Although various functions of CD99 have been reported, such as apoptosis and homotypic aggregation of thymocyte and transendothelial migration of immune cells, biochemical/molecular natures of CD99 are still elusive. Using mouse CD99 gene, we show that CD99 forms homodimer through its extracellular domain. Expression of mouse CD99 is up-regulated on T cells after CD3-mediated activation, like the case for human CD99. The potential of CD99 to form homodimer was tested with a recently developed bimoleular fluorescence complementation analysis (BiFC). In BiFC analysis, the dimerization-induced fluorescence was strong near the perinuclear region and was faded at the cell membrane. However, surface expression of CD99 was still detected by flow cytometry, suggesting that CD99 either in monomer form or in association with other molecules exists on the cell surface. In BiFC analysis using CD99 mutants with its extracellular, transmembrane, or cytosolic domains changed to corresponding human CD4 domains, the mutant replaced with human CD4-extracellular domain did not produce fluorescence. Purified soluble CD99-Fc fusion proteins bound to CD99-Fc immobilized onto the gold sensor chip in surface plasmon resonance analysis, confirming that the extracellular domain was responsible for dimer formation. Intracytoplasmic staining for CD99 expression in the thymocytes and mature T cells showed that most of the cells, even the cells with low surface level of CD99, contained the molecule inside the cell. Our results suggest that majority of CD99 homodimers may exit in the cell and be exported to the cell surface, dissociating from each other, after a certain regulatory signal is delivered.
Animals ; Antigens, CD/chemistry/*isolation & purification ; Cell Adhesion Molecules/chemistry/*isolation & purification ; Flow Cytometry ; *Fluorescence ; Luminescent Measurements/*methods ; Mice ; Molecular Biology/*methods ; T-Lymphocytes/immunology

OBJECTIVETo analyze the interaction of PD-1 and PD-L1 recombination protein and to know their bioactivity and affinity.

METHODSStick the PD-1 protein on the surface of CM5 sensor chip by the method of Ammine coupling after being preconsentrated. Dilute the PD-L1 protein step by step and reject it to the passage on CM5 sensor chip which had been stick by PD-1. The time of combination is 3 minutes and of separation is 15 minutes, respectively. Observe the procession and analyze data by BIA Evaluation software 4.

RESULTSOn the consistency of 40 microg/ml, pH 4.5, the PD-1 protein could couple steady on the surface of CM5sensor chip, RU is 3300. On the density of 200 mmol/ml PD-L1 could combine with PD-1 specifically, RU = 150, K(D) = 3.5 x 10(-6).

CONCLUSIONThe PD-1 and PD-L1 recombination protein which we expressed by prokaryotic system have good affinity and bioactivity. The results could provide basic condition for later study.

Antigens, CD ; chemistry ; genetics ; isolation & purification ; metabolism ; Apoptosis Regulatory Proteins ; chemistry ; genetics ; isolation & purification ; metabolism ; B7-H1 Antigen ; Escherichia coli ; genetics ; metabolism ; Gene Expression ; Humans ; Kinetics ; Programmed Cell Death 1 Receptor ; Protein Binding ; Recombinant Proteins ; chemistry ; genetics ; isolation & purification ; metabolism

OBJECTIVETo construct the programmed cell death 1 ligant 1 (PD-L1) recombination expression vector, express the fusion protein in prokaryotic and analyze the biological action of express product.

METHODSThe whole PD-L1 gene sequence was synthesized after codon optimized. Construct the thioredoxin-(PD-L1) recombination expression vector and express the fusion protein in E. coli. Purified the target protein and analyze the conjugated ability of protein by ELISA.

RESULTSThe PD-L1 recombinant expression vector has been constructed correctly. The target protein has been obtained with which expressed in high efficiency and production. The target protein can conjugate specifically with the PD-1, its specific receptor.

CONCLUSIONWe have obtained the PD-L1 recombinant protein success with high biological activity. The result provide the basic condition for further study on antibody and mutually action between PD-L1 and chronic virus infectious.

Antigens, CD ; chemistry ; genetics ; isolation & purification ; metabolism ; B7-H1 Antigen ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Gene Expression ; Humans ; Molecular Weight ; Recombinant Fusion Proteins ; chemistry ; genetics ; isolation & purification ; metabolism

After years of development, biosensors based on imaging ellipsometry and biosensors based on total internal reflection imaging ellipsometry have been successfully implemented in various engineering systems. Their experimental setups, detection principles, and biological and clinical applications are briefly reviewed.
Antibodies ; analysis ; immunology ; Antigens, CD ; analysis ; immunology ; Bacteria ; chemistry ; isolation & purification ; Biomarkers ; analysis ; Biosensing Techniques ; instrumentation ; methods ; Humans ; Ligands ; Microfluidic Analytical Techniques ; instrumentation ; methods ; Microfluidics ; instrumentation ; methods ; Molecular Imaging ; instrumentation ; methods ; Protein Array Analysis ; instrumentation ; methods ; Viruses ; chemistry ; isolation & purification

OBJECTIVETo clone human PD-1 gene, construct a prokaryotic expression plasmid and express in E. coli.

