Cloning and Characterization of a 7 Transmembrane Receptor from the Adherent Cells of Chicken Peripheral Blood Mononuclear Cells

Abstract

A cDNA encoding a 7 transmembrane (7TM) receptor gene from the adherent cells of chicken peripheral blood mononuclear cells (PBMC) was cloned and characterized. The open reading frame of the chicken-7TM (Ch-7TM) receptor gene was 1008 nucleotides long, encoding a protein of 335 amino acid residues with a molecular mass of approximately 37.1 kDa. Hydrophobic stretches indicated the presence of 7 TM domains. Moreover, the complete nucleotide sequences encoding 7TM of duck (Du-7TM) and goose (Go-7TM), corresponding to the open reading frame of Ch-7TM, were determined. Each of the Du- and Go-7TM encoding regions comprised 990 nucleotides, representing an 18-nucleotide deletion in alignment with the Ch-7TM encoding region, resulting in a 6-amino-acid deletion at the 3′-end. No signal peptides were predicted. Six phosphorylation sites were predicted and conserved for all three 7TMs. The proteins of the three 7TMs were similar, with 11 conserved cysteine residues. No glycosylation sites could be predicted. The results of the pairwise comparisons indicated that the Ch-7TM encoding region and Ch-7TM protein were the least similar to those of Du- and Go-7TMs. These results were in accordance with those of the phylogenetic analysis, which indicated that the Du- and Go-7TM encoding regions clustered, but were separated from the Ch-7TM encoding region. Monoclonal antibody B28D5 was prepared from spleens of mice immunized with the bacterially expressed N-terminal (55 amino acid residues) region of the Ch-7TM protein for further use. Double staining with B28D5 and KUL01 suggested that Ch-7TM was expressed in subsets of the adherent cells, among which a subset that was recognized with both antibodies was likely of monocyte and macrophage lineage. However, the fluorescence intensities of B28D5 and, particularly, KUL01 decreased after the adherent cells were incubated for additional 48 h.

Silencing the Chicken Seven Transmembrane (Ch-7TM) Receptor Leads to Apoptosis in Chicken Mononuclear Phagocytes

“…The candidates of siRNA sequences and target sites were predicted with BLOCK-iT™ RNAi Designer. The HeLa-E5E8 stable clone [16] was treated with various siRNA (siRNA 40, 113, 312, 627, 984, 1681, and cocktail) respectively.…”

“…The HeLa stable cell line E5E8 clone carrying the Ch-7TM-(G4S)3-enhanced green fluorescent protein (eGFP) gene [16] was cultured in Dulbecco modified Eagle medium (DMEM) with 10% FBS in a 96-well plate at 37 °C with 5% CO2. The HeLa…”

“…The newly identified chicken 7TM receptor (Ch-7TM) has been cloned and characterized in chicken MNPs derived from PBMC [16]. However, the functions of Ch-7TM in MNPs remain unclear.…”