CN109160938A - 一种寡肽及其应用 - Google Patents
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- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
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- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/10—Preservation of living parts
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- A01N1/125—Freeze protecting agents, e.g. cryoprotectants or osmolarity regulators
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Abstract
本发明公开了一种寡肽及其应用。目前，达氏鲟精子冻存时通常是使用稀释剂和抗冻剂，稀释剂采用0.7％氯化钠+0.02％氯化钾+3.5％葡萄糖，抗冻剂采用二甲亚砜等，但是这些抗冻剂对达氏鲟精子有不同的毒性作用，保存后精子寿命降低。研究结果表明，寡肽A、B或C作为抗冻剂显著优于现有技术，可以显著提高精子冻存复苏后的寿命，本发明寡肽A、B或C可以用作抗冻剂提高达氏鲟精子抗冻能力。
Description
技术领域
本发明属于化学领域，具体涉及一种寡肽及其应用。
背景技术
达氏鲟是一种较大型淡水定居性鱼类，是我国特有种，达氏鲟的肉和卵味道鲜美，富含营养，尤其是鱼卵可加工成鱼子酱。由于兴修水利、过度捕捞等导致生态环境急剧变迁，达氏鲟野生种群资源日趋减少。
低温冷冻技术不仅能够保护达氏鲟这一珍稀物种、提高其繁殖潜力和进行异地或异时受精成为可能以外，还能为达氏鲟种质资源保护和生物多样性的保护开辟了新途径，为达氏鲟的遗传育种和现代生物技术的研究长期不间断的提供生物材料等方面也具有重要的意义。
鱼类精子在精巢和精浆中受到抑制不活动，若抑制条件被解除则会被激活激烈运动起来，激烈运动的精子如果处于抑制环境，其活动又受到抑制，抑制条件被解除后将再次被重新激活，可以如此反复。添加稀释剂可以保持精细胞的活力，延长其保存时间，不同鱼类精子运动的诱导机制不尽相同，淡水鱼类精子的激活是由淡水环境的低渗所诱发，而海水鱼类精子运动则是由海水的高渗所刺激。
目前，达氏鲟精子冻存时通常是使用稀释剂和抗冻剂，稀释剂采用0.7％氯化钠+0.02％氯化钾+3.5％葡萄糖的稀释剂，抗冻剂采用二甲亚砜等，但是这些抗冻剂对达氏鲟精子有不同的毒性作用，保存后精子寿命降低。
发明内容
本发明旨在克服现有技术不足，提供一种合成寡肽及提高达氏鲟精子抗冻能力的用途。
本发明上述目的通过如下技术方案实现：
一种寡肽，序列如Sequence NO.1所示。
上述寡肽用作达氏鲟精子抗冻剂的用用途。
一种达氏鲟精子抗冻液，由上述抗冻剂和稀释剂组成。
优选地，所述稀释剂为0.7％氯化钠+0.02％氯化钾+3.5％葡萄糖的去离子水溶液。
一种寡肽，序列如Sequence NO.2所示。
上述寡肽用作达氏鲟精子抗冻剂的用用途。
一种达氏鲟精子抗冻液，由上述抗冻剂和稀释剂组成。
优选地，所述稀释剂为0.7％氯化钠+0.02％氯化钾+3.5％葡萄糖的去离子水溶液。
一种寡肽，序列如Sequence NO.3所示。
上述寡肽用作达氏鲟精子抗冻剂的用用途。
一种达氏鲟精子抗冻液，由上述抗冻剂和稀释剂组成。
优选地，所述稀释剂为0.7％氯化钠+0.02％氯化钾+3.5％葡萄糖的去离子水溶液。
本发明提供的寡肽A、B或C可以用作抗冻剂提高达氏鲟精子抗冻能力。
附图说明
图1为各组精子寿命。
具体实施方式
下面结合附图和实施例具体介绍本发明实质性内容，但并不以此限定本发明的保护范围。
一、实验材料
寡肽A～D由上海黙悉生物科技有限公司合成，纯度不低于95％，序列如表1所示。
表1寡肽A～D序列
氯化钠、氯化钾、葡萄糖、DMSO均为常规的分析纯试剂。去离子水自制。
二、实验方法
1、精子采集及预处理
2018年4-5月，对四川省农业科学院水产研究所培育的性成熟达氏鲟雄鱼进行人工催熟催产。用干净的干毛巾擦去体表和生殖孔周围的水及杂物，用手轻轻挤压腹部使精液自然流出，用干净干燥的塑料封口袋收集无水、无血和无粪便的精液并充氧待检，镜检精子活力，选取精子活力在95％以上的精液作为试验材料，将其放置于4℃的冰箱中备用。
