饲料中添加侧孢短芽孢杆菌FAS05对凡纳滨对虾生长、抗病及免疫力的影响

侧孢短芽孢杆菌(Brevibacillus laterosporu)是一种重要的生物防治益生菌,但在水产养殖中鲜见报道。为研究饲料中添加侧孢短芽孢杆菌FAS05对凡纳滨对虾(Litopenaeus vannamei)生长、抗病及免疫力的影响,以体重为(1.00±0.08) g的凡纳滨对虾为研究对象,开展了28 d的养殖实验。实验分为4组,每组3个重复,每个重复50尾对虾,分别饲喂添加0 (C组,作为对照组)、10⁵ (BL1组)、10⁷ (BL2组)和10⁹ CFU/g(BL3组)侧孢短芽孢杆菌FAS05的实验饲料。结果显示,各组存活率无显著差异(P>0.05),BL1和BL2组对虾的体长、体重和特定生长率显著高于C组(P<0.05);与C组相比,BL1～BL3组养殖水体的弧菌数显著降低(P<0.05);侵染副溶血弧菌(Vibrio parahemolyticus)后,BL1～BL3组存活率显著高于C组(P<0.05),BL1～BL3组之间的差异不显著(P>0.05);与C组相比,BL1～B...     

侧孢短芽孢杆菌溶藻活性代谢产物对虾池颤藻的溶藻效果

为改善对虾养殖水环境,研究了不同阶段的侧孢短芽孢杆菌无菌滤液和颤藻对溶藻效果的影响.通过测定溶藻过程中藻体干重、叶绿素a含量以及藻蓝蛋白含量,探究了溶藻效果的最佳作用阶段及其作用机理.结果显示,稳定期和衰亡期的无菌滤液对颤藻的溶藻效果极为显著,7d后颤藻干重分别减少了51.77％、47.04％,叶绿素a含量分别降低了67.60％、59.13％,藻蓝蛋白含量分别增加了33.51％、30.97％;溶藻细菌无菌滤液对延滞期颤藻的溶藻效果极显著,7d后颤藻干重减少了63.90％,叶绿素a含量下降了69.72％,藻蓝蛋白含量升高了54.17％.结果表明,侧孢短芽孢杆菌培养至稳定期的无菌滤液对延滞期颤藻的溶藻效果最好.     

侧孢短芽孢杆菌(Brevibacillus laterosporus)溶藻活性代谢产物对虾池颤藻(Oscillatoria sp.)溶藻效果研究

颤藻(Oscillatoria sp.)为对虾养殖池塘中的一种常见的有害蓝藻。本实验以侧孢短芽孢杆菌(Brevibacillus laterosporus)的除菌体滤液和颤藻为基础,研究各自在不同阶段的溶藻效果,通过测定藻体干重、叶绿素a含量以及藻蓝蛋白含量,探究溶藻效果的最佳作用阶段及其作用机理,为改善对虾养殖水环境提供一定的理论基础。实验结果表明：稳定期和衰亡期的除菌体滤液对颤藻的溶藻效果均影响极显著(P<0.01)：7天后颤藻干重分别减少了51.77%、47.04%,叶绿素a含量分别降低了67.60%、59.13%,藻蓝蛋白含量分别增加了33.51%、30.97%;溶藻细菌除菌体滤液对延滞期的颤藻溶藻效果影响极显著(P<0.01)：7天后颤藻干重减少了63.90%,叶绿素a含量下降了69.72%,藻蓝蛋白含量升高了54.17%。因此,本研究显示侧孢短芽孢杆菌培养至稳定期的除菌体滤液对延滞期颤藻的溶藻效果最好。     

金属蛋白酶组织抑制剂在皱纹盘鲍抗弧菌免疫中的作用

由弧菌感染引起的疾病是影响鲍存活率的主要因素,实验以皱纹盘鲍为研究对象,探讨金属蛋白酶组织抑制剂(tissue inhibitor of metalloproteinase,TIMP)在皱纹盘鲍抗弧菌免疫中的功能及其与基质金属蛋白酶1 (matrix metalloproteinase 1,MMP-1)的相互作用关系....     

