Molecular cloning and sequencing of canine T-cell costimulatory molecule (CD28)

T-cells express CD28 and CTLA-4, and through binding to their shared ligands (CD80/CD86) on antigen presenting cells, provide a potent co-stimulatory signal for T-cell activation and proliferation. To investigate the role of CD28 in canine immune system, we hereby report the molecular cloning and sequencing of the full-length complementary DNA (cDNA) coding for canine CD28, from pokeweed mitogen stimulated canine peripheral blood lymphocytes. The cloned cDNA contains an open reading frame of 663 nucleotides, encoding for a polypeptide of 221 amino acids. The amino acid sequence of the canine CD28 showed 91.9, 80, and 79.6% similarities with those of the cat, cattle, and human counterparts, respectively. Five sequence motifs of TATT or ATTTA involved in the regulation of gene expression by influencing mRNA stability are found in the 3' untranslated region. The hexapeptide motif (MYPPPY), five cysteine residues, a potential N-glycosylation site and a cytoplasmic phosphatidylinositol 3-kinase binding site in canine CD28 molecule are completely conserved in canine CTLA-4. The availability of full length canine CD28 will provide a useful molecule for studying its role in dog immune system.     

Molecular cloning and sequencing of feline CD7

Human CD7 is one of the earliest molecules to appear in T cell development. In this study, putative feline CD7 cDNA was identified based on its similarities with human and mouse CD7 genes. The feline CD7 cDNA contained an open reading frame consisting of 630 nucleotides. The amino acid sequence of feline CD7 had 47.7% identity with that of human CD7, and 52.9% with that of mouse CD7. In addition, the feline CD7 protein fused with histidine tag was expressed in 293T cells. The expression was confirmed by indirect immunofluorescence assay.     

Molecular cloning and expression analysis of pig CD7

CD7 is an integral membrane protein which mediates an important signal to mediate the differentiation, activation, and regulation of some T cells and NK cells. However, only human and mouse CD7 have been identified and studied among mammalian species. In this study, we cloned pig CD7 cDNA and determined its complete cDNA sequence. Pig CD7 cDNA contained an open reading frame (627 bp) encoding 208 amino acids with well conserved motifs involved in signal transduction within cytoplasmic tail among mammalian species. Pig CD7 mRNA was detected by RT-PCR in mainly lymphoid tissues, indicating the conserved functions of CD7 in pigs. Moreover, we generated soluble pig CD7 fusion immunoglobulin (pig CD7Ig) containing extracellular domain of pig CD7 to test whether pig CD7 binds to pig galectin-3. Flow cytometry and immunohistochemistry analyses indicated that soluble pig CD7Ig can bind to galectin-3 expressed in macrophages and epithelial cells of small intestine. These results help to analyze the structural relationship between CD7 and its ligand transferring signal transduction among mammalian species.     

Molecular cloning and sequencing of the cDNA encoding the feline FcgammaRIIIA (CD16) homologue

We amplified the cDNA encoding the feline FcgammaRIIIA (CD16) homologue from peripheral blood mononuclear cells by polymerase chain reaction and cloned two forms of FCGR3A cDNA. Sequencing analysis revealed that the open reading frame of feline FCGR3A cDNA consists of 750 or 747 base pairs encoding 250 or 249 amino acid residues, respectively. Comparison of the predicted amino acid sequence of feline FCGR3A cDNA with those of other mammalians' homologues revealed that the extracellular domain has a relatively low homology. However, the cytoplasmic domain contained an 8-amino acid motif, Leu-Phe-Val-Val-Asp-Thr-Gly-Leu, which was considered to interact with an accessory molecule such as the gamma chain of Fc receptors for IgE to form heterodimeric complexes.     

Molecular cloning and characterization of Duck CD25

The IL-2Ralpha chain (CD25, Tac) is an essential component of high affinity IL-2Rs, playing critical role for the immune specificity of antigen-activated T-cell clonal expansion. Up to now, no duck cytokine receptor has been described. Here, the cDNA segment of a duck cytokine receptor (duCD25), encoding a 226 aa precursor protein with a 20 aa signal peptide, was isolated. Then a novel mouse monoclonal antibody (mAb) was generated using the prokaryotically expressed duCD25 protein as immunogen. Using this mAb, the endogenous duCD25 molecule was localized on the surface of duck lymphocytes, and the duck IL-2-induced lymphocyte proliferation was further inhibited. Furthermore, flow cytometry analysis showed that duCD25 positive cells were upregulated in ducks infected with avian influenza virus (H9N2). Our findings confirm that duCD25 is a receptor of duck interleukin-2, and duCD25 positive cells play a potential role in H9N2 virus infection.     

Molecular cloning and functional characterization of porcine IFN-beta promoter stimulator 1 (IPS-1)

The IFN-beta promoter stimulator 1 (IPS-1), also known as MAVS/VISA/Cardif, is an adaptor molecule for the retinoic-acid-inducible protein I (RIG-I) or melanoma-differentiation-associated gene 5 (MDA5) that recognizes intracellular double-stranded RNA (dsRNA) and triggers a signal for producing type I IFN. In the present study, porcine IPS-1 cDNA was cloned, using RT-PCR coupled with rapid amplification of cDNA ends (RACE)-PCR, from porcine peripheral blood mononuclear cells. The open reading frame of porcine IPS-1 consists of 1575bp encoding 524 amino acids. The putative porcine IPS-1 protein contains a N-terminal CARD-like domain, a central proline-rich domain, a C-terminal transmembrane domain, and exhibits similarity to mouse, rat, monkey, human and cattle counterparts, ranging from 59% to 79%. Semi-quantitative RT-PCR showed that porcine IPS-1 mRNA was widely expressed in different tissues. Porcine kidney (PK-15) cells transfected with a DNA construct encoding porcine IPS-1 produced type I IFN, and activated IRF3 and NF-kappaB. Deletion mutant analyses further revealed that both the CARD-like domain and transmembrane domain are essential for these functions. In addition, poly(I:C)-induced porcine IFN-beta promoter activation in PK-15 cells was significantly reduced by siRNA targeting IPS-1, indicating that IPS-1 is an important immunoregulator in the porcine innate immune system. The availability of porcine IPS-1 and establishment of its function in the type I IFN signaling pathway provides a useful molecule for defining its role during the course of pig infectious diseases.     

