Molecular cloning of canine interleukin-31 and its expression in various tissues

The newly discovered cytokine, interleukin-31 (IL-31), belongs to the short-chain cytokine group. It was reported that transgenic expression of IL-31-induced pruritus, similar to atopic dermatitis, in mice, further, excessive amounts of IL-31 was also expressed in the skin from human patients with atopic dermatitis as compared to that from normal people. In this study, canine IL-31 was molecularly cloned from concanavalin A-stimulated canine peripheral blood mononuclear cells (PBMCs), and its nucleotide sequence was determined. Canine IL-31 contains 4 alpha-helix structures characteristic of the IL-31 family, and the amino acid identity of canine IL-31 with those of human or mouse is 54% and 28%, respectively. Furthermore, we detected low levels of canine IL-31 in the thymus, testis, spleen, and kidneys, but not in the skin of atopic dogs.     

Evaluation of interleukin-2 production and interleukin-2 receptor expression in dogs with generalized demodicosis

Abstract The experimental hypothesis testéd was that dogs with generalized demodicosis have significantly lower levels of interleukin-2 (IL-2) production and IL-2 receptor expression as compared with normal dogs. The specific objective of this study was to compare the production of IL-2 and IL-2 receptor expression of peripheral blood mononuclear cells in normal dogs and dogs with generalized demodicosis. Ten dogs with juvenile-onset generalized demodicosis were evaluated. Dogs with generalized demodicosis had a significantly lower in vitro lymphocyte blastogenesis response (P < 0.006), fewer cells expressing IL-2 receptors (P < 0.03), and decreased IL-2 production (P < 0.01) than control dogs. Resumen La hipótesis experimental fue que los perros con demodicosis generalizada tienen niveles significativamente inferiores de producción y expresión de receptores de interleuquina-2 (IL-2) comparado con perros normales. El objetivo especifico de este estudio fue el de comparar le producción y expresión de receptores de IL-2 en células mononucleares de sangre periférica en perros normales y en perros con demodicosis generalizada. Se evaluaron diez perros con demodicosis generalizada iniciada en edad temprana. Los perros con demodicosis generalizada tenian in vitro una respuesta blastogénica linfocitaria inferior (P < 0.006), menos células con expresión de receptores de IL-2 (P < 0.03) y una disminución en la producción de IL-2 (p<0.01) respecto a los perros control. [Lemarie, S.L., Horohov, D.W. Evaluation of interleukin-2 production and interleukin-2 receptor expression in dogs with generalized demodicosis. (Evaluación de la producción y expresión de recepetores de interleuquina-2 en perros con demodicosis generalizada.) Veterinary Dermatology 1996; 7 : 213–219.] Résumé L'hypothèse expérimentale est que les chiens à démodécie généralisée présente une diminution significative du taux d'interleukine 2 (IL-2) et de l'expression des récepteurs de l'interleukine 2 comparativement à des chiens sains. L'objectif de cette étude est de comparer la production d'I12 et l'expression des récepteurs de l'IL-2 par des lymphocytes sanguins périphériques de chiens témoins et de chiens atteints de démodécie généralisée. Dix chiens avec une démodécie généralisée juvenile ont été testés. Les chiens à démodécie généralisée présentent une transformation lymphoblastique plus basse (p< 0.006), moins de cellules exprimant les récepteurs IL-2 (p<0.03) et une diminution de la production d'IL-2 (p<0.01) par rapport aux chiens témoins. [Lemarie, S.L., Horohov, D.W. Evaluation of interleukin-2 production and interleukin-2 receptor expression in dogs with generalized demodicosis. (Evaluation de la production d'interleukine 2 et de l'expression des récepteurs de l'interleukine 2 chez des chiens à démodécie généralisée.) Veterinary Dermatology 1996; 7 : 213–219.] Zusammenfassung Es wurde die experimentelle Hypothese getestét, daß Hunde mit generalisierter Demodikose über signifikant niedrigere Spiegel an Interieukin2-(IL-2)Produktion und IL-2-Rezeptorexpression verfügen im Vergleich zu gesunden Hunden. Der wesentilche Gegenstand dieser Untersuchung war, die Produktion von IL-2 und die IL-2Rezeptorexpression in den mononukleären Zellen des peripheren Blutes bei gesunden Hunden und solchen mit generalisierter Demodikose zu vergleichen. Zehn Hunde mit juvenilem Beginn von generalisierter Demodikose wurden untersucht. Hunde mit generalisierter Demodikose hatten eine signifikant niedrigere in vitro-Reaktion der Lymphozytenblasogenese (P < 0, 006), weniger Zellen, die IL-2Rezeptoren zeigten (P < 0,03) und eine verminderte IL-2Produktion (P < 0,01) als die Kontrollhunde. [Lemarie, S.L., Horohov, D.W. Evaluation of interleukin-2 production and interleukin-2 receptor expression in dogs with generalized demodicosis (Untersuchungen über die Interleukin2-Produktion und Interleukin2-Rezeptorexpression bei Hunden mit generalisierter Demodikose) Veterinary Dermatology 1996; 7 : 213–219.]     

