Molecular cloning and expression of feline CD28 and CTLA-4 cDNA

Feline CD28 and CTLA-4 (CD152) cDNA were cloned from Con-A stimulated feline peripheral blood mononuclear cells (PBMC) by rapid amplification of cDNA end-PCR (RACE-PCR). Both CD28 and CTLA-4 proteins belong to the immunoglobulin superfamily (Ig SF) and are composed of a signal sequence, an extracellular domain, a transmembrane domain and a cytoplasmic domain. The open reading frame (ORF) of CD28 cDNA encoded a predicted protein of 221 amino acids and that of CTLA-4 cDNA encoded a predicted protein of 223 amino acids. The B7 ligands binding motif MYPPPY hexamer was found on the extracellular Ig V-like domains of both receptors and phosphatidylinositol 3-kinase (PI 3-kinase) binding motifs pYMNM for CD28 and pYVKM for CTLA-4 were identified in the cytoplasmic domains. Comparisons of amino acid sequences of feline proteins with known sequences of other species indicated that rabbit CD28 and CTLA-4 were most closely related and mouse molecules were the least conserved with feline molecules. Comparison of each domain of both molecules with that of other animals showed that the cytoplasmic domain of CTLA-4 was 100% conserved and that of CD28 was the most conserved domain. The cloned CD28 and CTLA-4 cDNA could be expressed in transfected mammalian cells. Expression of feline CD28 and CTLA-4 mRNA in freshly isolated feline PBMC was demonstrated by RT-PCR. Stimulation of PBMC with Con-A similarly increased the expression of both CD28 and CTLA-4 mRNA.     

Molecular cloning of a cDNA coding for feline liver xanthine dehydrogenase

A cDNA coding for feline liver xanthine dehydrogenase (XDH, EC 1.1.204) was amplified by RT-PCR and cloned for determining the sequence. The clones contained an open reading frame of 4002 base pairs encoding 1333 amino acid residues. The calculated molecular weight and isoelectric point were approximately 146 kDa and 7.0. Comparison of the deduced amino acid sequences indicated remarkable high homology, i.e., the amino acid residues of feline XDH shared approximately 90%, 87%, 87% and 86% identity with those of human, bovine, rat and mouse, respectively. The anino acid sequences of two putative iron-sulfur centers, one NAD binding site and one molybdenum binding site were well conserved among mammalian animals.     

Molecular cloning and identification of full-length cDNA encoding high affinity Fc receptor for bovine IgG (Fc gamma RI)

The receptor I for the Fc region of immunoglobulin G (Fc gamma RI) is a member of the Ig superfamily with a high affinity, and it mediates antibody-dependent cellular cytotoxicity and immune complex clearance. In this study, a cDNA encoding the bovine Fc gamma RI was cloned. The full-length cDNA sequence is 1050 bp long with a short 5'- and long 3'-untranslated end regions, which codes for 349 amino acids and contains a signal peptide, an extracellular region with three Ig-like domains, and transmembrane and intracytoplasmic domains. Five potential N-linked glycosylation sites are recognized in this sequence. Compared with the sequences of human and mouse Fc gamma RI, the homologies of nucleotide sequences are 80 and 69% and homologies of deduced amino acid sequences are 66 and 55%, respectively. It is shown that the sequences of the monomeric IgG binding domain in these three species of Fc gamma RI are highly conserved.     

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.     

