摘要
体外合成全长为71个碱基的随机ssDNA文库,采用SELEX技术,以玉米赤霉烯酮单克隆抗体为靶蛋白,微孔板为筛选介质进行筛选。通过对比不对称PCR法和生物素-链霉亲和素磁珠2种分离方法制备ssDNA文库的效果,确定了以生物素-链霉亲和素磁珠分离方法制备ssDNA为优选方法。ssDNA文库扩增为双链DNA(dsDNA)文库的PCR反应条件为:退火温度50℃,循环数20次。不对称PCR退火温度为50℃,最佳循环数为30次,引物Ⅰ与引物Ⅱ的比例为80∶1。此反应条件下,ssDNA文库PCR扩增的条带清晰、稳定、特异性高。生物素-链霉亲和素磁珠法为有效筛选特异性强的玉米赤霉烯酮单克隆抗体适配子的优选技术方案,为适配子SELEX筛选奠定基础。
A 71 bp of random DNA library was synthesized in vitro, anti-zearalenone monoclonal antibodies were regarded as target protein,and the screening process for aptamer was performed with microwell plate by SELEX method. We compared the separation effects of two methods using asymmetric PCR or streptavidin-coated magnetic beads adsorption to separate and prepare for ssDNA. The method of streptavidin-coated magnetic beads adsorption to separate and prepare for ssDNA was determined. The optimal combination of PCR reaction system for random ssDNA pool was obtained. PCR conditions for ssDNA amplified as double-stranded DNA （dsDNA） were that annealing temperature 50 ℃ and the optimal number of cycles 20. For asymmetrical PCR,the optimal annealing temperature was 50℃and the optimal number of cycles was 25, the proportion of upstream primer and downstream primer was 80.. 1. Under the above conditions, clear and stable bands with high specificity for ssDNA pool were amplified. The method of streptavidin-coated magnetic beads adsorption to separate and prepare for ssDNA was anoptimized technical proposal to effectively screen anti-zearalenone monoclonal antibodies aptamer with high specificity,and therefore lay a foundation for aptamer screening by SELEX.
出处
《上海交通大学学报（农业科学版）》
2013年第5期54-60,共7页
Journal of Shanghai Jiaotong University(Agricultural Science)
基金
国家863计划项目(2007AA10Z424)
