高通量筛选孤儿受体和膜结合配体的MACS/FACS方法的建立

本文报道了一种用于分离、筛选孤儿受体和膜结合配体的高通量的表达克隆系统(expression cloning system).该系统是以高效表达的逆转录病毒载体为核心，以高通量的磁力分离法（magnetic cell sorter，MACS）和高精确度分选型流式细胞仪（FACS）相结合的独特的筛选方法.具体方法是将配体通过生物反应对固定在磁粒上 （如biotin/streptavidin， Fc/protein A等）.该磁粒的特点是非常微小，相当一个病毒颗粒.当配体蛋白结合到表达受体的BaF3细胞上时，该细胞将停留在MACS磁场中，从而与非受体表达细胞分开.MACS的优点是效率高，可同时操作上亿细胞.这些细胞是无法直接在分选型流式细胞仪上操作的.当这些阳性细胞增殖后，再次用同一配体染色，用更精确的分选型流式细胞仪分离，直至完全纯化.受体基因将用PCR方法，用载体引物克隆.利用该系统可以从1000 000个细胞中筛选出低至2个表达配体/受体的阳性细胞.为验证该系统的可行性，应用该系统在相应的cDNA文库中筛选到2个已知基因的细胞受体.应用诱饵B7-1从人T淋巴细胞cDNA文库中筛选其功能受体CD28.应用B7-H2作为诱饵，从活化的人T淋巴细胞cDNA文库中筛选到ICOS.最终，从纯化的受体表达细胞中分离出受体的cDNA编码序列.这些结果表明，改进的表达克隆系统适合于大规模分离孤儿受体和膜结合配体，以及在后基因组时代用于研究新蛋白的功能.

MACS/FACS Expression Cloning Method for High Throughput Screening of Orphan Receptor and Its Membrane-bound Ligands

An improved expression cloning system is reported for the identification of orphan receptors and membrane-bound ligands.The system is mediated by an efficient retrovirus-expression vector in combination with a high throughput screening procedure， involving high throughput magnetic cell sorter (MACS) enrichment and real time fluorescence activated cell sorter (FACS) selection.As low as two ligand/receptor-expressing cells can be recovered from one million background cells via this approach.To validate the system， two known receptors were isolated from cell libraries， namely， the T cell co-stimulator receptor CD28 from BaF3 cells expressing a human T cell cDNA library using B7-1 protein as the bait， and the T cell inducible co-stimulator (ICOS) receptor from BaF3 cells expressing an activated human T cell cDNA library using B7-H2 protein as the bait.Finally，the isolated cDNAs coding for these receptors from the purified cells were determined.These results indicated that the improved system was suitable for large-scale screening for orphan receptors or membrane bound ligands，therefore，might be useful for the functional assessment of new proteins in the post-genomic era.