中国明对虾Kazal型丝氨酸蛋白酶抑制剂基因(Fc-Kazal)的重组表达及活性分析

Kazal型丝氨酸蛋白酶抑制剂可以通过精确调控丝氨酸蛋白酶的活力,在生物体的防御应答等众多生物过程中发挥重要作用。以前期克隆的中国明对虾Kazal型丝氨酸蛋白酶抑制剂基因(Fc-Kazal, GenBank注册号为DQ318856)为基础,对其功能结构域进行序列比对和进化分析;组织表达分析结果表明,该基因在血细胞、鳃和淋巴器官等组织中高水平表达,而在眼柄、神经和肌肉中无表达;利用原核表达系统对该基因成熟肽区域成功进行了重组表达,纯化后的目的蛋白最终得率为0.4 g/L培养液;活性分析结果显示,复性后的rFc-Kazal对鳗弧菌、金黄色葡萄球菌、杀鲑气单胞菌、苏云金芽孢杆菌有明显的抑菌作用。     

长牡蛎中I84蛋白酶抑制因子家族的扩张和功能分化

蛋白酶抑制因子是极其多样的蛋白质或多肽,能抑制蛋白酶的水解活性。研究发现蛋白酶抑制因子能通过抑制病原蛋白酶活性,从而抑制病原的入侵。I84蛋白酶抑制因子家族是MEROPS数据库中新增的一个家族,其部分成员在免疫防御过程中的作用得到了一定的揭示。为探究I84蛋白酶抑制因子家族在长牡蛎中的分布和功能状况,实验鉴定了长牡蛎中23个潜在的I84家族基因,根据系统进化树分析,挑选了5个同源基因进行时空表达和功能分析。首先,利用克隆技术验证了长牡蛎中5个I84家族同源基因CgSi3、CgSi5、CgSi6、CgSi16和CgSi19可表达性。结果显示,5个基因在外套膜、闭壳肌、性腺、血淋巴细胞、肝胰腺和鳃等6个组织中均表达,但肝胰腺中表达量显著高于其他组织。同时,5个基因在长牡蛎幼体不同发育阶段表达模式不同,其中各基因在受精卵时期均不表达,CgSi3表达量则在眼点幼虫期显著上升后下降,而CgSi6在壳顶幼虫期开始表达后,表达量随长牡蛎发育而持续增加。另外,对牡蛎进行病原相关模式分子(PAMPs)注射后,5个基因也表现出不同的表达模式,其中LPS和PGN注射后CgSi6表达量变化明显,而poly (...     

血管紧张素转换酶抑制肽的分离

采用截留分子量分别为30 ku、10 ku、6 ku、3 ku的中空纤维超滤膜对扇贝酶解产物进行分级分离,并结合凝胶柱层析分离技术测定了各分离组分的分子量分布,同时还测定了各分离组分对血管紧张素转换酶(ACE)的抑制活性。实验结果表明,不同截留分子量超滤膜可以实现酶解产物按其分子量大小的分级分离,不同分离组分的ACE抑制力活性差异较大,总趋势是截留分子量相对较小的超滤膜透过液的ACE抑制活性较强。其中,截留分子量为3 ku超滤膜的透过液具有最强的ACE抑制活性,其ACE抑制率I(%)=96.17%,半抑制浓度IC_(50)= 0.078 mg·mL~(-1)。通过对截留分子量为3 ku超滤膜的透过液依次经过Sephadex G-25及Sephadex G-15凝胶柱层析分离纯化后,分离出了ACE抑制活性最强的2个组分活性肽,其平均分子量分别为1 300 u和900 u左右,其IC_(50)分别为0.026 mg·mL~(-1)和0.012 mg·mL~(-1)。     

泥蚶基质金属蛋白酶组织抑制因子3基因的克隆及镉免疫表达研究

通过已构建的泥蚶转录组文库EST,利用SMART RACE技术扩增得到泥蚶Tg-TIMP-3基因全长c DNA序列,并对其进行生物信息学和组织表达相关分析。结果显示,泥蚶Tg-TIMP-3 c DNA全长为804 bp,其中开放阅读框为570 bp,编码189个氨基酸;氨基酸序列比对发现其氨基酸序列与其他动物的相似性并不高,与长牡蛎的相似性仅为35.8%,但TIMP基因的两个功能域(N端和C端)相对保守。qRT-PCR检测结果显示:Tg-TIMP-3 mRNA在鳃中表达最高,外套膜和肝胰腺次之,表达量最低的是血液。在不同浓度重金属镉(Cd)攻毒后,泥蚶鳃中的Tg-TIMP-3 mRNA表达量先略微下调后显著上调达到最高,其中25μg/L和250μg/L镉攻毒后在6 h时表达量达到最高(P<0.01),500μg/L镉攻毒后在9 h时表达量达到最高,48 h后各浓度组Tg-TIMP-3基因表达受到不同程度抑制。研究表明,Tg-TIMP-3对重金属Cd免疫防御或解毒中可能起重要作用,而泥蚶鳃组织中的Tg-TIMP-3 mRNA表达对不同浓度Cd的抗逆免疫反应的灵敏度和时序上存在一定差异。     

