神经生长因子高亲和力受体互补核糖核酸探针的研究  

Study on complementary RNA probe of digoxigenin labeled nerve growthfactor high affinity receptor

作　　者：姜声扬[1] 丁斐[2] 沈爱国[2] 严美娟[2] 沈宓[2] 

机构地区：[1]南通医学院卫生毒理学教研室,江苏南通226001 [2]南通医学院神经科学研究所

出　　处：《卫生毒理学杂志》2003年第4期220-222,共3页Journal of Health Toxicology

基　　金：江苏省高校自然科学研究指导性计划项目 (0 1KJD330 0 0 1 )

摘　　要：目的　制备用地高辛标记的神经生长因子高亲和力受体 (TrkA)正、反义互补核糖核酸 (cRNA)探针的研究。方法　设计TrkA引物 ,构建pGEM TEasy TrkA重组质粒 ,分别用ApaⅠ和SacⅠ进行酶切得到线性DNA片段 ,以SP6和T7RNA聚合酶转录合成带有地高辛标记的高比活度的正、反义单链cRNA探针。结果　经斑点杂交证实 ,该探针敏感性高、特异性强。TrkA ApaⅠ探针稀释 12 5 0 0倍后的浓度为 40ng μl;TrkA SacⅠ稀释 1∶2 5 0 0倍后的浓度为 8ng μl;。 结论　成功制备了地高辛标记的TrkAcRNA探针 ,为毒理学中有关TrkAmRNA在组织。Objective To construct digoxigenin labeled nerve growth factor (NGF) high affinity receptor(Trk A) (+/-) cRNA probe.Methods A recombinant plasmid pGEM-T Easy-Trk A was constructed by molecular cloning techniques for the preparation of digoxigenin labeled Trk A cRNA probe.The recombinant plasmid was lined by ApaⅠ or SacⅠ restriction enzyme separately,and then the inserted fragment of Trk A cDNA was used as templates to synthesize the digoxigenin labeled cRNA probes by means of transcription procedure in the presence of SP6 or T7 RNA polymerase.Results Sequencing showed that recombinant plasmid pGEM-T Easy-Trk A did contain the DNA fragment of Trka gene.After the digoxigenin labeled Trk A (+/-)cRNA probe was constructed,it was identified by dot hybridization with purple-blue presentation on the positive labeled sites.The concentration of Trk A-ApaⅠ probe was 40 ng/μl when it was diluted 12 500 times and the concentration of Trk A-SacⅠ probe was 8 ng/μl when it was diluted 2 500 times.Conclusion Digoxigenin labeled Trk A (+/-) cRNA probe has been successfully prepared and identified.It can be used for further toxicological research on the distribution,expression and functions of Trk A in cells and tissues.

关 键 词：神经生长因子 受体 NGF 互补核糖核酸 eRNA 地高辛 

分 类 号：R971[医药卫生—药品]