RHD mRNA替代剪接区域研究

摘要

目的 2007年国内报道一例弱D型个体存在第4～9外显子选择性剪接的转录子,我们探讨正常Rh(D)阳性个体的RHD基因mRNA的选择性剪接区域.方法 随机选择3名Rh(D)阳性个体,从新鲜全血中提取总RNA,通过特异性引物,采用"一步法"逆转录-PCR(RT-PCR),扩增RHD mRNA第1～7外显子区域,以及第6～10外显子区域,然后cDNA琼脂糖凝胶电泳和成像分析.结果 未发现第1～7外显子区域存在mRNA的选择性剪接条带,仅存在由特异性引物所扩增的第1～7外显子全长的序列条带;而第6～10外显子区域观察到5种替代剪切条带,序列分析显示分别为无缺失片段,以及完整缺失第7、第9、第7和9、第7～9外显子5种RHD转录子.结论 正常Rh(D)抗原阳性个体的RHD基因mRNA的选择性剪接仅存在于第7～9外显子区域.%Objective It was reported in 2007 that the alternative splicing regions between ex-ons 4-9 exists in a weak D type individual. Here we sought to investigate the regions in RHD gene mRNA in normal Rh-positive individuals. Methods Total mRNA from whole blood was isolated from three normal Rh ( D ) -positive individuals. The region of RHD mRNA from exon 1 to 7 and the region from exon 6 to 10 were amplified through a one-step RT-PCR method. Then cDNA were separated through electrophoresis. Results A single cDNA band for the full length region of exon 1-7 was observed in all three D-positive individuals. However, 5 alternative bands were detected for the region of exon 6-10, which were fragments representative for the sequence with no deletion, and the sequences with full deletion of exon 7, exon 9, exons 7 and 9, exons 7-9. Conclusion Our data suggested that the RHD mRNA alternative splicing regions in normal D-positive individual occur in exons 7-9.