Cloning and sequence analysis of the complementary DNA for feline preproparathyroid hormone

OBJECTIVES: To clone and sequence the cDNA for feline preproparathyroid hormone (preproPTH) and to compare that sequence with other known parathyroid hormone (PTH) sequences. SAMPLE POPULATION: Parathyroid glands from 1 healthy cat. PROCEDURES: A cDNA library was constructed in lambda phage from feline parathyroid gland mRNA and screened with a radiolabeled canine PTH probe. Positive clones were sequenced, and nucleic acid and deduced amino acid sequences were analyzed and compared with known preproPTH and PTH sequences. RESULTS: Screening of approximately 2 X 10(5) recombinant plaques revealed 3 that hybridized with the canine PTH probe; 2 clones comprised the complete sequence for feline preproPTH. Feline preproPTH cDNA consisted of a 63-base pair (bp) 5'-untranslated region (UTR), a 348-bp coding region, and a 326-bp 3'-UTR. The coding region encoded a 115-amino acid peptide. Mature feline PTH consisted of 84 amino acids. Amino acid sequence analysis revealed that feline PTH was > 83% identical to canine, bovine, swine, equine, human, and macaque PTH and 69, 71, and 44% identical to mouse, rat, and chicken PTH, respectively. Within the region responsible for hormonal activity (amino acids 1 to 34), feline PTH was > 79% identical to other mammalian PTH sequences and 64% identical to the chicken sequence. CONCLUSIONS AND CLINICAL RELEVANCE: The amino acid sequence of PTH is conserved among mammalian species. Knowledge of the cDNA sequence for feline PTH may be useful to investigate disturbances of calcium metabolism and alterations in PTH expression in cats.     

Characterization of a feline homologue of the alphaE integrin subunit (CD103) reveals high specificity for intra-epithelial lymphocytes

The characteristics of a feline homologue of the alphaE integrin (CD103), defined by two murine monoclonal antibodies, Fe7.1B8 (IgG1) and Fe7.2D8 (IgG1), are described. These antibodies recognized 75% of intra-epithelial (range 59-88%) and 40% of lamina proprial (range 28-46%) T cells of the intestinal mucosal tissue of the small intestine in contrast with approximately 2% of peripheral blood lymphocytes. Both antibodies immunoprecipitated a 180 kDa protein from biotinylated feline intra-epithelial mucosal leukocytes consistent with the alphaE integrin subunit in conjunction with a 120 kDa protein consistent with the beta7 subunit. The nucleotide sequence of feline alphaE integrin, generated from molecular cloning of the feline alphaE encoding cDNA, is also reported. This feline molecule shares 72% sequence homology with human and 69% homology with murine and rat counterparts. Homology includes the presence of an X (extra) domain, that appears unique to alphaE molecules as described for human, rat and mouse, as well as areas of homology common to other alpha integrins. Of note is a typical I (inserted) domain, the presence of seven repeat regions, and highly conserved sequences in the cytoplasmic tail. Transfection studies demonstrated that both antibodies recognized an extracellular component which encompassed the X and I domains of the cloned alphaE integrin subunit. These studies demonstrate that the pattern of tissue distribution, biochemical characteristics, and cDNA sequence of the feline alphaE integrin subunit are largely similar to that described for other species.     

Cloning of feline cDNA encoding the cytotoxic T lymphocyte-associated antigen 4 (CTLA-4)

Cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) is a CD28 homologue which down-modulate T cell responses rather than augment them. To investigate its biological role in feline immune system, we cloned and sequenced full-length feline CTLA-4 (fCTLA-4) cDNA by RT-PCR from pokeweed mitogen stimulated peripheral blood lymphocytes. The fCTLA-4 contains an open reading frame of 669 nucleotides, coding for a polypeptide of 223 amino acids. The predicted fCTLA-4 amino acids sequence shows the homology of 86.6%, 87.0%, and 76.2% with human, bovine, and murine molecules respectively. The hexapeptide motif (MYPPPY) within the extra-cellular domain of CTLA-4 molecule, which is believed to be responsible for interaction with the B7 family members, is completely conserved in all the species.     

