跨膜型CD59分子的构建及其在小鼠NIH3T3和EL-4细胞表达的研究

RECOMBINATION AND EXPRESSION OF TRANSMEMBRANE FORM OF HUMAN CD59 ON MOUSE NIH3T3 AND EL 4 CELLS

应用ＲＴ－ＰＣＲ方法，从Ｕ９３７细胞总ＲＮＡ中扩增得到编码人ＣＤ４６分子跨膜区和膜内区ｃＤＮＡ片段，用ＰＣＲ方法扩增得到编码成熟的ＣＤ５９胞外区蛋白的ｃＤＮＡ片段，连接并构建了跨膜型的ＣＤ５９分子ｃＤＮＡ。快速克隆于ｐＧＥＭ－ＴＥａｓｙ载体进行序列测定，证实了其阅读框的完整和序列的正确性。进一步将ｃＤＮＡ重组于逆转录病毒ｐＬＸＳＮ载体，电穿孔转染ＰＡ３１７细胞，用病毒上清感染小鼠ＮＩＨ３Ｔ３、ＥＬ－４细胞，经ＦＡＣＳ检测筛选获得表达重组ＣＤ５９ＴＭ分子的阳性细胞克隆。本研究为更深入地研究ＧＰＩ锚固的ＣＤ５９分子与细胞活化信号转导的关系建立了可靠的细胞模型；同时也为探讨应用跨膜型的ＣＤ５９分子对ＰＮＨ进行基因治疗奠定了良好的基础。

In this paper,the transmembrane form of CD59(CD59 TM) cDNA was constructed by linking the extracellular portion of CD59 to the transmembrane and cytoplasmic domains of MCP,which was amplified by RT PCR method from total RAN of U937 cells.The fused cDNA fragement was cloned into pGEM Easy plasmids and sequenced.The recombinant CD59 TM cDAN was further subcloned into retroviral vector pLXSN and after transfected into packaging cell line PA317,mouse fibroblasts and thymotase cell line EL 4 were infected with the virus resulting in stable expression of CD59 TM on the cell surface.The success of construction and expression of CD59 TM molecule on NIH3T3 and EL 4 cells provides a useful model to study the significance of the GPI anchor in si gnal transduction,and also pave a way for retroviral gene therapy for PNH patients.