重组人pLXSN-CD80逆转录病毒载体的构建及在CHO和PA317细胞中的表达

CD80是表达于抗原提呈细胞表面的分化抗原,它提供T细胞活化的协同刺激信号,并在抗肿瘤免疫应答中起着重要作用。我们在克隆了CD80全长。cDNA的基础上,将其与逆转录病毒pLXSN表达载体连接,构建成表达质粒,并用磷酸钙沉淀法将其转染PA317和CHO细胞,经G_(418)筛选获得抗G_(418)的PA317和CHO细胞克隆。以RIA,FACs和Westernblot检测CD80分子在PA317和CHO细胞上的表达、分布和分子量,结果显示,pLXSN-CD80转染的CHO细胞可表达较高丰度的CD80分子,其表观分子量为40kD。pLXSN-CD80转染的PA317和CHO细胞经5个月连续传代培养,无论存在G_(418)的选择压力与否,仍然持续表达CD80分子,表明我们构建的pLXSN-CD80表达载体具有应用于试验性治疗的潜能。

CONSTRUCTION OF HUMAN pLXSN-CD80 RETROVIRUS VECTOR AND EXPRESSION IN CHO AND PA317 CELLS

CD80, a molecule on the antigen presenting cells, provide costimulatkm signals for T cell activation and play a key role in tumor immune. Based on our previous work of human CD80 full length cDNA cloning, a retrovirus expression vector pLXSN-CD80 was constructed. CD80 expression cells were selected by G418 from pLXSN-CD80 transfected PA317 and CHO cells by calcium phosphate. Expression, distribution and molecular weight (MW) of CD80 were mea-sured by RIA, FACs and western blot. pLXSN-CD80 transfected CHO cells expressed relatively high level of CD80 protein (approximately the same as Raji cells) with an apparent MW of 40 kD. In the presence of G418 or not, pLXSN-CD80 transfected PA317 and CHO cells maintained CD80 expression for five months of passage. The results indicate that our construct is potent for experimental use in gene therapy.