CN117466976A - 一种花斑副沙鳅促性腺激素释放激素类似物及复合催产剂及应用 - Google Patents
一种花斑副沙鳅促性腺激素释放激素类似物及复合催产剂及应用 Download PDFInfo
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Abstract
本发明涉及渔药领域，更具体涉及一种花斑副沙鳅促性腺激素释放激素类似物及复合催产剂及应用。本发明提供的花斑副沙鳅促性腺激素释放激素类似物pfGnRHa具有显著的促进花斑副沙鳅卵细胞和精细胞成熟的作用。修饰设计的pfGnRHa是一种高效的、特异针对花斑副沙鳅人工催产的活性肽。利用该活性肽与地欧酮、绒促性素复配而成的复合催产剂，可显著促进卵子或精子成熟及流出，该复合催产剂运用花斑副沙鳅人工繁育生产具有明显的经济效益。
Description
技术领域
本发明涉及渔药领域，更具体涉及一种花斑副沙鳅促性腺激素释放激素类似物及复合催产剂及应用。
背景技术
花斑副沙鳅(Parabotia fasciata)隶属鲤形目，鳅科，沙鳅亚科，沙鳅属，是我国特有鱼类，长江流域各水系分布丰富。花斑副沙鳅是生活在水质良好的流水或缓流水环境中的底栖鱼类。花斑副沙鳅肉质鲜美、营养丰富，四川、重庆、湖北宜昌等地居民喜食该鳅，经济价值高。市场上销售的花斑副沙鳅基本来源于野生捕捞。近年，由于水土流失、水利工程建设等影响，使其原有的产卵场和索饵场受到破坏，加之过度捕捞导致江河资源急剧下降。长江全面禁捕后需要开展花斑副沙鳅的人工繁育和养殖，才能满足我国居民对花斑副沙鳅的消费需求。花斑副沙鳅需要建立人工催产繁育技术。目前，市场上没有针对花斑副沙鳅的催产剂，难以开展花斑副沙鳅的人工繁殖。
针对上述问题，申请人针对花斑副沙鳅内源诱导精、卵成熟的关键调因子促性腺激素释放激素的核心活性肽编码蛋白进行分析，设计并合成了高效类似物。基于花斑副沙鳅促性腺激素释放激素类似物研制出特异复合催产剂，并运用于花斑副沙鳅全人工催产繁殖。
发明内容
本发明的目的在于提供了一种花斑副沙鳅促性腺激素释放激素类似物，所述的类似物为Asn-Thr-Gly-Pro-Tyr-[d-Arg]-Leu-Ser-Pro-Net。
本发明的另一个目的在于提供了花斑副沙鳅复合催产剂，所述的复合催产剂包括花斑副沙鳅促性腺激素释放激素类似物、地欧酮、绒促性素。
本发明的还有一个目的在于提供了花斑副沙鳅促性腺激素释放激素类似物在制备花斑副沙鳅复合催产剂中的应用。
本发明的还有一个目的在于提供了利用上述复合催产剂花斑副沙鳅人工催产方法，方法简单，催产效率高。
本发明最后一个目的在于提供了上述类似物、复合催产剂或催产方法在花斑副沙鳅育种或人工养殖中的应用。
为了达到上述目的，本发明采取以下技术措施：
一种花斑副沙鳅促性腺激素释放激素类似物，所述的类似物为Asn-Thr-Gly-Pro-Tyr-[d-Arg]-Leu-Ser-Pro-Net(天冬酰胺-苏氨酸-甘氨酸-脯氨酸-酪氨酸-右旋精氨酸-亮氨酸-丝氨酸-脯氨酸-乙胺基)。
花斑副沙鳅复合催产剂，包括花斑副沙鳅促性腺激素释放激素类似物、地欧酮、绒促性素。
以上所述的复合催产剂，优选的，所述的类似物pfGnRHa、地欧酮DOM、绒促性素HCG的比例为：0.2-0.5μg：0.1-0.5mg：30-70IU。
以上所述的复合催产剂，优选的，所述的类似物pfGnRHa、地欧酮DOM、绒促性素HCG的比例为：0.25μg：0.3mg：50IU。
一种花斑副沙鳅人工催产方法，包括将上述复合催产剂注射至花斑副沙鳅繁殖期，性腺成熟的雄鱼或雌鱼体中。
本发明的保护范围还包括，上述催复合催产剂或催产方法在花斑副沙鳅育种或人工养殖中的应用。
本发明与现有技术相比，具有以下优点和效果：
市场上缺乏花斑副沙鳅的催产剂，现有的商业化的鱼类催产剂对花斑副沙鳅的催产效果不明确。本发明提供的修饰设计的促性腺激素释放激素类似物pfGnRHa具有显著的促进花斑副沙鳅卵子成熟排出以及精子成熟的作用。修饰设计的pfGnRHa是一种高效的、特异针对花斑副沙鳅人工催产的活性肽。该催产剂运用花斑副沙鳅人工繁育生产具有明显的经济效益。
附图说明
图1为注射复合催产剂促进花斑副沙鳅繁殖获得受精卵以及胚胎发育示意图。
