Nucleotide sequence of the canine alphaIIb gene from platelet-derived cDNA

OBJECTIVE: To determine the nucleotide sequence of the alphaIIb gene from canine platelet-derived cDNA. ANIMALS: 3 adult dogs. PROCEDURE: First-strand cDNA was prepared from total RNA isolated from canine platelets. The cDNA was amplified, using specific primers in polymerase chain reaction (PCR), and the nucleotide sequence was obtained from purified PCR products. RESULTS: Except for the nucleotide at position 694, results of all sequencing reactions of alphaIIb were identical for canine platelet-derived cDNA. Canine alphaIIb had 3 fewer codons than alphaIIb of humans. The nucleotide and deduced amino acid sequences of full-length canine alphaIIb shared > or = 83% similarity with the sequences established for humans. Segments of canine alphaIIb nucleotide and deduced amino acid sequences were > or = 78% similar to alphaIIb associated with 7 functional domains (extracellular, transmembrane, cytoplasmic, and 4 calcium-binding domains) in humans, with the highest degree of similarity correlating with the sequences of the 4 calcium-binding domains. Amino acid residues associated with development of alloantibodies in humans (Met837, Val837, Ile843, Ser843) are not encoded by canine alphaIIb. CONCLUSIONS AND CLINICAL RELEVANCE: The nucleotide variation at position 694 of canine alphaIIb may represent a polymorphism. The species differences in the alphaIIb sequence may contribute to variations in receptor-li gand interactions. The high degree of alphaIIb sequence conservation of the 4 calcium-binding domains implies functional importance. Some disorders associated with alphaIIbbeta3 in dogs are clinically analogous to diseases in humans, and results indicate that dogs are an appropriate model for the evaluation of gene therapy and other treatments of platelet-associated disorders.     

Molecular cloning and expression of the canine metallothionein-III gene.

We have isolated and determined the complete nucleotide sequence of canine metallothionein-III (MT-III) cDNA. The predicted amino acid sequence of the canine MT-III showed a high homology (93%, 87% identity) to that of human and mouse MT-III. The canine MT-III had 2 insertions relative to known mammalian MT-I and MT-II: a threonine after the 4th amino acid and a block of 6 amino acids near the carboxyl terminus. Expression of the canine MT-III mRNA was found exclusively in the central nervous system, where neurons in the olfactory bulb, hippocampus and cerebral cortex showed predominant signals.     

Cloning of canine cDNA encoding tektin

Tektins are a group of proteins that form filamentous polymers in the walls of ciliary microtubules. The cloning of canine cDNA encoding tektin, was carried out and identified from the testis of beagle dog. Canine tektin cDNA is 1,523 bp in length, has an open reading frame of 1,281 bp nucleotides encoding a protein of 426 deduced amino acids. The predicted amino acid sequence has 77% and 33-50% of homology with the murine tektin and the sea urchin tektins. The amino acid sequence RPNVELCRD and four cysteine residues were conserved in the dog, mouse and sea urchin, suggesting the functional significance of this protein domain and the amino acid residues in the tektin proteins.     

cDNA of Canine Heat Shock Protein 70 (HSP70)

Full-length canine HSP70 cDNA was sequenced and the expression of HSP70 mRNA was investigated. The full-length cDNA sequence of the HSP70 gene (2322 bp) contained a single long open reading frame (1920 bp) coding a protein of 640 amino acids. The amino acid sequence of the canine HSP70 gene shared about 90-95% sequence similarity with bovine, human and mouse HSP70 proteins. Southern blot analysis with HSP70 probe gave three distinct bands of 9.4 kb, 5 kb and 4.4 kb in BamHI digests and two distinct bands of 19 kb and 4 kb in EcoRI digests. Canine HSP70 mRNA was detectable in canine peripheral blood mononuclear cells and stomach but not in liver, kidney, spleen, small intestine, large intestine and skin of dogs.     

