title: Establishment of a new cell line for high-throughput evaluation of chemokine CCR5 receptor antagonists.
abstract: Chemokine receptors belong to a superfamily of proteins that signal through coupled heterotrimeric G proteins. Chemokine receptor CCR5 is the major co-receptor essential for HIV entry into host cells, and now chemokine CCR5 receptor has become an important target in searching for anti-HIV drugs. Here, we describe the establishment of a human embryonic kidney (HEK) 293/CCR5-HA cell line stably expressing CCR5 receptor with influenza hemagglutinin (HA) tag at the N termini on the membrane surface of HEK293 cells. Plasmid pcDNA3.0-CCR5-HA was transfected into HEK293 cells, and monoclonal HEK293 cell lines expressing CCR5 receptor were generated under G418 selection. The expression of functional CCR5 receptor was tested by GTP?S assay, and the results showed about 5 monoclonal HEK293 cell lines expressed functional CCR5 receptor, and of which No.7 monoclonal cell line is the best. The FACS analysis results further confirmed that CCR5 receptor was positive in 96.89% of No.7 monoclonal HEK293/CCR5-HA cell line. Further results showed that RANTES significantly stimulated GTP?S binding in the dose-dependent manner, and CCR5 antagonist Sch-351125 inhibited RANTES stimulated GTP?S binding in the dose-dependent manner in No.7 monoclonal HEK293/CCR5-HA cell line. Our results suggest that the established HEK293/CCR5-HA cell line is suitable for highthroughput screening and is feasible to identify CCR5 receptor antagonists.
authors.affiliation: Key Laboratory of Standardization of Chinese Medicines of Ministry of Education, Shanghai Key Laboratory of Complex Prescription, Institute of Chinese Materia Medica, Shanghai University of Traditional Chinese Medicine, Shanghai, China.
authors.forename: L L
authors.initials: LL
authors.lastname: Ji
authors.forename: Y C
authors.initials: YC
authors.lastname: Sheng
authors.forename: L
authors.initials: L
authors.lastname: Chen
authors.forename: Z T
authors.initials: ZT
authors.lastname: Wang