Meanwhile, tyrosine phosphorylation sites were found to be present in the YMNT motif

Meanwhile, tyrosine phosphorylation sites were found to be present in the YMNT motif. stimulation alone and the control group, and a higher level of IL-2 was detected in the culture medium. Meanwhile, anti-CD28 Abs increased the percent of CD28+ cells (10.41 1.35%), CD4+ T lymphocytes (18.32 2.15%), and CD28+/CD4+ double-positive cells (6.24 1.52%). This effect also resulted in significant variations in the genes of cell membrane-bound molecules, cytokines, and related signaling pathways in cultured leukocytes, with significant changes in the genes of and in the early stages of culture, and the expression of other molecules increased over time. These results proved the localization of the CD28 molecule on T lymphocytes in flounder, and anti-CD28 may act as the B7 ligand involved in T cell activation after antigen stimulation. These data provide a basis for a more ORM-15341 in-depth study of the mechanism of the CD28 costimulatory pathway in T cell activation. in humans (20C23), although many non-specific mitogens like PHA (phytohemagglutinin) (24, 25) or Con A (Concanavalin A) also provide activation signals to T cells in different ways (26). Given the fundamental position of fish in the vertebrate phylogeny, the study of their immune system has gained more attraction. Current studies have demonstrated that the basic components of the mammalian immune system (B and T lymphocytes, MHC, CDs, cytokines, etc.) are also present in fish (27, 28). Recently, more CD28 homologs had been identified in teleost including Half-smooth tongue single (describe in their study that CD28 molecules can bind to CD80/86 molecules at the protein level in tilapia (30). In half-smooth tongue single, the CD28 polyclonal antibody was able to proliferate head kidney ORM-15341 lymphocytes and cause upregulation of IL-2 expression (29). Therefore, given these observations, it has been suggested that CD28 molecules may play a similar function as a CD28 homolog in mammals. However, these studies have not delineated the distribution characteristics of the CD28 molecule in different types of lymphocytes in fish and their response characteristics to KLH, PHA, and LPS which are usually distinguished by the need for T-cell involvement Rabbit Polyclonal to SIRPB1 in the induction of an immune response (35, 36). In the present study, we aim to elucidate the structural features, distribution of lymphocytes, and importance in T/B ORM-15341 cell immune response. Here we cloned the CD28 homolog from flounder (were used to search the flounder transcript database published around the National Center for Biotechnology Information (NCBI; through BLASTn or BLASTp. Based on the partial sequence searched, specific primers were designed by Primer Premier 5.0 (listed in Table?1 of the Supplemental Material ) to extend the 3 and 5 untranslated region (UTR) using cDNA from the spleen by the rapid amplification of cDNA ends (RACE) method. All PCRs were performed in a 50-l reaction containing Ex Taq 0.25 l, 10 Ex Taq buffer 5 l, dNTP mixture 4 l, forward primer 1.5 l (10 M), reverse primer 1.5 l (10 M), cDNA 2 l, and DEPC H2O 35.75 l. The thermocycling program was 98C for 1?min, 35 cycles of 98C for 10 s, 60C for 30 s, and 72C for 1?min, followed by a final extension period of 72C for 5?min. The PCR products were electrophoresed on 1% agarose gels, and the expected segment was extracted using EasyPure? Quick Gel Extraction Kit (TransGen, China) and cloned into the pClone007 Simple Vector (Tsingke, China). Following transformation into qualified DH5 cells (TransGen, China), positive clones were screened by ampicillin selection and colony PCR and then sequenced by Tsingke Biological Technology (Qingdao, China). Sequence Analysis The full-length CD28 cDNA was assembled by DNAman and mapping the intron/exon composition of the CD28 molecule by comparing the genomic CD28 sequence with the CD28 cDNA sequence obtained by cloning. The potential open reading frame (ORF) was analyzed with the Finder program ( The protein analysis was conducted with the ExPASy tools ( The signal peptide and the TM domain name of the deduced protein sequences were predicted with the programs SignalP ( and TMHMM (,.

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