Molecular cloning and characterization of lingual antimicrobial peptide cDNA of Bubalus bubalis

Antimicrobial peptides form a crucial component of innate immune system, making it a highly effective first line of defense in animals. In the study, lingual antimicrobial peptide cDNA of Bubalus bubalis has been characterized. The characterized cDNA has complete ORF of 195 bases. The signal sequence of buffalo LAP comprised of N-terminal 1-20 amino acids and mature peptide from 23-64 amino acids. The percentage of similarity of buffalo LAP and buffalo EBD at nucleotide and amino acid level was 96.4% and 92.3% respectively. The identity of buffalo LAP with cattle LAP and TAP at nucleotide level was 92.8% and 90.3%. Both at nucleotide and amino acid level buffalo LAP is closer to buffalo EBD followed by cattle LAP and TAP. Phylogenetic tree at nucleotide and amino acid level also showed close relationship of buffalo LAP with buffalo EBD, cattle LAP and TAP. The synthesized LAP fragment had antibacterial activity.     

Molecular cloning of canine thymus and activation-regulated chemokine (TARC) gene and its expression in various tissues

Thymus and activation-regulated chemokine (TARC) is known as a functional ligand for CC chemokine receptor 4 (CCR4), which is selectively expressed on Th2 lymphocytes and induces selective migration of the cells to allergic lesions. In this study, we cloned canine TARC cDNA from canine thymus by RT-PCR with rapid amplification of cDNA ends (RACE) method. The canine TARC clone contained a full-length open reading frame encoding 99 amino acids and included four cysteine residues characteristic to CC chemokine family. The canine TARC cDNA showed 77.5%, 67.4%, and 68.5% amino acid sequence similarity with human, mouse and rat homologues, respectively. Expression of TARC mRNA was detected not only in thymus but also in spleen, lymph node, lung and heart of the various normal dog tissues examined. TARC cDNA clone obtained in this study will be useful for further investigation on allergic diseases in dogs.     

Molecular properties of the matrixprotein(M) gene of the lapinized rinderpest virus

The nucleotide sequence of the matrixprotein (M) gene of the lapinized rinderpest virus (RPV-L) was determined. The full-length cDNA of the RPV-L M gene is composed of 1460 base pairs and is supposed to contain an open reading frame of 1005 nucleotides encoding on M protein of 335 amino acids. The homology of the predicted amino acid among congeneric morbilliviruses such as RPV Kabete 'O' strain (wild strain of RPV), RPV RBOK strain (vaccine strain of RPV for cattle), measles virus (MV), and canine distemper virus (CDV), is approximately 94%, 93%, 87% and 77%, respectively. In the present study, all coding regions of the RPV-L strain have been determined.     

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.     

Cloning of bovine CD34 cDNA

CD34 is a leukocyte antigen that is expressed in various cell types including hematopoietic cells. Monoclonal antibodies against human, murine, and canine CD34 proteins have been used for the identification of lymphohematopoietic stem/progenitor cells. The cDNA encoding bovine CD34 was cloned, and its nucleotide sequence was determined. The identity of the deduced amino acid sequence of the encoded protein to those of human, murine. and canine CD34 proteins was 61.1%, 56.0%, and 66.1%, respectively. Northern blot hybridization with the cDNA as a probe detected CD34 RNA expression in the cerebrum, spleen, heart, and lung of a fetal calf.     

Cloning of canine Toll-like receptor 7 gene and its expression in dog tissues

Toll-like receptor 7 (TLR7) is activated by single strand RNA and imidazoquinoline compounds, and induces interferon production. In this study, canine TLR7 cDNA was cloned and sequenced. The full-length cDNA of canine TLR7 gene was 3419bp, encoding 1032 amino acids. The similarities of canine TLR7 with human and mouse TLR7 were 84 and 80% at the nucleotide sequence level, and 86 and 79% at amino acid sequence level, respectively. Further, the expression of TLR7 mRNA was investigated in canine normal tissues by semiquantitative RT-PCR analysis. The common expression level of TLR7 mRNA in tissues from three dogs examined was in large intestine, lung, pancreas, small intestine and skin, though the expression level in each tissue was varied among these healthy dogs. In other tissues (kidney, liver, lymph node, spleen, adrenal gland, and PBMCs), the level of TLR7 mRNA expression was different in individuals.     