其中：左图：花斑副沙鳅受精卵；右图：花斑副沙鳅胚胎发育图，数字编号为不同发育时期。
具体实施方式
本发明所述技术方案，如未特别说明，均为本领域的常规方案；所述试剂或材料，如未特别说明，均来源于商业渠道。
实施例1：
花斑副沙鳅促性腺激素释放激素类似物的获得：
A、分析花斑副沙鳅内源促性腺激素释放激素编码基因结构，获得发挥生物活性的促性腺激素释放激素核心十肽，其氨基酸序列为：Asn-Thr-Gly-Pro-Tyr-Gly-Leu-Ser-Pro-Gly。
B、针对核心十肽的特定位点进行修饰设计：第六位Gly转换为d-Arg，第十位Gly转换为NEt。
C、人工合成获得修饰设计的pfGnRHa活性肽，其结构式为：Asn-Thr-Gly-Pro-Tyr-[d-Arg]-Leu-Ser-Pro-Net，即天冬酰胺-苏氨酸-甘氨酸-脯氨酸-酪氨酸-右旋精氨酸-亮氨酸-丝氨酸-脯氨酸-乙胺基。
实施例2：
花斑副沙鳅复合催产剂的获得：
将花斑副沙鳅促性腺激素释放激素类似物pfGnRHa、地欧酮DOM、绒促性素HCG按照剂量单位比例1：1000：(1000-1500)混合，既得。
所述的花斑副沙鳅促性腺激素释放激素类似物结构式为：Asn-Thr-Gly-Pro-Tyr-[d-Arg]-Leu-Ser-Pro-NEt。
在本实施例中，花斑副沙鳅促性腺激素释放激素类似物、地欧酮、绒促性素的比例为0.25μg：0.3mg：50IU。
实施例3：
不同的催产药物用于花斑副沙鳅人工催产：
实验过程如下：
以市售的鱼类催产剂促黄体素释放激素LHR-A2、绒促性素HCG、地欧酮(DOM)(购自宁波三生生物科技有限公司)混合物为对照，对比分析本发明设计的pfGnRHa促进雌性花斑副沙鳅卵子成熟和雄鱼精子成熟的效果差异。
花斑副沙鳅繁殖期，选取性腺成熟的雌性亲本20尾(体重约30克/尾)，雄性亲本20尾(体重约20克/尾)。雌雄亲本鱼随机分为2组，每组含10尾雌鱼和10尾雄鱼。放置于水温26℃的水箱，微流式充氧，适用2天后，注射复合催产剂。
对照组：复合催产剂为0.25μg LHR-A2、0.3mg DOM、50IU HCG溶解于0.2毫升的生理盐水，胸鳍基部注射到雌鱼。雄鱼采用相同的注射方法，但是药量减半。注射催产剂的花斑副沙鳅放回水箱，保持26℃水温待产。
实验组：实验组催产剂为0.25μg pfGnRHa(委托南京金斯瑞生物科技有限公司合成)和0.3mg DOM、50IU HCG溶解于0.2毫升的生理盐水，胸鳍基部注射雌性花斑副沙鳅。雄鱼采用相同的注射方法，但是药量减半。注射催产剂的花斑副沙鳅放回水箱，保持26℃水温待产。
注射后7.5-8小时，对照组有6条雌鱼卵子成熟，实验组8条雌鱼卵子成熟，轻压腹部流出成熟的卵子。对照组和实验组雄鱼挤压腹部都流出精液，实验组雄鱼的精液量明显多于对照组。综上所述，注射含pfGnRHa的复合催产剂促进花斑副沙鳅精卵成熟的效果明显优于现有市售的鱼类催产剂。
Claims (8)
1.一种花斑副沙鳅促性腺激素释放激素类似物，所述的类似物为天冬酰胺-苏氨酸-甘氨酸-脯氨酸-酪氨酸-右旋精氨酸-亮氨酸-丝氨酸-脯氨酸-乙胺基。
2.权利要求1所述的类似物在制备花斑副沙鳅复合催产剂中的应用。
3.一种花斑副沙鳅复合催产剂，所述的花斑副沙鳅复合催产剂包括权利要求1所述的类似物、地欧酮和绒促性素。
4. 根据权利要求3所述的复合催产剂，所述的类似物、地欧酮、绒促性素的比例为：0.2-0.5 μg：0.1-0.5 mg：30-70 IU。
5. 根据权利要求4所述的复合催产剂，所述的类似物、地欧酮、绒促性素的比例为：0.25 μg：0.3 mg：50 IU。
6.一种花斑副沙鳅人工催产方法，包括将权利要求3所述的复合催产剂注射至花斑副沙鳅繁殖期的性腺成熟的雄鱼或雌鱼体中。
7.权利要求1所述的复合催产剂、权利要求3所述的复合催产剂或权利要求6所述的催产方法在花斑副沙鳅育种中的应用。
8.权利要求1所述的复合催产剂、权利要求3所述的复合催产剂或权利要求6所述的催产方法在花斑副沙鳅人工养殖中的应用。
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