HSP90抑制剂根赤壳菌素对斑马鱼胚胎发育的影响

为研究热休克蛋白90(heat shock protein 90,HSP90)在斑马鱼(Danio rerio)胚胎发育中的作用,本实验采用2μmol/L、5μmol/L、10μmol/L的HSP90抑制剂根赤壳菌素(radicicol)对斑马鱼发育期胚胎进行处理,监测斑马鱼胚胎不同发育时期两个HSP90功能抑制标志基因BAG3(BCL2-associated athanogene3)和HSPB1(heat shock protein beta-1)的mRNA的表达水平,并观察不同发育时期的胚胎发育状况。结果如下:(1)实时荧光定量PCR结果显示,BAG3和HSPB1 mRNA水平在根赤壳菌素处理胚胎12 hpf(hours post-fertilization,hpf)或24 hpf后显著增高,Western blot检测到5μmol/L根赤壳菌素处理胚胎24 hpf后HSP70表达上调,证明该实验条件下HSP90功能受到抑制;(2)根赤壳菌素处理后斑马鱼胚胎发育变缓,胚胎成活率统计显示:2μmol/L、5μmol/L、10μmol/L根赤壳菌素处理24 hpf胚胎成活率分别是95%、77%、35%,成活率随根赤壳菌素浓度的增高而降低;(3)5μmol/L根赤壳菌素处理72 hpf可见部分个体色素沉积减少、心包膜增大、肌肉萎缩等发育畸形。研究结果证明HSP90在斑马鱼胚胎发育过程中发挥重要的作用,根赤壳菌素在5μmol/L时已达到较佳抑制效果且成活率较高,而且胚胎发育出现了各种形态学变化,为后期研究HSP90调节斑马鱼胚胎发育奠定了基础。     

Identification of oyster-derived hypotensive peptide acting as angiotensin-I-converting enzyme inhibitor

Angiotensin-I-converting enzyme (ACE) plays a crucial role in the crisis of hypertension. Some peptides that originate from protease hydrolysates are known to suppress ACE activity in vitro and in vivo. Here, we investigated whether trypsin hydrolysate of oyster Crassostrea gigas showed hypotensive activity and ACE inhibition. The hydrolysate significantly suppressed systolic blood pressure and ACE activity in spontaneously hypertensive rats following a one-shot oral administration and a long-term feeding experiment lasting 9 weeks. Each hydrolysate from oyster tissue showed ACE inhibitory activity, indicating the hypotensive effect was due to synergism. One potent ACE inhibitory peptide, Asp-Leu-Thr-Asp-Tyr, was identified from the hydrolysate of the striate muscle, and the peptide exhibited hypotensive activity in vivo. Protease digestion analysis suggested that Asp-Tyr could be the real effector of this penta-peptide in vivo.     

Effects of a nonsteroidal aromatase inhibitor on gonadal differentiation of bluegill sunfish Lepomis macrochirus

In the present study, the efficacy of Letrozole, a potent nonsteroidal aromatase inhibitor (AI), on gonadal sex differentiation and sex reversal was examined in bluegill sunfish (Lepomis macrochirus). In Experiment 1, using AI diet treatments (50, 150, 250 and 500 mg kg⁻¹) from 30 to 90 days posthatch (dph), AI interrupted ovarian cavity formation at a dose of 500 mg kg¹ diet and one intersex fish was identified in this group. The proportions of males in all the treated groups were significantly higher than those in the control group. In Experiment 2, using AI immersion treatments (250, 500 and 1000 μg L⁻¹) during 30–50 dph, the treated groups of 500 and 1000 μg L⁻¹ produced significantly more males than the control and 250 μg L⁻¹ groups. Histological examination revealed no differences in ovary or testis tissue between control and AI‐treated fish. There were no significant differences detected in body weight and length among the AI treated and control groups (P>0.05) for both experiments. The results from these two experiments suggest that inhibition of aromatase activity by AI could influence sex differentiation in bluegill sunfish.     