Canine CD20 gene

The human CD20 antigen, a 35kDa cell surface nonglycosylated hydrophobic phoshpoprotein is expressed consistently on almost all human B-cells, and its monoclonal antibody is used for the therapy on human B-cell lymphoma. In the present study, canine CD20 gene was cloned and sequenced, and the expression of CD20 mRNA was investigated in canine peripheral blood mononuclear cells (PBMCs), and lymph nodes from healthy dogs, and canine lymphoma cells. Using canine cDNA as a template, full-length of canine CD20 gene was sequenced by 5'-RACE and 3'-RACE methods. The full-length of the cDNA sequence of canine CD20 was 1239bp encoding 297 amino acids. The amino acid sequences of canine CD20 showed 73 and 68% sequence similarities with those of human and mouse, respectively. Canine CD20 was predicted to contain domains of amino acid sequences consisting of two extracellular domains (EM), four transmembrane domains (TM), and three intracellular domains (IC) as in human CD20. Canine CD20 mRNA was detected in PBMCs and lymph node from healthy dogs, and B-cells of canine lymphoma, but not in T-cell lymphoma cells and non-T and non-B-cell lymphoma cells by RT-PCR analysis. From these results, canine CD20 might be targeted for monoclonal antibody therapy against B-cell lymphoma of dogs.     

Cloning and characterization of cDNAs encoding four different canine immunoglobulin γ chains

cDNAs encoding four different canine immunoglobulin G (caIgG) γ chains were identified in this study. One of these IgG γ chain cDNAs, (caIgG-A), represents 92.5% of the IgG γ chain cDNAs in a dog spleen cell cDNA library; a second partial IgG γ chain cDNA (caIgG-B) was also identified in the library. The other two IgG γ chain cDNAs (caIgG-C and caIgG-D) were RT-PCR amplified from canine lymphoma samples. Comparison of the four different canine IgG γ chain cDNAs showed homologies from 83.6 to 89.2% and from 73.1 to 81.8% at nucleotide and amino acid sequence levels, respectively. Despite the high similarity in CH1, CH2 and CH3 domains among the different caIgG γ chains, the hinge regions were distinct, sharing only 19.0–35.2% homology at the amino acid level. No multiple duplication of the hinge region, as reported for human IgG1 and IgG3, was detected in any of the canine IgG γ chains. The numbers of cysteines in the putative hinge regions were found to be 3, 2, 7 and 3 for the four canine IgG heavy γ chains (A, B, C and D), respectively. Specific primers were designed based on caIgG γ chain hinge region DNA sequences and were used in RT-PCR for measuring different caIgG γ chain mRNA levels in canine PBMC samples.     

Differential detection of infectious bursal disease virus serotypes, using cDNA probes to VP2 coding region.

Two nonoverlapping clones, pOH405 and pOH632, containing cDNA inserts in the VP2 coding region of genome segment A were selected from a cDNA library prepared from the double-stranded RNA genome of the OH strain of infectious bursal disease virus (IBDV) of serotype 2. Clone pOH405, which is located in the hypervariable segment of VP2, is 328 base pairs long, has nucleotide sequence homology of 72 to 73%, and amino acid sequence homology of 64 to 67% with IBDV strains of serotype 1. Clone pOH632, which is located in the highly conserved C-terminal part of VP2, is 230 base pairs long, has nucleotide sequence homology of 87 to 88%, and amino acid sequence homology of 100% with IBDV serotype 1. The lower detection limit of 32P-labeled probes prepared from both clones was 10 ng of OH-IBDV double-stranded RNA, using high-stringency conditions of hybridization (54 C, 50% formamide) and washing (55 C, 0.015M NaCl, 0.0015M trisodium citrate, pH 7.0, with 0.1% sodium dodecyl sulfate), and autoradiography for 24 hours. Under these conditions, the dot-blot hybridization assay for detection of serotype 2 IBDV double-stranded RNA, was 1,000 times more sensitive, using probe pOH632, but only 10 times more sensitive, using probe pOH405, compared with the assay for IBDV serotype 1, using the same probes. Thus, probe pOH632 could differentiate between the 2 IBDV serotypes by nucleic acid hybridization.     