Nucleotide sequence and expression of the feline vascular endothelial growth factor

Vascular endothelial growth factor (VEGF) is an angiogenic factor which targets vascular endothelial cells. In this study, cDNA encoding a feline VEGF (fVEGF) isoform was cloned from a feline lymphoid tumor cell line and sequenced. The fVEGF cDNA contained an open reading frame of 567 nucleotides coding for a polypeptide of 163 amino acids with a putative signal peptide of 26 amino acids. The predicted fVEGF amino acid sequence shared 98.4, 94.2 and 94.2% homology with the sequences of canine, bovine and human VEGF, respectively. Though predicted fVEGF polypeptide was two amino acid residues shorter than human VEGF165, a potential glycosylation site and regions critical for receptor binding were conserved in all the species examined. Transient expression of fVEGF in mammalian cells resulted in secretion of VEGF which could be detected by antibodies against human VEGF165. Furthermore, wide expression of fVEGF mRNA was observed in various feline tissues using RT-PCR methods.     

鸭RIG-1基因的PCR扩增及其序列分析

维甲酸诱导基因1(RIG-1)是细胞质中侦测病原相关分子模式的识别受体。论文旨在扩增北京鸭RIG-1编码基因,并对其进行序列分析。通过PCR方法,从北京鸭脾脏中扩增得到RIG-1基因cDNA,并使用LaserGene分子生物学分析软件进行序列分析。结果发现北京鸭的RIG-1基因cDNA开放阅读框全长2 802bp,编码933个氨基酸。结构预测发现鸭RIG-1结构与哺乳动物类似,N端有两个串联CARD区,中间为DExD/H解旋酶区,C端为抑制区,各功能区起重要作用的氨基酸较为保守。同源性及进化树分析发现鸭RIG-1与鹅和斑马鱼的同源性最高分别为93.7%、78.4%,与哺乳动物同源性高于50%,与其他鱼类的同源性低于50%。成功扩增出北京鸭RIG-1基因cDNA编码序列,为鸭RIG-1的深入研究奠定了基础。     

Molecular cloning and sequence analysis of feline interferon-stimulated gene 15

The interferon-stimulated gene 15 (ISG15) is induced by type I interferon (IFN). Recent studies have revealed that like ubiquitin, ISG15 is conjugated with target proteins. In this study, the feline ISG15 (FeISG15) gene was cloned from feline IFNomega (FeIFNomega)-stimulated feline kidney epithelial (CRFK) cells. According to gene sequence results, cDNA was 474bp long and encoded a protein of 157 amino acids. The putative amino acid sequences showed 62.5-72.1% identity with those of other mammalian ISG15s. Similar to human and mouse ISG15, FeISG15 included tandem ubiquitin-like domains; its homology with feline ubiquitin was 36.3-39.5%. The LRLRGG conjugating motif was located only in the carboxyl terminal ubiquitin-like domain. FeISG15 also lacked the carboxyl terminal extension after the LRLRGG motif, which is present in mouse and human ISG15. Recombinant FeISG15 protein was expressed as a His-tagged fusion protein in Escherichia coli and purified by ion-exchange chromatography followed by affinity chromatography. Monoclonal anti-FeISG15 antibodies revealed free FeISG15 and FeISG15 conjugated with target proteins in cells after IFNomega stimulation by Western blotting analysis. Furthermore, mRNA of IFNgamma was detected from peripheral blood mononuclear cells (PBMCs) after stimulation with rFeISG15 extracellularly by RT-PCR. Taken together, these results suggested that FeISG15 had ubiquitin- and cytokine-like activity, as in other species.     

