鲎素的研究及开发利用

鲎是一种栖生于海洋的低等无脊椎动物。鲎素为鲎血液中抗菌肽的总称,研究表明鲎素具有抗菌、抑病毒、检测内毒素等多种作用。鲎素对人胃癌BGC823细胞、人肝癌SMMC7721细胞、人肺腺癌SPCAi细胞、人早幼白血病HL60细胞的增殖都有明显的抑制作用。对鲎素的研究为细菌耐药性、药物残留和抗HIV等医学难题的解决提供了新途径。近年来,由于对鲎资源的过度开发利用,这一资源已濒临灭绝。现在,对鲎资源的保护工作正在展开。     

鲎抗菌肽polyphemusinⅡ基因的改造、克隆及其在大肠杆菌中的表达

中国鲎(Tachypleus trideutatus)血细胞产生的抗菌肽(Antibacterial peptide)—鲎素,具有广谱的杀菌、抑病毒和抗肿瘤细胞的作用。实验根据已报道的美洲鲎的抗菌肽polyphemusinⅡ氨基酸序列,设计了一对引物,并对原抗菌肽基因进行改造,在基因开放阅读框末端添加了Asn密码子,使抗菌肽羧基末端酰胺化,旨在提高其稳定性和抗菌活性。PCR扩增获得改造过的目的片段,连接于载体pMD18-T,测序后,与pEI-28c(+)连接,构建表达质粒pET28c-rr,测序鉴定正确后,转化大肠杆菌E.coli.BK21(DE3)。表达菌株经终浓度为1mmol/L的IPTG诱导后以包涵体的形式表达,在体外表现出明显的抑菌活性。     

鲎的人工养殖及鲎试剂的制备

鲎试剂可用来检测细菌内毒素或热原，具有操作简便、灵敏度高、特异性强、反应迅速等优点，受到普遍欢迎，应用范围日益广泛，尤其是抢救危急病人，检测、诊断，制药工艺流程中内毒素检测，食品工业、水质污染检测等。因此鲎试剂成为国际上价格昂贵，又十分热门的商品。 1鲎的生活习惯与繁殖 鲎生活在温暖的海洋中，冬季需到较深的海域越冬。每当春末夏初，水温上升之际，鲎从深海游向沿岸沙滩。雌雄抱合，在中潮带泥沙中挖穴产卵，受精卵依靠太阳能量孵化，经 5～ 6周时间，幼虫出膜，称三叶幼虫，雌鲎一生中要蜕皮 18次，雄鲎 19次，大约 …     

中国鲎幼鲎人工养殖饵料初步研究

选用沙蚕、菲律宾蛤仔肉、明虾肉、明虾肉海带混合、海带和初孵卤虫6组饵料投喂2龄期中国鲎幼鲎。试验结果表明:投喂初孵卤虫组2龄期幼鲎存活率最高,为71.43%,其他投喂组存活率不足20%;投喂初孵卤虫组的35只2龄幼鲎有12只蜕壳发育成3龄期幼鲎,其他投喂组均未出现蜕壳;投喂初孵卤虫组幼鲎湿重和头胸部宽增加明显,其他投喂组湿重与初始湿重相比略有增加,但头胸部宽无明显变化。说明初孵卤虫是2龄期幼鲎人工养殖的适宜饵料。     

中国鲎微卫星引物扩增圆尾鲎DNA的PCR反应体系优化

采用正交设计法,从dNTPs浓度、引物浓度、Mg2＋浓度、Taq DNA聚合酶用量4个因素3个水平出发,优化设计圆尾鲎DNA的PCR反应体系（引物为中国鲎微卫星引物）.并采用直观分析方法分析正交试验结果,最终建立了圆尾鲎SSR-PCR最佳反应体系：总体积20μL,Taq DNA聚合酶1.5 U、dNTPs 0.16 mmol/L、引物0.2μmol/L、Mg2＋2.0 mmol/L;并通过PCR梯度实验进一步优化模板DNA质量浓度、退火温度及退火时间,获得最佳反应条件：模板DNA质量浓度为30 ng/μL,退火温度为48℃,退火时间为20～25 s.对最佳反应体系和反应条件进行了检验,结果显示该反应体系稳定性高、重复性好.     

底质对东方鲎幼体存活的影响

研究以人工授精培育的鲎幼体为材料,在模拟的海区生态环境下观察鲎幼体在海沙和海泥中的成活情况,并比较幼鲎的生长发育情况,结果发现:幼鲎具有钻入沙或泥的生活习性,且幼鲎在含沙或泥的海水比在普通海水中成活率显著提高,生长发育情况较天然水体更好.     