禽流感病毒WUDI-H9N2株的分离鉴定及 HA基因的分子特征研究

本研究于2011年8月从山东省某鸡场鸡群中分离一株病毒,通过对分离株进行病毒血凝试验及血凝抑制试验、RT-PCR、动物回归试验等方法证实已分离到禽流感病毒(avian influenza virus,AIV) H9N2,命名为WUDI-H9N2株。根据GenBank上发表的AIV H9病毒的HA基因序列,用Oligo 6.0生物学软件设计引物,对分离株WUDI-H9N2株HA基因进行扩增、测序及序列分析。将分离的AIV H9毒株与其他血清型代表参考毒株分别进行核苷酸和氨基酸系统发生进化关系分析。WUDI-H9N2株 HA基因全序列长为1708 bp,无核苷酸的插入和缺失,cDNA包含了1个完整的开放阅读框(ORF),编码区由1683 bp组成,共编码560个氨基酸。HA序列与国内参考毒株的核苷酸序列的同源性在92.9%~98.1%之间,其中与安徽分离株A/chicken/China/AH-10-012010(H9N2)同源性为98.1%。     

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.     

犬乙肝病毒S基因遗传变异研究

应用人乙肝诊断试剂盒，对检测出来的“三阳（HBsAg,HBeAg,抗-HBc)犬的乙肝病料进行了病毒分离和形态学鉴定，并以人乙肝病毒S基因两侧的保守区域作为模板，采用PCR方法扩增出犬乙肝病毒部分基因，序列分析结果，其核苷酸序列为1157bp，编码385个氨基酸，遗传变异分析结果表明，在S基因保守区内有27个编码氨基酸与人的不同，其中16个为性质相反或不同的氨基酸置换，核苷酸和氨基酸序列同源性分别为96.5%和93.4%，系统发育进化树比较表明二者的遗传关系较远，揭示犬乙肝病毒与人乙型肝炎病毒S基因保守区之间存在很大差异；而与鸡，羊乙肝病毒S基因保守区序列相比较，遗传变异更大。     

Canine Bcl-xL gene and its expression in tumor cell lines

The canine Bcl-xL gene was cloned and sequenced. Canine Bcl-xL cDNA clone was 1252 bp in length, and encoded 233 deduced amino acides. The predicted canine Bcl-xL amino acid sequence shared 99.6%, 97.0%, 97.9%, 98.7% and 98.3% homology with that of human, mouse, rat, sheep and pig Bcl-xL, respectively. RT-PCR analysis revealed that canine Bcl-xL mRNA was constitutively expressed in CL-1 (canine lymphoma) and GL-1 (canine B cell leukemia) cell lines.     

Expression of CC chemokine receptor 4 (CCR4) mRNA in canine atopic skin lesion

CC chemokine receptor 4 (CCR4) is a G protein-coupled seven transmembrane receptor that is selectively expressed on Th2 cells and plays an important role in the trafficking of Th2 cells into inflammatory sites. In this study, a full-length canine CCR4 cDNA was cloned and characterized in order to examine the potential role of CCR4 in allergic responses that produce skin lesions in canine atopic dermatitis (AD). The canine CCR4 cDNA reported in this study contained an open reading frame of 1083 nucleotides encoding 360 amino acids. The predicted amino acid sequence of canine CCR4 showed 91.9, 85.3 and 84.5% similarity with those of the human, mouse and guinea pig counterparts, respectively. Expression of CCR4 mRNA was detected in various tissues including thymus, spleen, heart, small intestine and lymph node. Furthermore, it was found that CCR4 mRNA was preferentially expressed in lesional skin of dogs with AD, together with the mRNA of thymus and activation-regulated chemokine (TARC), which is a ligand for CCR4. The present study demonstrates that CCR4 contributes strongly to the immunopathogenesis of canine AD.