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1.
Bovine interleukin-21 (IL-21) cDNA was cloned and sequenced from bovine peripheral blood lymphocytes (PBLs) stimulated with 10 microg/ml concanavalin A (ConA), 10 microg/ml phytohemagglutinin (PHA), and 50 ng/ml phorbol 12-myristate 13-acetate (PMA) for 48 h. The open reading frame of the bovine IL-21 cDNA is 459 bp in length and encodes 152 amino acids. The predicted amino acid sequence is 78.2 and 58.5% homologous to the human and murine IL-21 amino acid sequences, respectively. Recombinant bovine IL-21 was expressed by a baculovirus expression system. The bovine IL-21 was processed to the mature form in insect cells and secreted to the supernatant confirmed by N-terminal amino acid sequencing. The recombinant bovine mature IL-21 induced the proliferation of human IL-2-dependent cells, ILT-MAT. The mRNA expression for bovine IL-21 was observed in the spleen, but not in the brain, heart, lung, liver, and kidney. The bovine IL-21 identified in this study may provide new methods for the enhancement of innate immunity in cows. 相似文献
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CD69 is rapidly inducible on various hematopoietic cells upon stimulation and is detectable as an early activation antigen. Although CD69 is well characterized in human and mouse, no information is available on bovine CD69. We report here that, bovine CD69 was cloned from a cDNA expression library prepared from activated peripheral blood lymphocytes. The full-length cDNA contained an 80bp 5' untranslated region, followed by a 600bp coding region and AU-rich motifs in a 3' untranslated region (GenBank accession number AF272828). Comparison of the bovine CD69 coding sequence reveals 69.4 and 78.2% nucleotide sequence identities with mouse and human CD69, respectively. The predicted amino acid sequence of bovine CD69 shares 56.3 and 62.3% sequence identity when compared with mouse and human CD69, respectively. Bovine CD69 has the highly conserved amino acid sequences found in the C-type lectin family, suggesting that the conserved residues may be important for conformation and binding to the, as yet unidentified ligand. In addition, the cytoplasmic tail of bovine CD69 has two casein kinase-2 (CK-2) phosphorylation sites. These data suggest that bovine CD69 plays an important role in the activation of lymphocytes. 相似文献
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Bovine interleukin 2: regulatory mechanisms 总被引：1，自引：0，他引：1
N S Magnuson A G Spies M S Nissen C D Buck A D Weinberg P J Barr J A Magnuson R Reeves 《Veterinary immunology and immunopathology》1987,17(1-4):183-192
A cDNA clone of the bovine interleukin-2 (IL-2) gene has been isolated and demonstrated to produce a functional bovine IL-2 protein when transfected into either CV-1 or COS-1 monkey cells. Homology comparisons of both the nucleotide and predicted amino acid sequences of bovine IL-2 with those of the human and mouse show extensive regions of sequence conservation between the species. The amino acid sequence of the mature bovine IL-2 protein shares about 60-63% homology with those of the human and mouse, but the 3' untranslated regions of the human and mouse gene share as much, if not greater, sequence homology with the 3' untranslated regions of the human and mouse genes. In particular, a tandemly repeated sequence (TATT), n, found in the 3' untranslated tail of the bovine IL-2 clone is also found in the 3' untranslated region of a large group of cytokine genes and other inducible genes of the lymphoid and immune response systems. This sequence may serve a specific regulatory function in the immune system. 相似文献
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羊草乙醛脱氢酶基因LcALDH的克隆与表达分析 总被引：1，自引：1，他引：0
采用RACE技术从羊草中克隆了乙醛脱氢酶基因LcALDH的cDNA(GenBank登录号EF492045)。长为1 712 bp的LcALDH cDNA序列含有编码500个氨基酸的1 503 bp的开放读码框、66 bp的5′非翻译区和144 bp的3′非翻译区。氨基酸序列中含有醛脱氢酶家族绝对保守的谷氨酸活性位点和半胱氨酸残基活性位点。分析LcALDH基因在不同胁迫条件下的表达情况,结果表明,LcALDH的表达受到低温、干旱、高盐和ABA的正调控作用。研究结果为进一步从分子水平探明羊草的抗逆机制,挖掘并利用植物抗逆基因奠定基础。 相似文献
