【摘要】 设计三条引物Ｐ１、Ｐ２、Ｐ３扩增全长ｈＦａｓ基因和只含有跨膜区和胞浆区的ｍＦａｓ基因。Ｐ２上设计一个ＢｓｔＸＩ位点，以与ｈＦａｓ信号肽之后的单酶切位点ＢｓｔＸＩ连接，获得带信号肽的跨膜区和胞浆区ＳｐｍＦａｓ基因。另外设计两条引物Ｐ４、Ｐ５扩增人β－雌二醇受体的激素结合结构域。扩增基因均经ＤＮＡ序列分析得到证实。Ρ３、Ｐ４均设计了ＫｐｎＩ位点，用于两基因融合，并将此融合基因插入真核表达载体ｐｃＤＮＡ３。脂质体法转入ＣＯＳ－７细胞进行瞬时表达，ＷｅｓｔｅｒｎＢｌｏｔ检测到约５７ＫＤａ的表达产物，与融合基因的估算分子量相符。更多还原

【Abstract】 Three primers P 1、P 2、P 3 were used to amplify the whole sequence and the transmembrane and cytoplasma fraction of hFas by PCR. A BstX I site was designed in P 2 to ligate with the BstX I site downstream hFas signal peptide to obtain SpmFas. Other two primers P 4、P 5 were used to PCR amplify the hormone binding domain of human β estrogen receptor. All amplified fragments were verified by sequencing analysis. The two genes were fused at Kpn I site designed in both P 3 and P 4 and then the fusion gene was inserted into pcDNAs.Using lipofectamine mediated gene transfer technique, the fusion gene was tranduced into COS 7 cells and a 57DKa expression band which is in agreement with the calculated Mr was detected by Westen blot analysis.更多还原