地高辛标记的大鼠p75RNA探针的制备和应用  

The preparation of p75 RNA probelabeled with digoxingenin

作　　者：沈宓[1] 丁斐[1] 

机构地区：[1]江苏省神经再生重点实验室南通医学院,江苏南通226001

出　　处：《中国临床解剖学杂志》2005年第5期518-520,共3页Chinese Journal of Clinical Anatomy

基　　金：国家自然科学基金资助(30270427);江苏省教育厅高校科研项目(00KJD31007)

摘　　要：目的:制备用地高辛标记的神经生长因子低亲和力受体(p75)的RNA探针。研究p75在海马组织中的表达。方法:设计p75引物,构建p75/pGEM-T重组质粒,分别用ApaⅠ和SacⅠ进行酶切得到线性化DNA片段,以Sp6和T7聚合酶转录合成酶合成地高辛标记的(dig-)正反义RNA探针。运用点膜杂交的方法检验探针的敏感度,运用该探针,通过原位杂交分析p75在海马中的表达。结果:构建了p75/pGEM-T质粒,获得高效价的正、反义dig-p75RNA探针,应用该探针发现p75mRNA在海马中的表达。结论:成功制备了地高辛标记的p75RNA探针,为进一步研究p75在海马中发育和损伤过程中的表达打下基础。Objective： In order to study the expression locations of low-affinity Nerve Growth Factor Receptor（p75）, the sense and anti-sense p75 RNA probes for the analysis of in situ hybridization were prepared in this study. Methods：The fi：agrnent of p75 was obtained by RT-PCR through total RNA of rat brains. Amplified cDNA fragment was subcloned into pGEM-T Easy. The plasmids were linearized with the restriction enzymes of Apa Ⅰ and Sal Ⅰ. Using SP6 and T7 RNA polymemse respectively, the digoxigenin （dig） labeled sense and anti-sense probes were produced in vitro by transcription according to its protocol. Rcsults： Certificated by titer measuring and specificity analysis, the sense and anti-sense dig-p75 RNA probes were prepared successfully. Conelusions： The sense and anti-sense RNA probes for the analysis in situ hybridization of p75 are prepared in this experiment, which will provide an approach to study further the location of p75 particularly in hippocampus.

关 键 词：神经生长因子(p75) 大鼠 地高辛标记p75RNA探针 原位杂交 

分 类 号：Q349.53[生物学—遗传学]