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Ahmed M Kimura K Soliman M Yamaji D Okamatsu-Ogura Y Ishioka K Makondo K Hagiwara K Saito M 《Veterinary journal (London, England : 1997)》2008,176(3):361-368
This study examined the mitogenic response of bovine peripheral T lymphocytes to leptin, a pleiotropic hormone regulating food intake and energy expenditure. Leptin alone slightly suppressed proliferation of T lymphocytes in the presence of concanavalin A (ConA). Leptin also inhibited proliferation of T lymphocytes induced by anti-CD3 antibody. ConA treatment activated some protein kinases, including p44/p42(MAPK) and Akt/PKB, while anti-CD3 antibody treatment increased mRNA expression of suppressor of cytokine signalling (SOCS) 3, interferon (IFN)gamma, interleukin (IL) 2 and IL4 in T lymphocytes. Leptin alone increased only SOCS3 mRNA expression. Simultaneous treatment with mitogens and leptin enhanced IFNgamma mRNA expression but decreased IL2 mRNA expression, without any synergistic effect on phosphorylation of protein kinases or mRNA expression of SOCS3 and IL4. These results suggest that leptin modulates bovine T lymphocyte functions. 相似文献
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在家蚕丝腺cDNA文库测序过程中,发现一个编码家蚕泛素结合酶的EST序列,利用3′RACE方法克隆了一个新的家蚕泛素结合酶基因cDNA全长序列,命名为BmUCE2 I(GenBank登录号为DQ219874)。家蚕BmUCE2 I基因全长cDNA由465 bp的开放阅读框序列(ORF)、97 bp的5′端非翻译区序列(5-′UTR)和237 bp的3′端非编码区序列(3-′UTR)组成,其编码的154个氨基酸与其他真核生物间具有较高的同源性。利用BmUCE2 I的EST片断作探针,通过筛选家蚕噬菌体基因组文库,获得了家蚕BmUCE2 I基因组序列和5′调控序列。BmUCE2 I基因由4个外显子和3个内含子组成,在5′端上游调控区域没有类似TATA盒元件,但在-219~-268 bp的区域存在一个50bp的启动子序列,此外还存在CF2-Ⅱ、FTZ、DFD、BRCZ2、DL、STAT、PRD-HD等多个转录因子结合位点。家蚕泛素结合酶新基因的克隆、基因结构及5′调控区的分析为进一步研究泛素蛋白水解酶复合通路相关基因的调控规律提供了重要依据。 相似文献
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H Nagahata H Nochi K Tamoto H Noda G J Kociba 《Canadian journal of veterinary research》1995,59(1):1-7
Expression of CD18 on bovine neutrophils in response to stimulation by zymosan activated serum (ZAS) and phorbol myristate acetate (PMA) and the effects of monoclonal antibodies (MAB) recognizing CD18 or bovine neutrophil surface antigens (S2G8 and S5F8G10) on adherence, chemotactic responses and phagocytosis of bovine neutrophils were evaluated. CD18 expression of neutrophils was increased after ZAS and PMA treatment by 12.2 and 54.2% respectively, and were significantly (p < 0.05, p < 0.01) different from those of untreated neutrophils. CD18 expression by neutrophils from a Holstein-Friesian heifer affected with leukocyte adhesion deficiency was within negative controls when stimulated by ZAS and PMA. Adherence, chemotactic responses, and phagocytosis were significantly decreased (p < 0.01) in neutrophils continuously treated with anti-CD18 MAB (MHM 23). Adherence was also significantly decreased in anti-CD18 pretreated neutrophils. Significant (p < 0.01) differences of chemotactic responses and phagocytosis of neutrophils were found between neutrophils pretreated and continuously treated with anti-CD18 MAB (MHM 23). Monoclonal antibodies to other surface antigens did not significantly alter neutrophil adherence, chemotaxis or phagocytosis. This study demonstrated that CD18 expression on bovine neutrophils is increased significantly by stimulation with ZAS and PMA and that the adhesion molecule CD18 plays an important role in adhesion-related functions. 相似文献
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丙酮酸脱氢酶(PDH)是丙酮酸脱氢酶复合体(PDC)中的前件酶,参与生成柠檬酸循环(TCA)的起始物乙酰辅酶A,决定生物体内营养成分的分配。利用电子克隆、RT-PCR和cDNA末端快速扩增(RACE)等方法克隆了与果蝇lethal(1)G0334基因类似的具有丙酮酸脱氢酶功能的家蚕Bm-l(1)基因。Bm-l(1)基因的cDNA全长为1 630 bp,由1 200 bp的完整ORF序列、186 bp的5'-UTR和207 bp的3'-UTR组成,含8个外显子和7个内含子,编码蛋白为399个氨基酸残基,分子质量43.93kD,pI 8.07。Bm-l(1)基因编码蛋白的69-365氨基酸残基为E1-dh结构域,该结构域为硫胺素焦磷酸依赖性脱氢酶所特有。蛋白质二级结构预测结果表明α螺旋占28.8%,β折叠占12.0%。采用Clustal W进行多序列比对发现,Bm-l(1)基因编码蛋白与赤拟谷盗等昆虫的PDH具有63%以上的序列相似性,且保守区域高度一致。Bm-l(1)基因mRNA在家蚕整个卵期、幼虫期、蛹期和刚羽化的成虫中,以及5龄3 d幼虫的头部、丝腺、生殖腺、脂肪体、中肠和血液6种组织中都有较高的转录水平,且存在较小的组织差异性。 相似文献
