RHD基因不同转录子表达载体的构建

Construction of Expression Vector from Different Transcripts of RHD Gene

RHD基因存在多种不同的另路剪接的转录子,本研究分别构建正常mRNA及DEL9和DEL89转录子的表达载体。从Rh(D)阳性新生儿脐血网织红细胞中提取总RNA,分别采用一步法和两步法RT-PCR获取完整目的转录子片段,然后分别克隆至pCR4 TOPO测序载体并测序验证和筛选。用正常RHD cDNA筛选质粒为模板,PCR扩增全长目的基因,然后亚克隆至pcDNA3.1/V5-His TOPO表达载体;DEL9和DEL89转录子直接克隆至表达载体,通过测序验证目的基因序列及插入方向。结果表明,序列分析证实了不同目的基因片段序列和方向正确,表明3种不同RHD转录子的表达载体构建成功。结论:RHD不同转录子表达载体已成功构建,为进一步研究Rh(D)抗原膜表达机理奠定了基础。

RHD gene has different alternative transcripts. This study was aimed to construct expression vector of normal mRNA, DEL9 and DEL89 transcripts from RHD gene. Total RNA was extracted from Rh（D） positive umbilical blood cells of newborn. Intact RhD cDNA, DEL9 and DEL89 transcripts were obtained by one-step and two-step RT-PCR, respectively. The obtained products were cloned into pCR4 TOPO sequencing vector for choosing the right transcript. RHD gene was amplified again from the sequencing plasmid DNA, and then subcloned into pcDNA3.1/VS-His TOPO expression vector; DEL9 and DEL89 were cloned into the expression vector directly. Gene sequence and direction were identified by sequencing. The results showed that the sequence and direction of target genes were right, thus these 3 different expression vectors were correctly constructed. It is concluded that expression vector is constructed from different transcripts of RHD eene. which lavs a foundation for further exnlorin~ the membranous nrotein exnression of Rh（D） antigen_