从鲎试剂生产废料中提纯鲎素的方法研究

目的 建立从鲎试剂生产废料中纯化鲎素的方法.方法 用酸处理和sephdexG-50凝胶层析法从鲎试剂生产废料中提纯鲎素.结果 经凝胶层析纯化样品在SDS-PAGE中显示单一蛋白质条带.鲎素分子量约3kD左右.提取的鲎素能显著抑制大肠杆菌的生长,IC50为3.9mg/L.进一步研究显示,提取到的鲎素不仅抑制普通大肠杆菌的生长,而且可以显著抑制耐氨苄青霉素或耐卡那霉素的大肠杆菌的生长.结论 本文建立的方法可成功地从鲎试剂生产废料中提取到纯化的鲎素.     

鲎C因子的研究进展

鲎C因子是鲎血细胞中一种对内毒素敏感的丝氨酸蛋白酶原,主要有6种结构域,能特异结合内毒素而被激活.对鲎C因子的结构与功能、基因工程研究及其应用前景进行了综述.     

成熟中国鲎卵的营养成分分析

分别采用凯氏定氮法、索氏脂肪抽提称量法、反相液相色谱法及和相色谱-质谱法测定了成熟中国鲎卵中粗蛋白、粗脂肪、氨基酸及脂肪酸的含量。结果表明,中国鲎卵粗蛋白和粗脂肪含量分别为61.90%和15.82%;鲎卵至少含有16种氨基酸,其中异亮氨酸(Ile)、酪氨酸(Tyr)、亮氨酸(Leu)、赖氨酸(Lys)和天冬氨酸(Asp)含量较高,占氨基酸总量的60%以上;鲎卵至少含18种脂肪酸,其中不饱和脂肪酸(UFA)有7种,高度不饱和脂肪酸(HUFA)4种,分别占总游离脂肪酸含量的60.71%和13.15%。     

紫外线诱导大肠杆菌产生对鲎素抗药性菌株的研究

探讨经过紫外线诱变能否筛选出抗鲎素的大肠杆菌突变菌株以及突变菌株对抗生素敏感性的变化.以大肠杆菌JM109和ATCC25922为研究对象,通过紫外线诱变技术对出发菌株进行诱变,以相应的出发菌株作对照,并通过鲎素浓度梯度平板法对大肠杆菌进行初筛后,对初筛到的抗鲎素菌株进行亚抑菌浓度鲎素的连续传代诱导和抗生素对初筛到的抗鲎素菌株进行敏感性测定.结果表明,采用紫外诱变和鲎素浓度梯度筛选的方法,从中筛选到低抗鲎素的大肠杆菌ATCC25922 3株;通过抗生素对此3种低抗鲎素菌株进行敏感性测定,表明抗鲎素菌株对苄星青霉素有显著抗药性,而对盐酸左氧氟沙星仍然敏感.对于大肠杆菌JM109菌株,经过紫外线诱变后,未诱导出抗鲎素菌株.     

中国鲎3个地理群体遗传多样性的等位酶分析

应用等位酶技术对漳浦、连江和温州3个地理群体的中国鲎遗传多态性进行分析.实验结果发现：6个酶系统的10个位点中有6个为多态性位点,共获得21个等位基因;大部分位点在3个群体中偏离Hardy-Weinberg平衡;在物种水平上,有效等位基因数为1.635,观察杂合度为0.456,期望杂合度为0.306,香侬指数为0.486,表明中国鲎的遗传多样性较高;F-统计量（FST）平均为0.0475,表明中国鲎遗传差异主要存在于群体内;遗传一致度平均为0.969,遗传距离平均为0.0315,表明中国鲎群体间遗传分化较低;基因流平均为5.0120,显示群体间存在较大的基因交流.由此认为3个群体属于同一个繁殖群体.在进行鲎物种保护的同时,要加强对其遗传多样性的保护.     

Limulus lateral eye: properties of receptor units in the unexcised eye

Single receptor units in the compound eye of the horseshoe crab were illuminated, and their impulse discharges were recorded without removing the eye from the animal. The receptors were spontaneously active in darkness and responded without saturation over a light intensity range of 10(10) to 1. When the eye was excised, the receptors did not discharge in darkness and had an intensity range of 10(5) to 1, as is usually found. Experiments show that these and other differences result from cutting off the blood supply to the eye when it is excised. In addition, the range and shape of the intensity characteristic suggest that more than one receptor mechanism encodes light intensity in this eye.     