赤魟软骨血管生成抑制因子的纯化及抗血管生成活性验证

以赤魟软骨为原料,通过盐酸胍抽提、离子交换层析、凝胶过滤、反相高效液相色谱等步骤,首次获得赤魟软骨血管生成抑制因子-I(Dasyatis akajei cartilage angiogenesis inhibitory factor-I,DCAIF-I)。12%的SDS-PAGE电泳-考染显示为一条带,根据蛋白质的相对迁移率计算,分子量约为62 ku。通过鸡胚绒毛尿囊膜活性抑制模型进行活性分析,结果表明,活性物质处理组(DCAIF-I组)与阳性对照组(CS组)抑制效果明显,CAM上的血管发生大面积褪色,血管结构模糊,分枝发生断裂,血管密度减少。PBS对照组血管网清晰,呈叶脉状、放射状生长。血管定量分析结果表明,随着活性物质处理组DCAIF-I含量的增加,CAM上血管的数量减少更加明显,当DCAIF-I含量为1μg时,对CAM上血管的抑制率达56%,结果表明,DCAIF-I对鸡胚绒毛尿囊膜新生血管生成具有明显的抑制效果,且抑制活性具有一定的浓度依赖性。     

芳香化酶抑制剂来曲唑对胡子鲇性腺分化及相关基因表达的影响

采用组织学和荧光实时定量PCR方法, 检测饲喂不同剂量芳香化酶抑制剂来曲唑(letrozole, 50、100和200 μg/g)对胡子鲇(Clarias fuscus)性腺组织学和性别比率的影响, 以及200 μg/g 来曲唑对性分化前后脑型芳香化酶基因(Cyp19a1b)和翼状螺旋/叉头转录因子2(Foxl2)基因表达的影响, 结果表明, 50 μg/g 剂量的来曲唑对胡子鲇性腺分化无显著影响; 但100 μg/g和200 μg/g剂量的来曲唑可促进胡子鲇精巢分化, 抑制卵巢分化, 卵巢腔最早出现时间和初级卵母细胞出现时间均分别推迟3 d和6 d, 而初级精母细胞最早出现时间则分别提前2 d和5 d, 且雄性率分别达65.8%和71.3%, 显著高于50 μg/L组和对照组(P<0.05)。胡子鲇性腺分化前(出膜后12 d), Cyp19a1b和Foxl2基因即开始表达, 性腺分化前后Cyp19a1b相对表达量无显著差异(P>0.05), 但Foxl2相对表达量则随性分化而逐渐降低, 且来曲唑显著抑制性腺分化过程中Cyp19a1b和Foxl2的表达(P<0.05)。结果表明, 100 μg/g 以上剂量的来曲唑可有效诱导胡子鲇分化为雄性; Cyp19a1b可能不直接参与胡子鲇性腺分化, 但Foxl2直接参与此过程。     

Effects of feeding soybean meal with varying trypsin inhibitor activities on growth of fingerling channel catfish

Uncooked hexane extracted soybean meal was heated for various lengths of time to produce meals with varying trypsin inhibitor activities. Fingerling channel catfish (Ictalurus punctatus) were fed 25 and 35% crude protein practical type test diets containing soybean meal with graded levels of trypsin inhibitor activity for 10 weeks. Growth rates and protein efficiency ratio (PER) values were reduced in fish fed raw and inadequately heated soybean meal at both protein levels. These effects were more severe at the lower dietary protein level. Growth rates and PER values improved in each study as the trypsin inhibitor activity of the soybean meal decreased to tolerable levels. Fish fed the 35% crude protein diets appeared to tolerate soybean meal with much higher trypsin inhibitor activity than fish fed the 25% crude protein diets. Even though growth rates and PER values were not significantly different over a rather wide range of dietary trypsin inhibitor activities, the best growth rates were not observed at either protein level until about 83% of the trypsin inhibitor activity in the soybean meal had been destroyed.     

Artificial sex reversal of white grouper (Epinephelus aeneus) utilizing aromatase inhibitor (Fadrozole)

The white grouper is a desirable aquaculture species that adapts to captivity, grows well and commands a high market price. However, little is known about reproductive biology or control of sex reversal of this protogynous hermaphrodite. In this study, female white groupers were implanted with one dose of 17α‐methyltestosterone (10 mg/kg body weight [BW], MT) and two doses of aromatase inhibitor, fadrozole (1 and 3 mg/kg BW, FD1 and FD3) once a month for 4 months (April–July). At the start of the study, the fish had gonads full of oocytes compared to the end of the experiment when the control group mature oocytes compared to the experimental groups MT, FD1 and FD3 that exhibited different stages of testicular tissue. Plasma levels of testosterone were significantly highest in the FD3 group and the highest 11‐Ketotestosterone levels were observed in the MT group. Plasma levels of oestradiol (E₂) were significantly lower in the FD implanted groups, compared to initial individuals and control groups. The use of aromatase inhibitor, FD for sex reversal both gives further insight into the mechanisms controlling sex differentiation and provides an alternative to steroid treatment.     