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Y Inoue T Itou T Sakai T Oike 《The Journal of veterinary medical science / the Japanese Society of Veterinary Science》1999,61(6):693-696
Using polymerase chain reaction (PCR), a bottle-nosed dolphin (Tursiops truncatus) interleukin-4 (IL4) cDNA was cloned and sequenced. IL4 specific primers were based on the 5' and 3' untranslated regions of the human and murine IL4 gene. The dolphin IL4 cDNA is 528 base pairs in length and contains an open reading frame of 402 nucleotides coding an IL4 precursor of 133 amino acids, with the putative signal peptide of 24 amino acids. Analysis of the mature amino acid sequence shows three potential N-linked glycosylation sites and three disulfide bonds. Comparison of the predicted amino acid sequence shows that dolphin IL4 shares 77, 74, 58 and 41% identity with the bovine, ovine, human and mouse IL4s, respectively. 相似文献
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A variety of commercial DNA arrays specific for humans and rodents are widely available; however, microarrays containing well-characterized genes to study pathway-specific gene expression are not as accessible for domestic animals, such as cattle, sheep and pigs. Therefore, a small-scale application-targeted bovine immune-endocrine cDNA array was developed to evaluate genetic pathways involved in the immune-endocrine axis of cattle during periods of altered homeostasis provoked by physiological or environmental stressors, such as infection, vaccination or disease. For this purpose, 167 cDNA sequences corresponding to immune, endocrine and inflammatory response genes were collected and categorized. Positive controls included 5 housekeeping genes (glyceraldehydes-3-phosphate dehydrogenase, hypoxanthine phosphoribosyltransferase, ribosomal protein L19, beta-actin, beta2-microglobulin) and bovine genomic DNA. Negative controls were a bacterial gene (Rhodococcus equi 17-kDa virulence-associated protein) and a partial sequence of the plasmid pACYC177. In addition, RNA extracted from un-stimulated, as well as superantigen (Staphylococcus aureus enterotoxin-A, S. aureus Cowan Pansorbin Cells) and mitogen-stimulated (LPS, ConA) bovine blood leukocytes was mixed, reverse transcribed and PCR amplified using gene-specific primers. The endocrine-associated genes were amplified from cDNA derived from un-stimulated bovine hypothalamus, pituitary, adrenal and thyroid gland tissues. The array was constructed in 4 repeating grids of 180 duplicated spots by coupling the PCR amplified 213-630 bp gene fragments onto poly-l-lysine coated glass slides. The bovine immune-endocrine arrays were standardized and preliminary gene expression profiles generated using Cy3 and Cy5 labelled cDNA from un-stimulated and ConA (5 microg/ml) stimulated PBMC of 4 healthy Holstein cows (2-4 replicate arrays/cow) in a time course study. Mononuclear cell-derived cytokine and chemokine (IL-2, IL-1alpha, TNFalpha, IFN-gamma, TGFbeta-1, MCP-1, MCP-2 and MIP-3alpha) mRNA exhibited a repeatable and consistently low expression in un-stimulated cells and at least a two-fold increased expression following 6 and 24 h ConA stimulation as compared to 0 h un-stimulated controls. In contrast, expression of antigen presenting molecules, MHC-DR, MHC-DQ and MHC-DY, were consistently at least two-fold lower following 6 and 24 h ConA stimulation. The only endocrine gene with differential expression following ConA stimulation was prolactin. Additionally, due to the high level of genetic homology between ovine, swine and bovine genes, RNA similarly acquired from sheep and pigs was evaluated and similar gene expression patterns were noted. These data demonstrate that this application-targeted array containing a set of well characterized genes can be used to determine the relative gene expression corresponding to immune-endocrine responses of cattle and related species, sheep and pigs. 相似文献
