Isolation and characterization of polymorphic microsatellite markers for the chub mackerel (Scomber japonicus) and cross-species amplification in the blue mackerel (S. australasicus).

In this study, 10 polymorphic microsatellite markers were developed in Scomber japonicus and were examined on 30 individuals collected from the North Pacific. The number of alleles per locus ranged from 4 to 17. The observed and expected heterozygosities per locus ranged from 0.2759 to 0.8621 and from 0.43071 to 0.9177, respectively. The polymorphism information content (PIC) was from 0.3931 to 0.8939. One locus showed moderate polymorphism (0.25 < PIC < 0.5), while the rest were highly polymorphic (PIC > 0.5). Two loci showed significant deviation from Hardy-Weinberg equilibrium after Bonferroni corrections (P < 0.005). No linkage disequilibrium was detected among the loci. Results of cross-species amplification showed that 10 microsatellite markers were successfully amplified in 29 individuals of S. australasicus and 9 indicated polymorphisms. These markers will be useful for investigating the genetic structure, gene flow, and species identification of S. japonicus and S. australasicus, its closely related species.

Molecular characterization and tissue-specific expression of invariant chain isoform in Muscovy Duck (Cairina moschata).

Invariant chain (Ii) isoform, through its thyroglobulin-like (Tg) domain, inhibits cysteine proteases during antigen presentation in vertebrates. In birds, the Ii of Muscovy Duck (MDIi) has 2 forms: MDIi-1 and MDIi-2 (MDIi isoform). To understand the genetic information and expression characteristics of MDIi-2, polymerase chain reaction, and bioinformatic analysis were performed for MDIi-2 from healthy adult Muscovy Duck. The full-length MDIi-2 cDNA sequence was found to be 1377-base pairs, encoding a 285-amino acid protein. MDIi-2 contains 63 amino acids with an insertion sequence in the Tg domain. MDIi-2 shares high identity (72.51-94.74%) with the same protein in other birds. The Tg domain of MDIi-2 is highly conserved and showed relatively high identity (96.83%) among all tested birds. The molecular structure of the Tg domain supports this conservation. MDIi-2 expression was measured in various tissues using real-time quantitative polymerase chain reaction. Similar to MDIi-1, MDIi-2 was detected in all tissues but at different levels. Higher expression level was observed in the spleen, intestinal mucosa, and bursa stipe (bursa of Fabricius stipe) than in other tissues. This suggests that MDIi-2, like MDIi-1, plays an essential role in all tissues and that its differential expression may be related to its functions in these tissues. The coexistence of 2 MDIi isoforms indicates that their functions are correlated in Muscovy Duck. This study improves the understanding of poultry immunology and may be used to improve measures to protect Muscovy Duck from disease.

N-terminal functional region of the invariant chain efficiently targets the binding of a CTL epitope to MHC class I molecules during cross-presentation.

Cross-presentation (CP) is important for priming T cell responses to many viral, bacterial, and tumor antigens. Here, we designed two Ii mutants, based on evidence that the invariant chain (Ii, also named CD74) binds newly synthesized MHC class I molecules with the class II-associated invariant chain peptide (CLIP) region of Ii, which occupies the peptide-binding groove. Specifically, we designed (1) Ii-O257, which is a CLIP-substituted Ii chimer, in which OVA257-264 (SIINFEKL) was substituted for CLIP, and (2) Ii-, also named CT257, which is a C-terminal truncated form of Ii-O257 that contains the N-terminal flanking region of Ii. We immunized C57BL/6 mice with these recombinant proteins. Real-time PCR detected that mice immunized with either Ii-O257 or Ii-CT257 recombinant proteins exhibited increased IFN-γ mRNA expression (approximately 11-fold and 13-fold, respectively) and increased IL-2 mRNA expression (approximately 9-fold and 11-fold, respectively), compared to mice immunized with the OVA257-264 peptide. In vivo cytokine analysis showed that recombinant Ii proteins were highly efficient at activating T cells. Confocal microscopy and co-immunoprecipitation showed that the 2 Ii-OVA257-264 chimers are associated intracellularly with H-2K(b) molecules. Thus, Ii-CT257 (amino acids 1-89) binds stably to MHC class I with high affinity, indicating that it is a minimal functional fragment of the Ii immune vector. In conclusion, the N-terminal functional region of the Ii fusion protein containing CTL epitopes might prove to be useful for developing peptide or DNA vaccines that use CP as the main mechanism for CD8(+) T cell stimulation.

Molecular characterization and tissue-specific expression of invariant chain in the muscovy duck (Cairina moschata).

The invariant chain (Ii) plays an important role as a chaperone for MHC II maturation and facilitates antigen presentation in vertebrates. We cloned, characterized and made a homology analysis of healthy adult muscovy duck Ii (MDIi), from a poultry farm in the suburban district of Hefei city in China, by rapid amplification of cDNA ends (RACE)-PCR and by measuring expression of the MDIi gene in various tissues by real-time quantitative PCR. A full-length cDNA sequence of MDIi was obtained, 1188-bp long, encoding a 222-amino acid protein. A comparison of the amino acid sequence of Ii between muscovy duck and other birds showed high similarity (66.3-95.3%). Characteristic functional domains found in Ii of other species, such as cytoplasmic domain, transmembrane domain, class II-associated Ii-derived peptide (CLIP) and trimerization domain, were identified in MDIi. Although all functional domains of Ii were found to be highly conserved, small differences in the CLIP sequence occur among the various species. Expression of MDIi was detected in all tissues at different levels. A higher expression level was found in the spleen, intestinal mucosa and the bursa stipe (bursa of Fabricius stipe) than other tissues. This tissue-specific expression suggests that MDIi plays an essential role in all tissues and differential expression may be a function of the innate structures and essential functions of these tissues.