Retrovirus-based Expression Cloning Method For Identification Of Novel Receptors

At the beginning of the 21st century, after the human genome sequence is completed, investigation of gene function is becoming increasingly important. Receptor of gene will be found to understand gene function and mechanism. Different from current methods like cell models and animal models, this study start from searching receptors in order to open the door of researching new gene function. Ligand and receptor will interaction in vivo, although the affinity of ligand/receptor is different, but in most cases ligand/receptor response could be detected by using labeled ligand or receptor.This study established the high-throughput expression cloning system which is a rapid, sensitive and high-throughput screen system. By using this system, people could separate and screen receptor and membrane-bound ligand. This 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. Based on ligand and receptor combination, we use the MACS/FACS screen system to isolate positive cells from many negative cells, and then amplify positive cells and identify receptor gene.A known ligand signed by Fc is as the bait and combined with Protein G beads, the beads are very small, equivalent to a virus particle, a large number of cells could not be directly separated in flow cytometry. After amplification of positive cells which were screened by MACS, incubate positive cells with ligand protein again, the positive cells are sorted by flow cytometry, and the receptor gene are identified by PCR.As low as two ligand/receptor-expression cells could be isolated from one million normal cells by use this approach. To validate the system, two known receptors were isolated from human T lymphocyte cell library. The T cell inducible co-stimulator (ICOS) receptor was screened from an activated human T lymphocyte cell cDNA library by using B7-H2 protein as the bait; the T cell co-stimulator receptor CD28 was screened from an activated human T lymphocyte cell cDNA library using B7-1 protein as the bait and finally receptor gene was isolated from the positive cells. These results indicated that this screen system was suitable for large-scale screen receptors and membrane bound ligands, therefore, this screen system might be useful for the functional assessment of new proteins in the post genomic era and it becomes possible to research gene function by screening receptor.