feeding trail was conducted to investigate the effects of increasing DL‐methionine (Met) supplementation on the success of fish meal (FM) replacement with plant proteins in practical diets for juvenile gibel carp, Carassius auratus gibelio. Twelve isoenergetic diets were formulated including two 150 g kg^?1 FM diets (Diet 1—positive control 1 reflecting a commercial diet and Diet 2—positive control 2 reflecting a commercial diet but with balanced essential amino acid (EAA) profile) and ten 50 g kg^?1 FM diets (negative controls) supplemented with graded levels (0–3.0 g kg^?1) of DL‐Met (Diets 3–12). Each diet was fed to triplicate groups of gibel carp, near satiation four times daily for 10 weeks. Diet 2 with balanced EAA profile produced better final weight, specific growth rate (SGR) and feed conversion ratio (FCR) than the negative control diet containing no supplemental Met (Diet 3), but did not significantly differ from Diet 1. However, DL‐Met supplementation (0.5–3.0 g kg^?1) in the negative control diets (Diets 4–12) produced growth performances similar to those fed the positive control diets (Diets 1 and 2). Based on quadratic regression analysis, the optimal dietary Met level with 5.2 g kg^?1 of dietary cysteine (Cys) was found to be 7.1 g kg^?1 dry diet for SGR and FCR. The corresponding total sulphur amino acid requirements (Met + Cys) of this species were calculated to be 12.3 g kg^?1 dry diet for SGR and FCR. DL‐Met supplementation in 50 g kg^?1 FM diets showed a decreasing trend in plasma cholesterol contents (p < .05). No significant differences were observed in whole‐body composition, plasma protein, triglyceride and free EAA contents among dietary treatments, while plasma aspartate transaminase, albumin and ammonia contents were significantly influenced by dietary Met levels. Juvenile gibel carp grew equally well on 150 g kg^?1 FM diet or 50 g kg^?1 FM diets balanced for EAA profile with supplemental amino acids. The results of this study overall indicate that balancing dietary amino acid levels with DL‐Met supplementation is a key strategy in successfully reducing FM levels in the diets of gibel carp.