Molecular cloning and physiological effects of brushtail possum interleukin-1beta

Interleukin-1beta (IL-1beta) was isolated from LPS-stimulated brushtail possum alveolar macrophages using PCR primers based on conserved regions of mammalian IL-1beta. The complete cDNA was cloned by 5' and 3' rapid amplification of cDNA ends (RACE). The predicted protein of 269 amino acids shared 4346% identity with several mammalian IL-1beta proteins. Constructs were made to express the mature IL-1beta in Escherichia coli and two recombinant IL-1beta proteins, rpIL-1beta1 and rpIL-1beta2, which differed in length by four amino acids at the N-terminus, were produced. Both proteins induced a weak proliferative response in a possum thymocyte assay. Possums injected intravenously with 100 microg of rpIL-1beta1 or rpIL-1beta2 showed profound changes in body temperature and numbers of circulating leukocytes. A sharp decrease in temperature occurred within 2 h of administration followed by an elevation of temperature peaking at 24 h. The smaller rpIL-1beta1 protein had a greater effect on temperature than rpIL-1beta2. Both rpIL-1beta proteins caused a marked decrease in number of neutrophils and lymphocytes at 2-6 h after injection. At 24 h after injection, neutrophil and lymphocyte numbers were elevated 6.0-fold and 2.6-fold, respectively in the possums injected with rpIL-1beta1 and 3.9-fold and 1.5-fold, respectively in the possums injected with rpIL-1beta2. Fibrinogen levels were elevated at 24 and 72 h after injection with both proteins. In comparison, neither recombinant bovine IL-1beta (rbIL-1beta) nor PBS had significant effects on body temperature or blood haematology. The studies have shown that the two recombinant forms of IL-1beta were biologically active in possums and that the IL-1beta with four fewer amino acids at the N-terminus was the more active.     

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.     

猪akirin2基因的克隆与真核表达

Trizol法从猪脾脏中提取总RNA,RT-PCR方法得到猪Akirin2的完整编码区,软件分析猪Akirin2的核酸序列与蛋白序列,进行染色体定位。将Akirin2构建到带有荧光标记的真核表达载体pEGFP-C1中,通过脂质体转染法将pEGFP-Akirin2转入猪脐静脉血管内皮细胞系(SUVEC)中进行表达。结果表明,该序列编码203个氨基酸,猪akirin2基因定位于猪1号染色体,含有4个外显子,猪与牛的Akirin2蛋白高度同源。重组表达质粒pEGFP-Akirin2经双酶切鉴定和测序分析,表明构建成功,pEGFP-Akirin2转染SUVEC细胞后,经荧光检测证实转入的akirin2基因得到表达。研究结果为探讨猪Aki-rin2蛋白在免疫调控中的功能提供了基础数据。     

Molecular cloning, sequencing, and expression of equine interleukin-6

Equine interleukin-6 (IL-6) cDNA was amplified from mitogen-stimulated equine peripheral blood mononuclear cells (PBMC) using consensus sequence primers. The 727bp amplified cDNA contains the entire coding region for equine IL-6 and includes 118 bases in the 3' non-translated region. The coding sequence translates to a protein of 208 amino acids with a predicted 28 amino acid leader sequence. The mature protein of 180 amino acids has a predicted molecular mass of 20471Da without post-translational modifications. The amino acid sequence of equine IL-6 displays between 46 and 84% similarity to other mammalian IL-6 sequences. Expression of equine IL-6 in Chinese hamster ovary (CHO) cells yielded a supernatant that supported the proliferation of B9 cells in a dose-dependent manner. Treatment of B9 cells with an anti-IL-6 receptor antibody ablated the response to the recombinant equine IL-6.     

