Generation And Identification Of Stable ChCD4-or ChCD8α-expressed Flp-In-293 Cell Lines

Chicken CD4 and CD8 are type I transmembrane glycoprotein found on subsets of T cells, and act as co-receptors for the T cell receptor (TCR) in recognition of antigens associated with MHC molecules. ChCD4+ T cells contain the majority of the helper/inducer T cells, and recognize antigens in the context with MHC class II, whereas T cells that express ChCD8 recognize antigens associated with MHC class I, and are capable of affecting cytotoxic function. As well as increasing the overall avidity of TCR-MHC-peptide binding and the adhesion of the T cell to its target cell, ChCD4 and ChCD8 co-receptors bind intracellularly to p56lck tyrosine kinase, trigger intracellular signaling, and thus play an integral part in T cell activation through the TCR/CD3 complex. They also have an essential role in thymocyte development as well as in cell-mediated immune defense in mature T cells. Better knowledge of ChCD4 and ChCD8 will undoubtedly facilitate our understanding of the role of these co-receptors in the poultry immune defense, can help in elucidating pathogenesis of pathogenic microorganisms and immune mechanisms of diseases and thus solving the problems in avian infectious diseases.Here, we report the generating of stable cell lines that expressing chicken CD4 or CD8α. It was used a commercially available Flp/Flp recombination target (FRT) recombination system. Flp-In-293 cells were cotransfected with the pcDNA5/FRT expression plasmids containing ChCD4 or ChCD8αwith or without C-terminally FLAG tag and Flp recombinase expression plasmid pOG44. Flp-recombinase-mediated site-specific recombination occured between the two specific FRT sites in pcDNA5/FRT vector and Flp-In-293 cell genome. The antibiotic Hygromycin B substantially facilitated the subsequent selection of transfectants. The stable cell lines obtained in this study offer the advantage to develop mAb directed against ChCD4 or ChCD8α.1. Construction and expression of eukaryotic expression vectors encoding chicken CD4 or CD8αWhite Leghorn chicken CD4 and CD8αfragments were obtained by digesting pcDNA3.1-CD4 and pcDNA3.1-CD8 plasmids constructed by previously with BamH I / Apa I and BamH I / Xho I respectively, and then inserted into pcDNA5/FRT to construct pcDNA5/FRT-CD4 and pcDNA5/FRT-CD8αexpression plasmids.ChCD4 and ChCD8αfragment without stop codon were amplified by PCR, digested with Kpn I and Xba I and ligated just in front of a FLAG epitope (DYKDDDDK) of pcDNA3.1(+)-FLAG vector. The FLAG epitope became the carboxyl terminus of recombinant ChCD4 and ChCD8αproteins. ChCD4-FLAG and ChCD8α-FLAG fragments were released as Kpn I-Apa I fragments and inserted into pcDNA5/FRT to construct pcDNA5/FRT-CD4-FLAG and pcDNA5/FRT-CD8α-FLAG plasmids.Flp-In-293 cells were transfected with the four constructs respectively. 48h after transfection, transient expression of ChCD4 and ChCD8αwere detected by fluorescence-activated cell scanning (FACS). The results indicate that these eukaryotic expression plasmids were correctly constructed, proteins expressed on cell surface, and the C-terminally FLAG tag didn't affect their expression.2. Generation of stable ChCD4- or ChCD8α-expressed Flp-In-293 cell linesFlp-In 293 cells were cotransfected with Flp recombinase expression plasmid pOG44 and the pcDNA5/FRT constructs respectively at a ratio of 9:1 (w/w) using lipofectamine reagent. Cells were passaged at a 1:10 dilution into selective medium containing 120μg/ml Hygromycin B 48h post-transfection. Cells were fed with selective medium every 4-5 days, 3-4 weeks later the Hygromycin-resistant foci were formed on dishes. Foci were picked and expanded, and screened for the expression of ChCD4 or ChCD8αby FACS. Six clones were identified to express ChCD4 or ChCD4-FLAG and named as C1, C2, C7 and D1, D6, D8, respectively; five clones were verified to express ChCD8αor ChCD8α-FLAG and designated as E1 and F1, F2, F3, F4, respectively.FACS, direct and indirect immunofluorescence staining confirmed the stable expression of ChCD4 or ChCD8αon Flp-In-293 cell surface, and FLAG tag expressed too.β-galactosidase activity and Zeocin resistance assay indicated that the lacZ-Zeo was a very stable protein and was still around during the selection. Four clones C1, D1, E1, F1 were chosen for long-term stability analysis under selective conditions and proved by FACS. F1, F2, F3, F4 were cultured under normal condition and serum-starving condition 20h, and total protein lysates were extracted and resolved by SDS-PAGE, and transferred to nitrocellulose membrane for immunoblotting. The designed proteins were detected with anti-FLAG mAb, using horseradish peroxidase conjugated goat anti-mouse IgG as secondary antibody. Signals were detected by SuperSignal west pico chemiluminescent substrate and Kodak X-ray films. The anti-FLAG mAb recognized a 35kDa polypeptide under reducing conditions. Clone E1 and Flp-In-293 recipient cells were used as negative control and no band appeared with longer exposure time. The culture conditions had no effect on the bands indicated that cells express ChCD8α-FLAG all the time with or without serum stimulation. The anti-ChCD8αmAb also identified a 35kDa polypeptide expressed by clone F1, F2, F3, F4.