Ultrasound as potential inhibitor of salmon louse infestation—A small‐scale study

The effect of ultrasound exposure during controlled infection with salmon lice, Lepeophtheirus salmonis, has been studied. Salmon were placed in tanks with salmon lice copepodids for 1 hr, while simultaneously being exposed to sound frequencies of 9.3, 21 or 54 kHz. The sound transducers were operated at maximum power levels, and the 9.3 kHz transducer generated the highest sound level (220.6 dB). Only the group exposed to 9.3 kHz displayed a significant reduction in louse infestation. However, the observed effect of ultrasound was relatively small, and in a practical implementation in sea cages, the sound intensity will be lower than that used in the experiments. It is also possible that the observed reduction in infestation is due to ultrasonic cavitation effects, which are only present at a very short range from the ultrasound source. We therefore do not consider ultrasound a feasible method for preventing attachment of salmon lice copepodids on salmon in cage farms.     

螯虾免疫相关基因的检出及丝氨酸蛋白酶抑制物基因分析

通过对抑制性差减杂交和cDNA芯片技术分离的 2 0 1个阳性克隆的测序 ,得到螯虾 (Procambarusclarkii)免疫相关基因 4 8个 ,其中正向克隆中 4 2个 ,反向克隆 6个 ,除正向克隆中SNAP - 2 5 (synatosome-associatedproteinof2 5ku)已报道外 ,其余均为新基因 ,均在GenBank登录。正向克隆中的同源基因有微管蛋白、超氧化物歧化酶前体、丝氨酸蛋白酶抑制物Ⅰ、精氨酸激酶、70kD伴侣蛋白同类物等。进一步用DotNorthernblot对克隆号PCI188进行鉴定 ,结果攻毒组的杂交信号是对照组的 3.2 4倍 ,与cDNA芯片结果相符。用快速扩增cDNA末端技术扩增克隆号PCI188基因的 5’端片段和 3’端片段 ,全长共 112 8bp ,编码的蛋白质有 2 77个氨基酸 ,分子量为 30 .2 7kD。与GenBank序列号X795 12软尾太平剌蛄 (P .le niusculus)的蛋白酶抑制物Ⅰ (为丝氨酸蛋白酶抑制物 )的基因同源性为 5 8.7% ,氨基酸同源性为 6 9.7%。该蛋白质有 5个重复的结构域 ,与丝氨酸蛋白酶抑制物Kazal家族的结构域相似     

大豆胰蛋白酶抑制剂对鲢丝氨酸蛋白酶的作用

研究了大豆胰蛋白酶抑制剂(STI)对鲢肌肉中肌原纤维结合型丝氨酸蛋白酶(MBSP)引起的肌原纤维蛋白降解作用的影响。大豆胰蛋白酶抑制剂经高度纯化后加入肌原纤维中以判断它对MBSP的抑制效果。在不添加STI的情况下,55℃加热30min即可观察到肌球蛋白重链的分解,延长加热时间则可检测到肌动蛋白及原肌球蛋白的降解。相反,在终浓度为0.75μg·mL-1的STI存在下,包括肌球蛋白重链在内的肌原纤维蛋白降解均能得到有效抑制,表明STI是MBSP的特异性抑制剂。由于肌球蛋白重链及肌动蛋白的完整性是构成鱼糜制品凝胶强度的主要因素,因此,STI的添加有可能提高鲢鱼糜制品的凝胶强度。     

A microplate technique to quantify nutrients (NO2−, NO3−, NH4+ and PO43−) in seawater

Many methods to measure nutrients (NO₂^?, NO₃^?, NH₄⁺, and PO₄^3?) in water are based on the formation of coloured complexes that are measured by spectrophotometry, frequently using large‐volume (10 mL) spectrophotometer cells. Miniaturization of the techniques using microplate readers and sample volumes as small as 250 μL is an affordable alternative for these determinations. This work describes the adaptation to microtechniques using a 96‐well microplate to measure nitrogen compounds and phosphate in seawater. They can be used as an inexpensive procedure for routine monitoring in aquaculture ponds because of the smaller sample, reagent requirements, and suitable ranges.