Multiple alignment analysis revealed that duCD4 had four potential N-glycosylation sites (NISF, NATA, NGTK and NYTV) (Physique 1A). vertebrates. Quantitative real-time PCR analysis showed that duCD4 mRNA transcripts are widely distributed in the healthy Cherry Valley duck, and the highest level in the thymus. During the virus infection, the obvious change of duCD4 expression was observed in the spleen, lung and brain, which suggesting that duCD4 could be involved in the host’s immune response to multiple types of viruses. Our research studied the characterization, tissue distribution, and antiviral immune responses of duCD4. CD4 from the NCBI (“type”:”entrez-nucleotide”,”attrs”:”text”:”XM_013096823.1″,”term_id”:”874465484″,”term_text”:”XM_013096823.1″XM_013096823.1). In addition, the expressions of duCD4 were quantified using qRT-PCR with the ChamQ SYBR qPCR Grasp Mix (Q311, Vazyme) and analyzed with the 7500 Fast Real-Time PCR system (Applied Biosystems, Carlsbad, CA). The specificity of PCR amplification for all those primer sets was verified from the dissociation curves. The MAP2K2 qRT-PCR consisted of 20 L volume and conditions as follows: one cycle of pre-denatured at 95C for 5?min, followed by 40 cycles of denaturation at 95C for 10?s and extension at 60C for 34?s, then a dissociation curve was analyzed (95C for 10s, 65C for 10 s, 97C for 1 s). Expression levels of the duCD4 and endogenous housekeeping gene -actin were analyzed using the 2 2?Ct and 2?Ct method (Pfaffl,?2001). All qRT-PCR reactions were performed in triplicate. Statistical Analysis Data were expressed as mean SD from 3 individual experiments. Significance was determined by one-way ANOVA using SPSS 19.0 (SPSS Inc., Chicago, IL, USA). For all those tests, 0.05 was considered statistically significant, 0.01 was highly significant, and 0.001 was extremely significant. RESULTS Cloning and Structural Analysis of duCD4 The full-length CDs of duCD4 were amplified using the primers duCD4-F and duCD4-R (Table 1), and it consisted of GSK1016790A a single ORF of 1449 bp which encodes 482 AAs. And the sequence has been uploaded to NCBI with the GenBank number of “type”:”entrez-nucleotide”,”attrs”:”text”:”KX588247.1″,”term_id”:”1198363740″,”term_text”:”KX588247.1″KX588247.1. Multiple alignment analysis revealed that duCD4 had four potential N-glycosylation sites (NISF, NATA, NGTK and NYTV) (Physique 1A). Our results also indicated that this duCD4 contained the conserved Lck motif (CXC) of the CD4 typical signature (Physique 1A). The protein domains of duCD4 were predicted using SMART program, and the results indicated that duCD4 contained 7 characteristic domains: one signal peptide at its N-terminus (AA1-27), 3 immunoglobulins (IG) (AA28-121, 126-214, 221-333) and one IG-like domain name (AA338-424) at the central, one transmembrane domain name (AA429-451), and one C-terminal domain name of the CD4 T cell receptor (AA455-482) (Physique 1B). Open in a separate window Physique 1 (A) Alignment of the deduced AA sequence of duCD4 with other animals. Black GSK1016790A shading indicates AA identity; gray shading indicates similarity (50% threshold). The red font indicated the four potential N-glycosylation sites. Lck represented the conserved CXC motif which may bind the tyrosine protein kinase p56lck. (B) Prediction of GSK1016790A duCD4 protein domains by the SMART program. DuCD4 contains one signal peptide at its N-terminus (AA1-27), three IG (AA28-121, 126-214, 221-333) and one IG-like domain name (AA338-424) at the central, one transmembrane domain name (AA429-451), and one C-terminal domain name of the CD4 T cell receptor (AA455-482). Abbreviations: Du, Cherry Valley Duck; Ga, CD4, but distant GSK1016790A from fish CD4 molecules. These results indicated that this duCD4 AA sequence has a closer relationship to the CD4s of birds. To have a further study of CD4 from the Cherry Valley duck, the AA sequence of duCD4 was compared with the other birds, fishes, mammals as well as reptiles. And the multiple sequence alignment analysis showed that duCD4 shared 85.4% identity with CD4, 62.2% identity with and CD4, 62.1% identity with CD4, 26.2% identity with Homo sapiens CD4, and 25.8% identity with CD4 (Determine 2B). The CD4 gene identity was conserved in mammalians, but significantly different between mammalian and duck, with only 26% homology (Physique 2B). Open in a separate window Physique 2 Phylogenetic analysis and sequence similarity of.