基于文库固定化的内毒素适配体定向筛选
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- 英文论文题名：Orientation selection of DNA aptamers against endotoxin based on library immobilization
- 论文作者：叶华 ; 徐婧 ; 王周平
- 英文论文作者：Ye Hua ; Xu Jing ; Wang Zhouping ; School of Food Science and Technology ; Jiangnan University ; State Key Laboratory of Food Science and Technology
- 年：2016
- 作者机构：江南大学食品学院;食品科学与技术国家重点实验室;
- 论文关键词：内毒素 ; 适配体 ; 定向筛选
- 英文论文关键词：endotoxin ; aptamer ; orientation selection
- 会议召开时间：2016-11-09
- 会议录名称：中国食品科学技术学会第十三届年会论文摘要集
- 英文会议录名称：Abstracts of the 13th Annual Meeting of CIFST
- 语种：中文
- 分类号：Q78
- 学会代码：ZGSP
- 会议名称：中国食品科学技术学会第十三届年会
- 会议地点：中国北京
- 主办单位：中国食品科学技术学会
- 学会名称：中国食品科学技术学会
- 页数：1
- 文件大小：56k
- 原文格式：O
- 会议级别：全国
摘要
内毒素(Endotoxin)又名脂多糖(Lipopolysaccharide),是革兰氏阴性菌细胞壁外膜主要成分,由类脂A、核心多糖及O-特异性多糖三部分组成,其中类脂A是活性中心,且位于内层,具有高度保守性,而多糖链位于外侧,具有多样性。有研究表明,大分子物质比小分子可能有更多的适配体结合位点。另外,固定靶标有可能会影响靶标物质的天然构象、增加空间位阻作用,从而限制了适配体在实际分析检测中的应用。鉴于内毒素的特殊结构,本试验以鼠伤寒沙门氏菌内毒素为靶标,将寡核苷酸文库通过生物素-亲和素作用固定在磁珠上,并游离靶标以保持内毒素的天然构象,以鼠伤寒沙门氏菌为定向物质进行定向筛选,以BSA为反筛物质进行反向筛选,经过15轮筛选,得到一条亲和力高同时具有一定的抑制内毒素活性,且能特异性结合内毒素的适配体。本试验建立的基于固定文库定向筛选方法,避免了固定靶标对天然结构构象变化的影响,验证了定向筛选的有效性,也为今后适配体-靶标相互作用机制研究奠定了良好的基础。
Endotoxin,also called Lipopolysaccharide(LPS),which layer in the outer membrane of all gram-negative bacteria,is consisted of three parts;lipid A,core polysaccharide and O-specific polysaccharide.Lipid A which is highly conservative that located in the inner layer is the active moiety of LPS,while polysaccharide chain with diversity is located in outer layer.Research has shown that macromolecular substances has more binding site than small molecules for aptamers,and immobilizing targets on a matrix may be affect its natural conformation and increase space steric effects,which limit the aptamers' s application.Due to these,we chose lipopolysaccharides from Salmonella typhimurium as SELEX target.After immobilizing ssDNA library on magnetic bead by biotin-avidin interactions,the Salmonella typhimurium were chose as target for orientation selection,and BSA as target for counter selection.After 15 rounds of selection,an aptamer against endotoxin with high affinity and specificity were captured.Further experiments showed that the aptamer has the ability of inhibiting bacterial endotoxin activity.The SELEX strategy in our experiment avoid the negative effects of immobilizing,and verify the effectiveness of the orientation selection.It may give us a good foundation for aptamer-target interaction mechanism research.
Endotoxin,also called Lipopolysaccharide(LPS),which layer in the outer membrane of all gram-negative bacteria,is consisted of three parts;lipid A,core polysaccharide and O-specific polysaccharide.Lipid A which is highly conservative that located in the inner layer is the active moiety of LPS,while polysaccharide chain with diversity is located in outer layer.Research has shown that macromolecular substances has more binding site than small molecules for aptamers,and immobilizing targets on a matrix may be affect its natural conformation and increase space steric effects,which limit the aptamers' s application.Due to these,we chose lipopolysaccharides from Salmonella typhimurium as SELEX target.After immobilizing ssDNA library on magnetic bead by biotin-avidin interactions,the Salmonella typhimurium were chose as target for orientation selection,and BSA as target for counter selection.After 15 rounds of selection,an aptamer against endotoxin with high affinity and specificity were captured.Further experiments showed that the aptamer has the ability of inhibiting bacterial endotoxin activity.The SELEX strategy in our experiment avoid the negative effects of immobilizing,and verify the effectiveness of the orientation selection.It may give us a good foundation for aptamer-target interaction mechanism research.
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