【摘 要】
：
芋螺毒素(Conotoxin,CTxs)是由生活在热带海洋中的肉食性软体动物芋螺分泌出来的一种麻醉猎物的小肽类毒素,在镇痛、戒烟戒毒和抗癌等方面具有广阔的应用前景.芋螺毒素的种类繁多,生物活性多样,目前全世界发现的芋螺约有800多种,但由于芋螺的采集费时费力,芋螺资源日渐稀少,建立稀有芋螺毒素基因资源库至关重要,以期长期保存和可持续开发利用.目前,基于PCR的文库筛选方法已经基本替代了传统的杂交筛
【机 构】
：
海南大学热带生物资源教育部重点实验室,海口市海洋药物重点实验室,海南海口570228;海南大学农学院,海南海口570228
【出 处】
：
第12届生物毒素研究及医药应用学术大会
论文部分内容阅读
芋螺毒素(Conotoxin,CTxs)是由生活在热带海洋中的肉食性软体动物芋螺分泌出来的一种麻醉猎物的小肽类毒素,在镇痛、戒烟戒毒和抗癌等方面具有广阔的应用前景.芋螺毒素的种类繁多,生物活性多样,目前全世界发现的芋螺约有800多种,但由于芋螺的采集费时费力,芋螺资源日渐稀少,建立稀有芋螺毒素基因资源库至关重要,以期长期保存和可持续开发利用.目前,基于PCR的文库筛选方法已经基本替代了传统的杂交筛选方法,适用于cDNA文库、基因组文库等各类型的文库筛选.Fosmid文库是一种新兴的基于PCR的文库构建方法,其构建简单,快速,在宿主菌中以单拷贝形式存在,稳定性好,可用于全基因组物理图谱构建、研究基因功能和表达调控、重要性状基因的图位克隆和基因结构及功能分析等.本研究利用Fosmid载体构建海南产芋螺基因组文库.分离芋螺毒腺组织,提取基因组DNA,按照动物基因组提取试剂盒(天根公司)操作说明书进行.提取后的DNA溶液用200μL吸头采用反复吹打的方法,对基因组DNA进行片段化处理,电泳检测DNA片段大小.然后对DNA断裂末端进行补齐和磷酸化修复.进行切胶回收,制备插入片段.将回收的DNA片段与Fosmid载体连接,随后将连接产物加入到包装蛋白中进行噬菌体包装.将包装产物梯度稀释,过夜培养,筛选阳性克隆并计算滴度.最后进行酶切与测序鉴定,甘油保存.
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