细胞凋亡相关新基因Apr-3转录剪接体的克隆、测序及亚细胞定位

Cloning,sequencing and subcellular localization of a transcript variant of a noval apoptosis-related gene Apr-3

目的克隆Apr-3基因的一个转录剪接体,并对其进行测序及亚细胞定位。方法培养HL-60细胞,提取HL-60细胞总RNA,应用RT-PCR获取Apr-3的转录剪接体的编码区cDNA序列,将该cDNA与质粒pcDNA3·1/myc-His(-)B连接,构建克隆载体,将其转化E.coli DH5α,进行测序,与GenBank中登录序列比对结果正确后将该cDNA与质粒pEGFP-C3连接,并将其转染COS-7细胞。结果对测序结果进行序列分析,与GenBank中登录的Apr-3编码区完全一致。pEGFP-Apr-3基因表达产物定位于COS-7细胞的细胞膜和细胞质。结论对Apr-3基因的一个转录剪接体进行克隆,构建了真核表达载体,首次发现Apr-3的该转录剪接体主要在真核细胞的细胞膜和细胞质表达,为后期对该基因的结构和功能的研究奠定基础。

Purpose To clone a transcript variant of the apoptosis-related gene Apr-3 and to ascertain the subcellar localization of Apr3. Methods Total RNA was isolated from the ceils of HL-60. The cDNA encoding the transcript variant of Apr-3 was amplified by reverse transcription-PCR （RT-PCR）. PCR products were cloned into pcDNA3.1/myc-His（ - ）B vector and sequenced. Subcellar localization of the transcript variant of Apr-3 was performed. Results The transcript variant of Apr-3 cDNA fragment of 690 bp was obtained. It was cloned into pcDNA3.1/myc-His（ - ）B vector and the sequence was confirmed. Gene products of the transcript variant of Apr-3 were located in the cell membrane and cytoplasm of COS-7 cells. Conclusions The transcript variant of Apr-3 expresses in the cell membrane and cytoplasm of eucaryotic ceils.