大鼠心肌细胞钙调磷酸酶Aβ基因的克隆与真核表达

Cloning and expression of calcineurinAβ gene of cardiocyte in eukaryotic expression system

目的从原代培养的大鼠心肌细胞中扩增钙调磷酸酶(Calcineurin,CaN)AβcD N A序列,构建重组真核表达质粒pcDN A3.1-CaN,并检测其表达情况,为进一步研究C aN及其在心肌损伤的信号转导途径中的作用奠定基础。方法用RT-PCR方法从原代培养的大鼠心肌细胞R N A中扩增出C aN AβcD N A序列,测序后将其用限制性内切酶双酶切,然后与真核表达质粒pcD N A3.1(+)进行连接,再转化D H 5α,将鉴定为阳性的真核表达质粒转染人胚肾293T细胞,用免疫荧光细胞化学技术检测其表达情况。结果①将鉴定为阳性的质粒进行测序后,所测序列结果与G eneBank的C aN Aβ基因序列同源性达到91.7%;②真核表达载体pcDN A3.1-CaN转染293T细胞后经免疫荧光细胞化学染色检测到钙调磷酸酶基因在人胚肾293T细胞中表达。结论正确克隆出了大鼠心肌细胞C aN Aβ基因并建立了CaNAβ基因的真核表达载体pcDN A3.1-CaN。

【Objective】 To construct the plasmid pcDNA3.1-CaN from rat culture cardiocyte and observe the expression of CaN Aβ in eukaryotic expression system.【Methods】 The CaNAβ cDNA amplified by RT-PCR from rat culture cardiocyte was digested with EcoRI、 BamHI and the fragment was clone into the eukaryotic expression vector pcDNA3.1（＋）.The pcDNA3.1-CaN was transformed into E.coli DH5α,which was detected by immumofluorescent cell chemical method.The positive one was transfected into 293T cell and the expression of CaN was observed by immumofluorescent cell chemical method.【Results】 ①The sequence of pcDNA3.1-CaN had 91.7% homology with the CaN Aβ of Genebank;②The pcDNA3.1-CaN was expressed in 293T cells.【Conclusion】 The CaN Aβ gene has been cloned and constructed into the eukaryotic expression vector pcDNA3.1-CaN successfully.