运用SELEX技术筛选特异性高亲和IVB型纤毛结构蛋白RNA适配子  

Selection of RNA Aptmer to Type IVB Pili Structural Protein of Salmonella typhi by SELEX Technique

作　　者：潘勤[1] 章晓联[1] 汪付兵[1] 何攀文[1] 吴红艳[1] 屈雪菊[1] 戚中田[2] 

机构地区：[1]武汉大学医学院免疫学系,武汉430071 [2]第二军医大学微生物教研室,上海200433

出　　处：《中国生物制品学杂志》2004年第5期280-283,共4页Chinese Journal of Biologicals

基　　金：国家基金委课题 ( 3 0 2 70 0 76) ;湖北省杰出青年基金 ( 2 00 2AC0 17) ;武汉大学科技创新基金 ( 3 0 12 70 0 5 5 )资助

摘　　要：目的　获得特异性高亲和IVB型纤毛结构蛋白prepilS的RNA适配子 (aptamers) ,为开发预防、治疗伤寒杆菌肠热症的新型药物试剂奠定基础。方法　采用SELEX技术 (SystematicEvolutionofLigandsbyExponentialEn richment)筛选IVB型纤毛结构蛋白prepilS的特异性适配子 ,通过体外构建一个长度为 88nt含有 30个随机序列的单链DNA(ssDNA)文库 ,PCR扩增为双链DNA随机文库后 ,体外转录构建RNA随机文库 ,以Sepharose 4B为筛选介质 ,筛选具有特异性高亲和力prepilS蛋白的RNA适配子。结果　经 8轮筛选获得具有特异性高亲和prepilS蛋白的RNA适配子库。结论　成功地筛选出具有特异性高亲和力的RNA适配子库 ,该RNA适配子库可显著抑制伤寒杆菌侵入THPObjective To obtain the RNA aptamer to type IVB pili structural protein prepilS of Salmonella typhi and lay a foundation of developing a new drug for the prevention and treatment of enteric fever caused by S. typhi.Methods Construct a single-stranded DNA (ssDNA) library containing 30 random sequences, at a length of 88 nt, in vitro. A double-stranded DNA library was amplified from the ssDNA library by PCR and used for the construction of a RNA random library by in vitro transcription. Select the RNA aptamer to prepilS, with a high affinity, by SELEX(Systematic Evolution of Ligads by Exponential Enrichment) technique using Sepharose 4B as a medium.Results A RNA aptamer library to prepilS, with high affinity, was obtained by 8 rounds of selection.Conclusion A RNA aptamer library to prepilS, with high affinity, was successfully selected and might be used for inhibiting the invasion of S.typhi into THP-1 cells.

关 键 词：伤寒杆菌 m型纤毛结构蛋白 RNA适配子 人体细胞 

分 类 号：R378.23[医药卫生—病原生物学]