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Tajima M 《Veterinary microbiology》2004,99(2):131-138
Phylogenetic analyses of bovine viral diarrhea virus (BVDV) were performed based on the nucleotide sequences of the 5' untranslated region (5'-UTR) and E2-coding gene. Thirty-six BVDV detected from naturally infected cattle in the northern region of Japan were divided into three genotypes, BVDV1a, BVDV1b and BVDV2, in a 5'-UTR phylogenetic tree. In a phylogenetic tree constructed from the E2-coding gene, BVDV1c was identified and the viruses classified in BVDV1c were included in BVDV1a in the 5'-UTR phylogenetic tree. Moreover, BVDV1a and BVDV1b in the E2-phylogenetic tree clustered closer together than in the 5'-UTR tree. These results suggested that phylogenetic analysis of the E2 gene was more useful for identification of subgenotypes within BVDV1. 相似文献
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Major histocompatibility complex (MHC) class I and class II molecules encode glycoproteins which mediate the specificity of the vertebrate adaptive immune response. In this study, MHC class IIB gene from the Chinese longsnout catfish (Leiocassis longirostris) was cloned and sequenced, which encoded a predicted protein of 248 amino acids (28.06 kDa) containing a signal peptide, a beta 1 domain, a beta 2 domain, a connecting peptide, a transmembrane region, and a cytoplasmic tail. Using PCR with primers designed from known fish MHC class IIB sequences followed by elongation of the 5' and 3' ends using rapid amplification of cDNA ends (RACE), the full-length cDNA of longsnout catfish MHC class IIB was identified to be 1293 bp, consisting of a 26 bp 5'-terminal untranslated region (UTR), a 520 bp 3'-UTR, and a 747 bp open reading frame (ORF) bearing characteristics of the immunoglobulin C-type 1 (IGc1) family. The deduced amino acid sequences of the Chinese longsnout catfish MHC class IIB gene had 58-75% identity with those of other fishes. Six class IIB alleles were identified from five individuals. At most two different alleles observed in each individual may infer the existence of a single locus of class IIB gene in the Chinese longsnout catfish genome. An extensive study of polymorphism was examined in 60 individuals. A total of 11 haplotypes of exon 2 were detected in the sampled Chinese longsnout catfish. The rates of nonsynonymous substitutions (d(N)) occurred at a higher frequency than that of synonymous substitutions (d(S)), suggesting the polymorphism of exon 2 seemed to be maintained by the balancing selection. By using long PCR technique, the genomic sequence was further identified to be 2345 bp in length, which contained six exons and five introns. Interestingly, a 98 bp intron 5 cut the 3'-UTR into two parts. Real-time quantitative RT-PCR demonstrated high expression of MHC IIB in gills, spleen, head kidney, and intestine, moderate expression in liver and stomach, and low or negligible expression in heart. Homology modelling has been applied to create a 3D model of longsnout catfish MHC class IIB, with features evaluated and discussed to investigate its interaction with CD4 participating in antigen recognition. The present findings will provide valuable information for understanding structure, function, expression, and molecular polymorphism of MHC class IIB in adaptive immunity of the Chinese longsnout catfish and teleost. 相似文献