Molecular cloning and expression of bottlenose dolphin CD34

In terrestrial mammals, the surface molecule CD34 is used as a marker to identify hematopoietic progenitor cells. To clarify whether CD34 expression can be used to confirm the undifferentiated state of hematopoietic-like cells isolated from the bone marrow of bottlenose dolphin, Tursiops truncates, we determined in this study the sequence of dolphin CD34 cDNA and analyzed its mRNA expression. Dolphin CD34 cDNA can be expressed as two forms, one that encodes a full-length version and a variant, truncated version of the gene. Both forms were detected in bone marrow mononuclear cells and in various tissues using RT-PCR. The truncated form was not detected in peripheral blood mononuclear cells, and neither form was detected in polymorphonuclear leukocytes. This is the first report on CD34 in marine mammals and our results suggest that dolphin CD34 may be a useful marker to identify hematopoietic progenitor cells.     

Molecular cloning and expression analysis of pig CD81

CD81, also known as TAPA-1 (target of antiproliferative antibody 1), is a member of the tetraspanin family of proteins and a component of the B cell co-receptor complex. Several studies have shown that CD81 plays significant roles in a variety of immune responses, including activation of B cells and T cells. In this study, we cloned pig Cd81 cDNA using RT-PCR coupled with rapid amplification of cDNA ends (RACE)-PCR and determined the complete cDNA sequence of pig Cd81. Pig Cd81 cDNA contains an open reading frame (711 bp) encoding 236 amino acids. The identity of pig CD81 with those of human, cattle, rat, and mouse are 90.30%, 92.26%, 86.22%, and 86.22%, respectively. Alignment of the CD81 amino acid sequence with those of mammalian species showed that the large extracellular loop (LEL) is the most divergent, whereas other domains are largely conserved. Pig Cd81 mRNA was detected by RT-PCR in a broad range of tissues, including lymphoid tissues as well as nonlymphoid tissues, indicated variety of cellular functions of CD81 in most pig tissues. Flow cytometry analyses demonstrated that human CD81 antibody recognizes a pig CD81 on the cell surface. Further, immunohistochemistry analysis using human CD81 antibody on pig spleen was revealed that CD81 expression is widely diffused in spleen tissue. Future study will be focused on defining the functional role of CD81 during the course of pig infectious diseases.     

Molecular cloning of the mRNA coding for the G protein of the viral haemorrhagic septicaemia (VHS) of salmonids

Viral haemorrhagic septicaemia virus (VHSV), a rhabdovirus, is a major threat for continental European trout fish farming. The development of a recombinant subunit vaccine could solve that problem. The neutralizing epitopes are located on the glycoprotein or G protein, the surface antigen. The G protein has a molecular weight of 65 kDa, reduced to 55 kDa by deglycosylation. cDNA was synthetized from mRNA of VHS virus infected cells, and cloned in E. coli. The viral cDNA was recognized by positive hybridization with a labelled probe made from infected cell RNA, and negative hybridization with labelled cDNA made from cellular RNA. The Northern blot hybridization with different clones on VHS infected cell RNA revealed two VHS mRNA whose lengths, 2.0 and 1.5 kb, were compatible with the mRNA length for G and N proteins respectively. This mRNA must contain about 400 bp of untranslated sequence.     

Molecular cloning and tissue distribution of uncoupling protein 1 (UCP1) in plateau pika (Ochotona dauurica)

Uncoupling protein 1 (UCP1) is present exclusively in brown adipose tissue, and contributes to body temperature control during cold exposure. We cloned UCP1 cDNA of plateau pika (Ochotona dauurica), a small, non-hibernating, diurnal lagomorph that inhabits in relatively cold climates and at high altitudes in Mongolia and in northern China. The nucleotide sequence of pika UCP1 was highly homologous to UCP1 of other species, and the deduced amino acid sequence had some common domains for UCP, including six mitochondrial carrier protein motifs and a putative purine-nucleotide binding site. RT-PCR and Western blot analyses revealed that both UCP1 mRNA and protein were expressed exclusively in the interscapular adipose tissue. These results suggest that pika UCP1 contributes to heat production in brown adipose tissue, as do those in other species.     

Molecular cloning and sequencing of the cDNA encoding the bat CD4

Chiroptera is thought to be a vector or a natural reservoir of various pathogenic microbes. However, there are few basic studies on the subject of chiroptera immune systems. This is the first report to determine the sequence of bat CD4 cDNA. Comparison with other animals' CD4 and phylogenetic analysis have shown that bat CD4 had a higher homology to cat and dog CD4 than to human and mouse CD4. Moreover, from the analysis of the structure of the CD4 Ig-like C-type 1 region, in bat CD4 there was an insertion of 18 extra amino acids. In addition, bat CD4 lacked cystein, which suggested that the disulfide bond could not be formed. Human, monkey and mouse CD4 have the cystein and the disulfide bond, but pig, cat, whale and dog CD4, like that of the bat, lacked the cystein. We conducted the present study in order to help elucidate the infectious diseases derived from the bat as well as bat immune systems.