2、稀释剂的配制
用去离子水、氯化钠、氯化钾和葡萄糖配制0.7％氯化钠+0.02％氯化钾+3.5％葡萄糖的稀释剂，均为质量体积百分含量，4℃保存备用。
3、分组及冻存
寡肽A组：以稀释剂为溶剂，寡肽A为溶质，配制成寡肽A的质量体积百分含量为5％的冻存液A，与精液以1:1体积比在1.8mL冷冻管中混合，将混合液置于4℃冰箱冻存；
寡肽B组：以稀释剂为溶剂，寡肽B为溶质，配制成寡肽B的质量体积百分含量为5％的冻存液B，与精液以1:1体积比在1.8mL冷冻管中混合，将混合液置于4℃冰箱冻存；
寡肽C组：以稀释剂为溶剂，寡肽C为溶质，配制成寡肽C的质量体积百分含量为5％的冻存液C，与精液以1:1体积比在1.8mL冷冻管中混合，将混合液置于4℃冰箱冻存；
寡肽D组：以稀释剂为溶剂，寡肽D为溶质，配制成寡肽D的质量体积百分含量为5％的冻存液D，与精液以1:1体积比在1.8mL冷冻管中混合，将混合液置于4℃冰箱冻存；
DMSO组(现有技术)：以稀释剂为溶剂，取DMSO配制成体积百分浓度为8％的冻存液E，与精液以1:1体积比在1.8mL冷冻管中混合，将混合液置于4℃冰箱冻存。
4、复活及寿命观察
冻存16h后分别观察精子寿命(从添加激活液的瞬间开始计算；90％以上的精子停止颤动，即认为精子死亡)。进行观察时，从冰箱中取出盛有精液混合液的冷冻管，用解剖针沾取少量的精液混合液到载玻片上，将精液混合液涂匀，放在显微镜下，用滴管取江水(激活液)，滴加在精液混合液上，立即开始计时。重复3-5次，取平均值。
5、数据处理
采用SPSS17.0软件处理数据。One-WayANOVA分析用于比较不同处理组细胞之间的差异。数据采用均值±标准差表示。以P＜0.05为差异具有统计学意义。
三、实验结果
各组精子寿命如表2和图1所示。结果表明，寡肽A、B或C作为抗冻剂显著优于现有技术，可以显著提高精子冻存复苏后的寿命。而寡肽D的抗冻效果反而不如现有技术。
表2各组精子寿命
综上可见，本发明提供的寡肽A、B或C可以用作抗冻剂提高达氏鲟精子抗冻能力。
上述实施例的作用在于具体介绍本发明的实质性内容，但本领域技术人员应当知道，不应将本发明的保护范围局限于该具体实施例。
序列表
<110> 魏清滨
<120> 一种寡肽及其应用
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 17
<212> PRT
<213> 人工序列(Artificial Sequence)
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Lys Trp Glu Gly Glu Phe Arg Arg Val Ile Glu Tyr Arg Glu Tyr Arg
1 5 10 15
Arg
<210> 2
<211> 17
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 2
Lys Trp Ser Phe Arg Lys Ser Tyr Lys Ser Tyr Arg Gly Arg Arg Ser
1 5 10 15
Arg
<210> 3
<211> 17
<212> PRT
<213> 人工序列(Artificial Sequence)
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Lys Trp Arg Gly Trp Phe Arg Trp Tyr Arg Val Trp Tyr Ile Arg Trp
1 5 10 15
Arg
<210> 4
<211> 17
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 4
Lys Trp Val Phe Arg Val Arg Arg Tyr Arg Gly Ile Val Tyr Val Val
1 5 10 15
Arg
Claims (4)
1.一种寡肽，其特征在于：序列如Sequence NO.2所示。
2.权利要求1所述的寡肽用作达氏鲟精子抗冻剂的用用途。
3.一种达氏鲟精子抗冻液，其特征在于：由权利要求2所述的抗冻剂和稀释剂组成。
4.根据权利要求3所述的抗冻液，其特征在于：所述稀释剂为0.7％氯化钠+0.02％氯化钾+3.5％葡萄糖的去离子水溶液。
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Application publication date: 20190108 |