中华皮肤科杂志 ›› 2008, Vol. 41 ›› Issue (10): 699-700.

• 技术与方法 • 上一篇    下一篇

体外SELEX法筛选淋病奈瑟菌适体方法的建立

许少涵 兰小鹏   

  1. 福建省皮肤病性病防治院 福州总医院皮肤科
  • 收稿日期:2007-12-14 修回日期:2008-01-19 发布日期:2008-10-15
  • 通讯作者: 许少涵 E-mail:fzxu1237@hotmail.com

In vitro SELEX as a method for screening of ssDNA aptamer against Nesseria gonorrhea

XU Shao-Han   

  • Received:2007-12-14 Revised:2008-01-19 Published:2008-10-15
  • Contact: XU Shao-Han E-mail:fzxu1237@hotmail.com

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摘要： 目的 建立运用SELEX技术从人工体外合成的随机ssDNA文库中筛选到针对淋病奈瑟球菌适体的方法 方法 取一定量体外合成随机寡核苷酸文库结合缓冲液中95℃加热5分钟，迅速4℃冷却10分钟，再投入适量的淋病奈瑟菌，室温摇匀30分钟后更换离心管离心分离，使用洗脱液悬起冲洗，反复4次，尽量洗去结合不强的片段。最后菌体和目的片段的复合体在99℃的蒸馏水加热3分钟后中释放出所筛选的目的片段，纯化目的片段，以之为模板,经PCR大量扩增，产物纯化后做为下轮的文库。在反复筛选10轮后，利用卡它莫拉氏菌，干燥奈瑟氏菌，灰色奈瑟氏菌进行了反筛工作，最终获得所需的针对淋病奈瑟菌的适体。 结果 筛选到针对淋病奈瑟球菌的特异性适体 结论 成功运用SELEX技术从人工体外合成的随机ssDNA文库中筛选到针对淋病奈瑟球菌的特异性适体，为建立快速而简便检测淋病奈瑟菌的实验室方法奠定基础。

Abstract: Objective This study aim to screen the aptamer with high affinity and specificity for N . gonorrhoeae and set a base for establishing relative easy and quick laboratory detection methods. Methods ssDNA pools are used in the selection process,ssDNA is heat-denatured at 95℃ for 5 min ,and then cooled immediately to 4℃ in binding buffer.Selection is performed by incubating ssDNA pools with bacterial at room temperature for 25-30 minute in binding buffer by gentle rotation.After 30 min the aptamers-bacterial antigen complex is centrifuging and washed four times with washing buffer.ssDNA retained on the bacterial is elute after heat by 99℃ ddH2O. Selected ssDNAs are amplified by PCR and used for the next round selection. After round 10th ,