猪G蛋白偶联受体12(Gpr12)基因克隆鉴定及其表达分析

为研究猪G蛋白偶联受体12(G protein-coupled receptor 12,Gpr12)基因的序列和组织表达特征,探索Gpr12 mRNA在卵泡发育过程中的表达规律,本研究利用克隆测序技术获取猪Gpr12基因的编码区序列,并利用生物信息学方法分析Gpr12基因序列和蛋白的潜在特征;RT-PCR技术检测Gpr12基因的组织表达模式;荧光定量PCR技术检测Gpr12 mRNA在猪卵泡发育过程中的表达规律。结果表明:猪Gpr12基因编码区序列长1 005 bp,编码蛋白含有典型的7次跨膜结构域和DRY基序,存在多个潜在的磷酸化和糖基化修饰位点;Gpr12基因在所检测的组织和细胞中均有表达,且在脑、垂体和卵母细胞中高表达;随着卵泡的发育,Gpr12 mRNA的表达量极显著升高(P<0.01),表明Gpr12基因可能参与了猪卵泡发育的调控过程。     

Molecular cloning of the canine c-Met/HGF receptor and its expression in normal and regenerated liver

The c-Met proto-oncogene is the receptor for hepatocyte growth factor (HGF), which is a member of the tyrosine kinase family. Activation of the HGF/c-Met signal pathway leads to cell proliferation, motility, regeneration, and morphogenesis. In this study, the complete nucleotide sequence of complementary DNA (cDNA) of canine c-Met was cloned, and its distribution was determined in tissues. The canine c-Met cDNA clone had an open reading frame of 4419 bp that encoded a putative polypeptide of 1383 amino acids. The c-Met mRNA was expressed in a variety of canine tissues including peripheral blood mononuclear cells (PBMC), bone marrow, liver, kidney, lung, stomach, uterus, testis, thymus, lymph node, small intestine, colon, adrenal gland, thyroid gland, heart, muscle, skin, pancreas, ovary, prostate, spleen, fat, cerebrum, and cerebellum. In addition, the c-Met mRNA expression in normal and regenerated liver was examined. The levels of the mRNA increased 2-fold in regenerated liver compared to that found in normal liver, indicating that c-Met is involved in various functions including remodeling of canine hepatocytes.     

Molecular cloning and expression of bottlenose dolphin (Tursiops truncatus) interleukin-8

The bottlenose dolphin interleukin (IL)-8 cDNA was molecularly cloned. The dolphin IL-8 has an open reading frame of 303-bp encoding 101 amino acids. The homology of the amino acid sequence with that of other species was: sheep, 89.1%; cattle, 88.1%; pig, 85.1%; dog, 85.1%; horse, 79.2%; human, 74.5%; and macaque, 72.3%. The amino acid sequence suggested that dolphin IL-8 was a CXC chemokine. The recombinant dolphin IL-8 protein was recognized with anti-ovine IL-8 monoclonal antibody.     

Molecular cloning and tissue-specific expression of Toll-like receptor 5 gene from turkeys

Toll-like receptors (TLRs), a family of transmembrane and cytosolic proteins, detect microbial patterns, initiating innate immune responses in various organisms. Although they are abundant, genetic characterization and functional differences of TLRs in economically important avian species such as chickens and turkeys have not been investigated in detail. In this study, the putative TLR5 coding region from turkey genome was sequenced, and its homology to other vertebrate species was analyzed. Secondary structure analysis revealed protein motifs typical of the chicken TLR5 protein structure, with 97% amino acid identity between them. mRNA expression profiling in adult turkeys revealed abundant TLR5 expression in a broad range of tissues. Stimulation with the TLR5 ligand flagellin resulted in the production of the inflammatory mediators interleukin (IL)-1beta, IL-6, and nitric oxide in peripheral blood mononuclear cells. To our knowledge, this is the first complete turkey TLR5 coding DNA sequence reported in sequence databases.     

Molecular cloning and expression analysis of the canine chemokine receptor CCR9

The chemokine receptor CCR9, which interacts with the thymus-expressed chemokine TECK/CCL25, contributes to the localization of lymphocytes to the small intestine, and is implicated in the development of human inflammatory bowel disease (IBD); however, their role in canine IBD is unknown. The objective of this study was to isolate cDNA encoding CCR9 and to investigate CCR9 expression in normal canine tissues and lymphoid cell lines. The complete open reading frame contained 1104 bp, encoding 367 amino acids, with 85% and 81% identity to human and mouse homologs, respectively. CCR9 mRNA was detected in all tissues investigated with the highest expression level in the small intestine. CCR9 mRNA was also expressed in GL-1, a canine B cell leukemia cell line, but not in CLBL-1, a canine B cell lymphoma cell line. Immunoblot and flow cytometry analyses of these cell lines using an anti-human CCR9 monoclonal antibody revealed that CCR9 protein expression was detected only in GL-1, indicating the cross-reactivity of the antibody. Using the antibody, flow cytometry showed that the proportions of CCR9(+) cells were small (mean, 4.88%; SD, 2.15%) in the normal canine PBMCs. This study will be useful in understanding canine intestinal immunity and the immunopathogenesis of canine IBD.     