Recent synchronous radiation of a living fossil

Modern survivors of previously more diverse lineages are regarded as living fossils, particularly when characterized by morphological stasis. Cycads are often cited as a classic example, reaching their greatest diversity during the Jurassic-Cretaceous (199.6 to 65.5 million years ago) then dwindling to their present diversity of ~300 species as flowering plants rose to dominance. Using fossil-calibrated molecular phylogenies, we show that cycads underwent a near synchronous global rediversification beginning in the late Miocene, followed by a slowdown toward the Recent. Although the cycad lineage is ancient, our timetrees indicate that living cycad species are not much older than ~12 million years. These data reject the hypothesized role of dinosaurs in generating extant diversity and the designation of today's cycad species as living fossils.     

Epidermal patterns of the lemma in some fossil and living grasses and their phylogenetic significance

Morphological study of fossil grass anthoecia of Berriochloa and Nassella collected from Miocene-Pliocene strata in Kansas has revealed well-preserved epidermal structure. This seems to be the first micromorphological information known from fossil grass floral bracts. The epidermal pattern on the lemma in the fossils and their living counterparts are evidence in support of the view that the North American species of Stipa of the section Hesperostipa Elias and species of Piptochaetium have a common ancestry in Berriochloa, and that species of both taxa have been distinct from species of the Nassella, Oryzopsis, and other Stipa since at least the Miocene or Pliocene.     

Ostracod "living fossils": new finds in the pacific

New ostracod "living fossils" have been discovered in samples of Recent marine sediment collected from several Pacific islands. The finds include a male anatomy having several primitive features which, in conjunction with primitive features in the shell, necessitate reconsideration of current opinions on the phylogeny of freshwater ostracods.     

Paleontology. Shaking the earliest branches of anthropoid primate evolution

Primates living today are believed to share a common ancestor that originated in either Africa or Asia. Fossil examples of such anthropoid ancestors have been found in both continents, so pushing back the origins to a single location has been controversial. In their Perspective, Jaeger and Marivaux discuss results reported in the same issue by Seiffert et al. that may put part of the controversy to rest. Seiffert et al. describe the earliest and most complete African anthropoid fossils from the Fayum desert region of Egypt. Cranial and dental fossils of two different small species were found, and their character, especially the features of the fossil teeth, suggests an ancient evolutionary history in Africa. At the same time, the phylogenetic analysis of Seiffert et al. is consistent with the view that African anthropoids immigrated from Asia at a very early date, probably before the late Paleocene (60 million years ago), possibly followed by later waves of immigration.     

Enhancement of flicker by lateral inhibition

Sinusoidal modulation of illumination on the compound eye of the horseshoe crab, Limulus, produces a corresponding variation in the rate of discharge of optic nerve impulses. Increasing the area of illumination decreases the variation at low frequencies of modulation, but unexpectedly enhances-or "amplifies"-the variation at the intermediate frequencies to which the eye is most sensitive. Both effects must result from inhibition since it is the only significant lateral influence in this eye.     

Shell use: an adaptation for emigration from the sea by the coconut crab

The coconut crab. Birgus latro (L.) emigrates from the sea during the postlarval glaucothoe stage. Glaucothoes show ancestral hermit crab behavior of living in empty gastropod shells which protect them during this vulnerable time.     

虾油和蟹油的理化特性研究

[目的]研究以螃蟹壳和虾头为原料提取的蟹油和虾油的理化特性。[方法]在常温和煎炸条件下,测定比较芝麻香油、豆油、虾油和蟹油的酸值、碘值和过氧化值,并对4种油的外观变化进行分析。[结果]试验表明,虾油和螃蟹油在常温下新鲜度高,在煎炸条件下氧化稳定性比豆油和芝麻香油稍差,且在煎炸40min后失去特征优势;在感官评价中,虾油和螃蟹油综合得分较高,但在油炸条件下优势不明显。[结论]研究可为虾油和蟹油的综合开发利用提供参考依据。     

中国鲎同工酶组织器官特异性表达

采用垂直聚丙烯酰胺凝胶电泳技术,分析了中国鲎6种组织器官（肝胰腺、心脏、肠、鳃、肌肉和黄色结缔组织）的乙醇脱氢酶（ADH）、山梨醇脱氢酶（SDH）、酯酶（EST）、过氧化氢酶（CAT）、天冬氨酸转化酶（AAT）、苹果酸脱氢酶（MDH）和苹果酸酶（ME）等7种同工酶的活性和分布,并对其酶谱的表型进行了分析.结果表明,中国鲎的同工酶系统存在不同程度的组织特异性.乙醇脱氢酶在鳃和黄色结缔组织中不表达;山梨醇脱氢酶在鳃中不表达;酯酶、过氧化氢酶和苹果酸脱氢酶在6种组织器官中均有表达,但表达活性存在差异;而天冬氨酸转化酶Aat-1位点在鳃中不表达.