Expressed sequence identification and characterization of the cDNA for interleukin-4 from the mitogen-stimulated lymphoid tissue of a marsupial, Macropus eugenii

Very few cytokines that are important to the understanding of T helper cell function are characterized in marsupials. Expression of a 645 bp cDNA product that codes for a predicted Interleukin-4 peptide of 157 amino acids was detected in the lymph node tissues of Macropus eugenii, the tammar wallaby. Using Rapid Amplification of cDNA Ends, both 5'- and 3'-untranslated regions were identified and a polyadenylation signal and three mRNA instability motifs associated with secreted cytokine molecules were also present. The translated cDNA sequence has a putative signal peptide of 24 amino acids, a predicted secondary structure that is consistent with the short-chain alpha-helical cytokine family and 82% conservation of residues associated with the Interleukin-4 family sequence motif. Comparisons of wallaby nucleotide and predicted peptide sequences with the coding domains of other vertebrate species demonstrate the diversity within this gene family; with nucleotide and amino acid identities of 74% and 59% with opossum, 52% and 32% with human and 38% and 19% with chicken homologues respectively. Despite these differences in sequence conservation, the putative Macropus eugenii Interleukin-4 mature peptide contains conserved structural motifs and predicted receptor-binding residues that suggest that it may retain functional properties associated with this important Th2 cytokine in other mammals.     

Molecular cloning of canine mucosal addressin cell adhesion molecule-1 cDNA and its expression in normal tissues

A cDNA encoding canine mucosal addressin cell adhesion molecule-1 (MAdCAM-1) was cloned. The entire open reading frame of canine MAdCAM-1 cDNA comprises 1137 bp, corresponding to 378 amino acid residues. The deduced amino acid sequence of canine MAdCAM-1 was 55.2%, 53.7%, and 52.4% identical to rat, mouse, and human MAdCAM-1, respectively. Canine MAdCAM-1 appeared to contain two immunoglobulin-like domains at the N-terminus, followed by a mucin-like domain and a third immunoglobulin-like domain. The structures of the dog, rat, and mouse proteins are likely similar because all of the cysteine residues in the immunoglobulin-like domains were conserved. Canine MAdCAM-1 mRNA was confirmed to express extremely in the mesenteric lymph node by RT-PCR.     

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 phylogenetic analysis of a cDNA encoding the cat (Felis domesticus) Ig epsilon constant region

A feline splenic cDNA library was screened with a (32)P-labelled cDNA probe encoding the canine IgE epsilon heavy chain subunit. A cDNA sequence of 1614 nucleotides encoding the complete feline IgE heavy chain, as well as a portion of a variable region, was identified. A search of the GenBank database revealed an identity of 82% at the nucleotide level and 76% at the amino acid level between the feline epsilon heavy chain sequence and the canine homologue. In a separate study, feline genomic DNA, isolated from whole feline embryo cells, was subjected to PCR amplification using primers based on known partial genomic DNA sequences for the feline C epsilon gene. Following removal of an intron from the 683 bp PCR product, the coding sequence yielded an ORF of 506 bp. The DNA sequence of this PCR clone differed by a single nucleotide from the cDNA clone. This difference is silent, and therefore the proteins encoded by the two sequences are identical over the regions cloned and sequenced. Phylogenetic analysis of the constant regions of nine immunoglobulin epsilon genes revealed that the feline cDNA is most similar to the canine homologue.     

Molecular cloning of feline interferon-gamma-inducing factor (interleukin-18) and its expression in various tissues

Interleukin-18 (IL-18) is a cytokine with potent interferon-gamma-inducing activity, and plays an important biologic role in the enhancement of the activity of natural killer cells and cytotoxic T-lymphocytes. In this study, feline IL-18 cDNA was cloned and characterized to establish a basis for the prospective cytokine therapy in small animal practice. The nucleotide sequence of feline IL-18 cDNA obtained in this study was 712bp long and contained its entire open reading frame encoding 192 amino acid residues. The predicted amino acid sequence of feline IL-18 cDNA showed 77.2, 84.8, 60.2 and 62.6% similarity with those of human, dog, rat and mouse counterparts, respectively. The feline IL-18 cDNA included a putative cleavage site of IL-1beta-converting enzyme (ICE) and IL-1 signature-like sequences identified in human and mouse IL-18 cDNAs. Expression of IL-18 mRNA was detected in various tissues including spleen, liver and cerebrum in the cat.