猪IFN-β的克隆与原核表达

根据已发表的AY687281基因序列,设计1对特异性引物,采用PCR方法从长白猪白细胞基因组中扩增到猪β干扰素基因,将PCR产物克隆到pMD18-T载体,进行序列测定。序列分析表明,获得的猪β干扰素基因全长561bp,编码186个氨基酸,与GenBank中AY687281基因序列同源性达99.2%,在28、34、128位核苷酸发生了点突变,由G变为A,由C变为G,由A变为G,由C变为T,导致10、12、43位氨基酸由A变为T,L变为V,E变为G,而在177位点核苷酸的突变没有改变氨基酸。将该基因克隆到pEGX-4T-1表达载体中,转化宿主菌Rosetta,对猪β干扰素进行原核表达。重组表达菌经IPTG诱导后,得到高效表达,所表达蛋白约为43 000,占总蛋白的39.3%,表达产物主要为包涵体。纯化后的包涵体具有抗病毒的生物学活性,能抑制口蹄疫病毒（FMDV）增殖。     

Molecular cloning of equine chromogranin A and its expression in endocrine and exocrine tissues

Chromogranin A (CGA) is a member of a family of highly acidic proteins co-stored and co-released with catecholamines in the adrenal medullary cells as well as in other neurons and paraneurons. The nucleotide sequence encoding equine CGA was determined using RT-PCR and rapid amplification of complementary DNA (cDNA) ends (RACE) techniques. A total 1,828 bp of the nucleotide sequence reveals that equine CGA is a 448-residue protein preceded by an 18-residue signal peptide. Comparison of the amino acid sequence of equine CGA with those of human, porcine, bovine, mouse, rat and frog CGA showed high conservation at the NH2-terminal 1-77 amino acids regions (94.8%, 93.5%, 92.2%, 81.8%, 83.1% and 66.2%, respectively) and COOH-terminal 314-430 amino acids regions (90.6%, 81.4%, 90.6%, 80.5%, 83.3% and 39.0%, respectively), as well as a potential dibasic cleavage site, whereas the middle portion showed marked sequence variation (52.5%, 49.1%, 38.9%, 26.6%, 27.9% and 6.2%, respectively). Northern blot analysis and RT-PCR elucidated the tissue distribution of equine CGA mRNA. Its expression was confirmed not only in the adrenal medullary cells but also in other organs (cerebrum, cerebellum, pituitary gland, spinal cord, liver, thyroid gland, striated muscle, lung, spleen, kidney, parotid gland and sublingual gland). Further, in adrenal chromaffin cells and pituitary cells of the anterior-intermediate lobe, the expression was confirmed by in situ hybridization with anti-sense CGA cRNA probe.     

Molecular cloning and characterization of beluga whale (Delphinapterus leucas) interleukin-1beta and tumor necrosis factor-alpha.

Interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) are cytokines produced primarily by monocytes and macrophages with regulatory effects in inflammation and multiple aspects of the immune response. As yet, no molecular data have been reported for IL-1beta and TNF-alpha of the beluga whale. In this study, we cloned and determined the entire cDNA sequence encoding beluga whale IL-1beta and TNF-alpha. The genetic relationship of the cytokine sequences was then analyzed with those from several mammalian species, including the human and the pig. The homology of beluga whale IL-1beta nucleic acid and deduced amino acid sequences with those from these mammalian species ranged from 74.6 to 86.0% and 62.7 to 77.1%, respectively, whereas that of TNF-alpha varied from 79.3 to 90.8% and 75.3 to 87.7%, respectively. Phylogenetic analyses based on deduced amino acid sequences showed that the beluga whale IL-1beta and TNF-alpha were most closely related to those of the ruminant species (cattle, sheep, and deer). The beluga whale IL-1beta- and TNF-alpha-encoding sequences were thereafter successfully expressed in Escherichia coli as fusion proteins by using procaryotic expression vectors. The fusion proteins were used to produce beluga whale IL-1beta- and TNF-alpha-specific rabbit antisera.