论文部分内容阅读
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。
RT-PCR method was used to amplify the cDNA encoding human CD46 transmembrane region and intramembrane region from the total RNA of U937 cells. The cDNA fragment encoding the mature CD59 extracellular region was amplified by PCR and ligated and constructed Transmembrane CD59 molecule cDNA. Rapid cloning in pGEM-TEasy vector sequence determination, confirmed the integrity of its reading frame and the correct sequence. The cDNA was further recombined into retroviral vector pLXSN and electroporated into PA317 cells. The NIH3T3 and EL-4 cells were infected with virus supernatant and positive clones expressing recombinant CD59TM were obtained by FACS. In this study, we established a reliable cell model to further study the relationship between GPI-anchored CD59 molecules and cell activation signal transduction, and laid a good foundation for further studies on the gene therapy of PNH using transmembrane-typed CD59 molecule.