METHODSThe human PD-1 cDNA was cloned by RT-PCR from the total RNA, which was extracted from peripheral blood lymphocyte cell of the patient with chronic hepatitis B. Recombinant PD-1 protein was been expressed and purified after the prokaryotic expression plasmid had been constructed. It was identified by SDS-PAGE, DNA sequencing and amino acid sequencing.

RESULTSThe PD-1 gene was cloned and confirmed by DNA sequencing. The recombinant protein was expressed in E. coli. The purified protein was obtained, then been confirmed by amino acid sequencing.

CONCLUSIONThe human PD-1 gene was successfully cloned and expressed in E. coli, which lays the foundation for further study on the function and application of PD-1.

Amino Acid Sequence ; Antigens, CD ; biosynthesis ; chemistry ; genetics ; isolation & purification ; Apoptosis Regulatory Proteins ; biosynthesis ; chemistry ; genetics ; isolation & purification ; Cloning, Molecular ; Electrophoresis, Polyacrylamide Gel ; Escherichia coli ; genetics ; Genetic Vectors ; genetics ; metabolism ; Humans ; Polymerase Chain Reaction ; Programmed Cell Death 1 Receptor ; Prokaryotic Cells ; metabolism ; Sequence Alignment ; Sequence Analysis, DNA

The study investigated the effects of red clover extract (RCE) on mouse T macrophages and lymphocytes in vitro. The cell toxic effect of RCE was estimated by MTT assay. Multiple-fluorescence staining plus flow cytometry were used to detect the effect of RCE on CD69/CD25/CD71 expression of mouse T lymphocytes stimulated by Con A; CFDA-SE staining plus flow cytometry were used to analyze the effect of RCE on proliferation of T lymphocytes activated by Con A; The effect of RCE on nitric oxide (NO) secretion of mouse macrophages stimulated by lipopolysaccharide (LPS) for 24 h was assayed by Griess reagent system. We found that RCE had potent anti-inflammatory effects on mice. RCE had little cell toxic effect on mouse lymphocytes and macrophages. RCE strongly inhibited the excessive production of inflammatory mediators (NO, CD69, CD25, CD71), in a dose-dependent manner, like cyclosporine A injection. RCE could inhibit proliferation of CD3+ T lymphocytes. These data suggested that RCE might exhibit anti-inflammatory effect by inhibiting the activation and proliferation of mouse lymphocytes and the NO secretion of mouse macrophages.
Animals ; Anti-Inflammatory Agents ; isolation & purification ; pharmacology ; Antigens, CD ; metabolism ; Antigens, Differentiation, T-Lymphocyte ; metabolism ; CD3 Complex ; analysis ; Cell Proliferation ; drug effects ; Cells, Cultured ; Drugs, Chinese Herbal ; isolation & purification ; pharmacology ; Interleukin-2 Receptor alpha Subunit ; metabolism ; Lectins, C-Type ; Lymphocyte Activation ; drug effects ; Macrophages ; cytology ; secretion ; Male ; Mice ; Mice, Inbred BALB C ; Nitric Oxide ; secretion ; Plants, Medicinal ; chemistry ; Receptors, Transferrin ; metabolism ; T-Lymphocytes ; cytology ; drug effects ; metabolism ; Trifolium ; chemistry

This study investigated the effect of arctigenin (Arc) on the cell activation, cytokines expression, proliferation, and cell-cycle distribution of mouse T lymphocytes. Mouse lymphocytes were prepared from lymph node and treated with Phorbol-12-myristate-13-acetate (PMA)/Ionimycin (Ion) and/or Arc. CD69, CD25, cytokines, proliferation and cell cycle were assayed by flow cytometry. The results showed that, at concentrations of less than 1.00 micromol x L(-1), Arc expressed non-obvious cell damage to cultured lymphocytes, however, it could significantly down-regulate the expression of CD69 and CD25, as well as TNF-alpha, IFN-gamma, IL-2, IL-4, IL-6 and IL-10 on PMA/Ion stimulated lymphocytes. At the same time, Arc could also inhibit the proliferation of PMA/Ion-activated lymphocytes and exhibited lymphocyte G 0/G1 phase cycle arrest. These results suggest that Arc possesses significant anti-inflammatory effects that may be mediated through the regulation of cell activation, cytokines expression and cell proliferation.
Animals ; Anti-Inflammatory Agents ; isolation & purification ; pharmacology ; Antigens, CD ; metabolism ; Antigens, Differentiation, T-Lymphocyte ; metabolism ; Arctium ; chemistry ; Cell Cycle Checkpoints ; drug effects ; Cell Proliferation ; drug effects ; Cytokines ; metabolism ; Female ; Furans ; isolation & purification ; pharmacology ; Interferon-gamma ; metabolism ; Interleukin-10 ; metabolism ; Interleukin-2 ; metabolism ; Interleukin-2 Receptor alpha Subunit ; metabolism ; Interleukin-4 ; metabolism ; Interleukin-6 ; metabolism ; Ionomycin ; pharmacology ; Lectins, C-Type ; metabolism ; Lignans ; isolation & purification ; pharmacology ; Lymphocyte Activation ; drug effects ; Mice ; Mice, Inbred BALB C ; Plants, Medicinal ; chemistry ; T-Lymphocytes ; cytology ; drug effects ; immunology ; Tetradecanoylphorbol Acetate ; pharmacology ; Tumor Necrosis Factor-alpha ; metabolism