RHD基因的克隆及其在K562细胞中的表达

Cloning of Human RHD Gene and Its Expression in K562 Cells

本研究目的在于克隆人类红细胞RHD基因,构建RhD表达载体,观察其在K562细胞中的表达。从脐血网织红细胞中提取总RNA,采用逆转录聚合酶链反应扩增RHD/CE基因,扩增产物通过TA连接克隆到pGEMT质粒,采用测序方法筛选多个克隆获得RHD基因。将获得的RHD基因进一步亚克隆构建pcDNA3.1(-)表达载体,重组表达载体通过superfect试剂盒转染K562细胞,最后观察K562细胞中RHD基因的转录和表达。结果表明:成功分离克隆到RHD基因,构建的pcDNA3.1(-)重组表达载体经酶切和测序证实RHDcDNA序列及插入方向正确。转染后的K562细胞内有相应的mRNA转录,细胞表面有RhD抗原表达。结论:RHDcDNA转染的K562细胞能表达RhD抗原,该表达体系有助于进一步研究各种RhD变异型的分子基础。

The aim of this study was to clone human RHD gene and to investigate its expression in transduced K562 cells. Total RNA was extracted from reticulocyte of cord blood. RHD and RHCE genes were amplified using RT-PCR method. The amplified products were cloned into pGEM-T plasmid by TA ligation and several clones were screened by direct sequencing method in order to obtain the RHD gene. RHD gene was subcloned into p cDNA3.1(-) expression vector, then the recombined plasmids were transduced into K562 cells with superfect transfection reagent kit. Finally transcription and expression of RHD gene in K562 cells were detected. The result showed that RHD gene has been cloned sucessfully, the inserted sequence and direction of RHD cDNA in its recombined p cDNA3.1(-) vector were identified using enzyme cutting and sequencing method. After transduced with recombined p cDNA3.1(-) vector, K562 cells could transcribe RHD mRNA in its cytoplasm and express RhD antigen on its membrane surface. In conclusion, RhD antigen can expressed in K562 cells with RHD cDNA transduction, and the expression system in vitro may be helpful to further investigate the molecular basis of RhD variants.