地高辛标记的血小板T细胞活化抗原1cRNA探针的制备与鉴定

目的制备用地高辛 (digoxigenin ,Dig)标记的血小板T细胞活化抗原1(plateletandTcellactivationantigen1,PTA1)cRNA探针。方法构建重组质粒 pGEM 3ZF( ) PTA1 ,并做序列分析证实PTA1基因的插入方向正确后 ,用EcoRI消化得到线性DNA片段 ,再用SP6RNA聚合酶转录合成带有Dig标记的高比活度的单链cRNA探针。结果经斑点杂交证实 ,该探针敏感性高、特异性强。结论地高辛标记PTA1cRNA探针的制备 ,为进一步研究PTA1mR NA在组织、细胞的表达和分布提供了有效的工具。

Preparation and identification of digoxigenin labeled platelet and T cell activation antigen 1 cRNA probe

Aim To construct digoxigenin labeled platelet and T cell activation antigen 1 cRNA probe. Methods A recombinant plasmid pGEM 3ZF( ) PTA1 was constructed by molecular cloning techniques for the preparation of digoxigenin labeled PTA1cRNA probe. Sequencing showed that recombinant plasmid pGEM 3ZF( ) PTA1 did contain the DNA fragment of PTA1 gene and insertion direction was correct. We used the plasmid as a template to synthesize the digoxigenin labeled RNA probes by means of transcription procedure in the presence of SP6 RNA polymerase. Results The probe was identified by dot hybridization with purple blue presentation on the positive lableled sites. Conclusion Digoxigenin labeled PTA1 cRNA probe could be used for further research on the distribution, expression and function of PTA1 in the cells and tissues.