人RHD基因的克隆和鉴定

Cloning and characterization of human RHD gene

目的克隆人RHD基因,并对其进行鉴定。方法以RhD阳性志愿者骨髓为材料,用TRIzol试剂提取总RNA;设计、合成人RHD基因扩增引物,RT-PCR方法扩增RHD基因片段;T/A克隆后将其亚克隆入pET28a(+)载体中,经酶切、PCR和测序对重组质粒进行鉴定。结果骨髓总RNA被成功提取;RT-PCR成功扩增出RHD基因片段,其大小与预期约509 bp基本一致;T/A克隆后再将其亚克隆,通过酶切和PCR证明RHD基因成功亚克隆入pET28a(+)载体中;基因测序结果比对显示,与已公布的RHD基因(GenBank登录号为NM016124)序列基本一致,同源性为98%。结论成功克隆了RHD基因,这将为进一步研究奠定基础。

Objective To clone RHD gene,and to identify it. Methods The RNA was picked up from bone marrow of an RhD-positive volunteer with the TRIzol reagent. The primers of the target gene were designed and synthesized, and then the fragment of RHD gene was amplified by RT-PCR. The amplified product was cloned by T/A and was then subcloned into pET28a vector. The recombinant plasmid was identified by restriction enzyme digestion, PCR and sequencing. Results The total RNA of bone marrow was successfully picked up. The RHD gene was amplified by RT-PCR and the size of amplified product was 509 bp. The gene was cloned, subcloned and inserted in plasmid correctly, which was verified by restriction enzyme and PCR. As a result,the pET28a expression vector carrying RHD gene was recombined correctly. The DNA sequencing of RHD gene showed 98% homology with NM016124. Conclusions The RHD gene was successfully cloned. This will establish foundation for further research.