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The study was aimed to obtain carp iNOS cDNA full-length sequence, and investigate the changes in peripheral blood leukocyte expression of iNOS in the stimulation of different mitogens.Based on EST sequences of iNOS that obtained from carp normal peripheral blood leukocytes cDNA library, full-length cDNA sequence of carp iNOS was successfully amplified by using gene library screening and rapid-amplification of cDNA 5'ends (5'-RACE) method.Carp peripheral blood leukocytes were divided into control and experimental groups and cultured.The experimental groups were stimulated by LPS (1.0 μg/mL) and ConA (1.0 μg/mL) for 4 and 12 h, respectively.And control groups were in the same cultured conditon time without mitogen stimulated.Real-time PCR was used to detect the expression of iNOS in peripheral blood leukocytes of each group.The results showed that the cDNA fragment was total 3 704 bp containing 74 bp 5'untranslated region (UTR), 246 bp 3'UTR and a 3 384 bp ORF encoding 1 127 amino acids.Sequence homology analysis showed that the amino acid sequence of carp iNOS shared 100% identity with Carassius auratus.After LPS and ConA stimulation, the iNOS expression of peripheral blood leukocytes in the experimental groups were increased, and the expression in the short time stimulation condition (4 h) was higher than the long time (LPS 12 h; ConA 24 h).In conclusion, iNOS expression of peripheral blood leukocytes was raised after LPS and ConA stimulation, suggesting that there were dynamic changes in the inflammation. 相似文献
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Relationship between the chemiluminescent response of bovine neutrophils and changes in intracellular free calcium concentration. 总被引：2，自引：1，他引：1
The relationship between luminol dependent chemiluminescent (LDCL) response and changes in intracellular free Ca2+ concentrations in the bovine neutrophils was evaluated. LDCL responses and changes in intracellular Ca2+ concentrations of neutrophils were clearly detected by the stimulation with opsonized zymosan (OPZ), concanavalin A(ConA), heat-aggregated IgG (H-agg.IgG) and phorbol myristate acetate (PMA). Patterns of LDCL responses and intracellular Ca2+ of neutrophils showed characteristic features for each stimulant. PMA was a weak stimulant of the intracellular Ca2+ concentration, whereas it was a strong stimulant of LDCL response. Con A strongly stimulated an increase in the intracellular Ca2+ concentration, but was a weak stimulant of LDCL response. LDCL response of intracellular Ca(2+)-depleted neutrophils treated with ionomycin, stimulated with each stimulant was inhibited markedly without extracellular Ca2+. The sustained phase of intracellular Ca2+ concentrations stimulated with OPZ was inhibited significantly (P < 0.05) by the preincubation with anti-CD18 antibody, whereas the transient phase of intracellular Ca2+ concentrations was not inhibited. These results indicate that LDCL response is regulated at least in part by the elevation of the intracellular Ca2+, and a rise in intracellular Ca2+ concentration, which may be mediated by specific receptors appears to be essential in the LDCL response of bovine neutrophils. 相似文献
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试验旨在获得鲤鱼诱导型一氧化氮合成酶(inducible nitric oxide synthase,iNOS)cDNA全长序列,并以此为基础探讨在丝裂原刺激下外周血白细胞中iNOS的表达变化。以从鲤鱼正常外周血白细胞cDNA文库中获得的iNOS的EST序列为基础,采用基因文库筛选和cDNA5'末端快速扩增技术(5'-RACE)相结合的方法,成功扩增出鲤鱼iNOScDNA全长序列,然后进行鲤鱼外周血白细胞原代培养,分成对照组和试验组,其中试验组分别为脂多糖(LPS,1.0μg/mL)刺激4、12h,刀豆蛋白A(ConA,1.0μg/mL)刺激4、24h,对照组为相同培养时间无丝裂原刺激的外周血白细胞,根据得到的iNOScDNA全长序列和鲤鱼β-actin序列分别设计特异性引物,应用实时荧光定量PCR方法检测各组外周血白细胞中iNOS在mRNA水平上的表达情况并进行分析。序列分析结果显示,最后获得的cDNA片段共3704bp,包含74bp的5'端非编码区,246bp的3'端非编码区,一个3384bp的完整的开放阅读框(ORF),共编码1127个氨基酸。序列同源性分析结果显示,该序列与鲫鱼iNOS基因同源性高达100%;实时荧光定量PCR结果显示,经LPS、ConA刺激的试验组外周血白细胞中iNOS表达量均升高,且短时间(4h)刺激的表达量高于长时间(LPS12h;ConA24h)刺激的表达量。综上所述,在LPS、ConA刺激过程中,iNOS在外周血白细胞中表达量均有上调,结果提示存在炎症反应的动态变化。 相似文献
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两种方法测定羊外周血淋巴细胞增殖的研究 总被引：3，自引：0，他引：3
本试验比较~3H胸腺嘧啶核苷(~3H-TdR)渗入法和BrdU-ELISA方法测定羊外周血淋巴细胞增殖。分离健康羊外周血淋巴细胞,与不同浓度的非特异刺激因子刀豆素A(concanavalinA,1.0,0.5,0.25,0.125,0.0625,0.0313,0.0156,0.0078,0.0039μg/孔)培养,分别用~3H胸腺嘧啶核苷(~3H-TdR)和5-溴-2’-脱氧尿苷(BrdU)标记细胞,测定刺激细胞和对照细胞的每分钟脉冲数cpm或光吸收值OD,计算刺激指数SI和光吸收值差△OD,统计学方法分析SI和△OD的相关程度。同样的方法测定羊试验感染肝片吸虫(Fasciola hepatica)后,在感染早期阶段(PIWO-PIW4)其外周血淋巴细胞在特异性抗原-片形吸虫分泌排泄产物(ESP,5μg/孔)刺激下的增殖。结果表明,刺激指数SI和光吸收值差△OD在两个试验中的相关系数分别为0.946和0.924,SI和OD用均呈强正线性相关。BrdU-ELISA方法无同位素放射物,且敏感、快速、方便,和~3H-TdR渗入法一样可以用于测定淋巴细胞增殖。 相似文献