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鹿茸尖端组织核心蛋白多糖基因全长cDNA的克隆及其表达分析 总被引：1，自引：0，他引：1
为了进一步了解鹿源DCN基因的结构与功能,揭示该基因在鹿茸尖端不同组织层的表达规律,本研究从梅花鹿鹿茸尖端组织cDNA文库中首次克隆具有完整编码区的DCN基因全长cDNA序列,并结合生物信息学方法和实时荧光定量RT-PCR技术对该基因的氨基酸序列结构和表达特征进行分析.结果表明,梅花鹿DCN基因cDNA全长为1 831 bp,编码360个氨基酸;其编码蛋白具有N端信号肽,相对分子质量为39.9 ku,理论等电点为8.8,其一级结构中亮氨酸所占比例最高(12.5%);梅花鹿与绵羊DCN氨基酸序列的相似性最高(98%).实时荧光定量RT-PCR分析表明,DCN在间充质层的表达显著高于其他3层组织,提示DCN的抗纤维化活性可能是维持鹿茸间充质层快速生长的重要调节因素. 相似文献
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Okui Y Kano R Maruyama H Hasegawa A 《Veterinary immunology and immunopathology》2008,121(1-2):156-160
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. 相似文献
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He W Ohashi K Sugimoto C Tsuji M Onuma M 《The Japanese journal of veterinary research》2005,53(1-2):27-35
A cDNA clone encoding a prohibitin-like protein (Toprh) was isolated from a piroplasm cDNA library of Theileria orientalis and its nucleotide sequence was determined. An open reading frame, encoding a polypeptide of 278 amino acid residues, was found in Toprh cDNA sequence. An intron of 89 bp was identified when this cDNA clone was compared with the Toprh gene in the genome of T. orientalis. The deduced amino acid sequence of Toprh shares 93.8, 93.1 and 69.1% identities with the prohibitins of T. parva (from chromosome 1), T. annulata (from chromosome 1), and Plasmodium falciparum, (from chromosome 10), respectively. By Western blot analysis, Toprh was found to be expressed in the piroplasm stage of the parasites. 相似文献
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OBJECTIVES: To determine the full-length complementary DNA (cDNA) sequence of equine retinal and pineal gland phosducin (PHD) and to clone these sequences. SAMPLE POPULATION: Samples of equine retinal RNA. PROCEDURE: A primer set was designed for use in identifying a fragment of the equine PHD nucleotide sequence, derived from retinal RNA samples, and subsequently for use to deduce specific primers for additional examination. The full-length cDNA was determined by the method of rapid amplification of cDNA ends (RACE). For full-length cDNA, newly designed primers were used. Nucleotide sequences were analyzed by use of computer software. The deduced amino acid sequence was compared with sequences of PHD reported for other species. In addition, the sequence of equine pineal PHD was cloned. RESULTS: The cDNA nucleotide sequence for equine PHD was 1,209 base pairs (bp) in length with an open-reading frame encoding a protein of 245 amino acids and a calculated molecular mass of 28.214 kd. Similarity with amino acid sequences of PHD from other species was 89 to 93%. Sequences of equine PHD from retina and pineal gland were identical. Equine PHD contained a peptide sequence with 100% homology to an uveitopathogenic peptide reported for rat PHD. CONCLUSIONS: Equine PHD is a highly conserved protein that has homology of immunologic interest with rat PHD. These results establish a basis for studying the role of PHD in ocular inflammation of horses. 相似文献
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本研究根据其他鱼类MHC-Ⅱα基因的保守序列设计兼并引物,运用同源克隆和末端快速扩增方法扩增了军曹鱼MHC-Ⅱα基因的全长cDNA序列,并对cDNA及其氨基酸序列进行了分析,比较了军曹鱼和其他物种的MHC-Ⅱα氨基酸序列的差异,分析了军曹鱼MHC-Ⅱα基因的组织分布及经LPS刺激后头肾组织中MHC-Ⅱα基因的表达变化。结果表明,军曹鱼MHC-Ⅱα cDNA全长998 bp,包括53 bp的5′末端非编码区（5′UTR）、234 bp的3′末端非编码区（3′UTR）及711 bp的开放阅读框（ORF）,编码236个氮基酸,其蛋白质分子质量约为25.94 ku,等电点为4.39;军曹鱼MHC-Ⅱα蛋白质序列具有一些重要的特征,包括前导肽、α1、α2、CP/TM/CYT区和保守的半胱氨酸等;军曹鱼MHC-Ⅱα与鼠、人及其它鱼类的氨基酸同源性在25.0%～69.5%之间。Real-time PCR检测结果显示,MHC-Ⅱα基因在正常军曹鱼组织中均有表达,但其表达量在各种组织中存在差异,其中较强表达于头肾、鳃,中等程度表达于脾脏、肠,在心脏、脑、肌肉中表达较弱;经LPS刺激后,头肾中MHC-Ⅱα基因表达下调。 相似文献
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本试验旨在克隆、鉴定猪硒蛋白P基因(Sepp1),并探明其在猪不同组织中的mRNA相对表达量,为以猪为模型研究硒蛋白P(Sel P)的功能奠定基础。根据表达序列标签(EST)序列设计引物,利用c DNA末端快速克隆(3'-RACE)技术从猪肝脏总RNA中扩增出含开放阅读框(ORF)至poly A片段,然后与EST序列进行拼接;采用荧光定量PCR技术考察Sepp1在猪9个组织中的mRNA相对表达量。结果显示:1)扩增出共1 707 bp的片段,测序后与EST拼接获得了2 109 bp的猪Sepp1序列,并提交至NCBI Gen Bank数据库,序列号为EF113596.2;该基因1 170 bp的ORF编码区和对应的氨基酸残基与人相应序列分别有83.72%和75.64%序列同源性,其编码390个氨基酸,含有14个硒代半胱氨酸(Sec)残基,分别位于第59、267、286、309、311、327、339、352、354、361、376、378、385和387位。2)Sepp1 mRNA在猪组织中广泛分布,在肝脏中具有最高分布,依次为甲状腺肾脏睾丸下丘脑脾脏垂体心脏肌肉。本试验成功克隆、鉴定了猪Sepp1,检测了其在猪不同组织中表达分布情况,为其进一步以猪为模型探讨其功能奠定了基础。 相似文献
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旨在克隆猪作用于RNA的腺苷脱氨酶2基因（ADAR2）全长cDNA序列,同时对该基因在猪不同组织中的表达规律进行探索。利用RACE （rapid-amplification of cDNA ends）对大白猪ADAR2基因mRNA全长序列进行克隆,并进行生物信息学分析;用荧光定量PCR方法检测35日龄大白猪心、肝、肺、肾、脾、脑、小肠、背最长肌和背部脂肪9种组织中ADAR2的表达水平。结果表明,猪ADAR2基因cDNA全长6 305 bp,共包含12个外显子,编码704个氨基酸,与人、黑猩猩、猕猴、长臂猿、黄牛、山羊和绵羊的CDS区核酸序列和氨基酸序列的一致性均在84%以上。该基因编码的蛋白含有2个双链RNA结合基序和一个脱氨酶结构域。猪ADAR2在检测的各组织中均表达,其中在肺中的表达量最高。综上所述,本研究成功克隆了猪ADAR2基因全长cDNA序列,并且发现其在猪体内广泛表达,为深入研究ADAR2的功能奠定了良好的基础。 相似文献
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Maeda S Mizuno T Yamashita K Kurata K Masuda K Ohno K Tsujimoto H 《The Journal of veterinary medical science / the Japanese Society of Veterinary Science》2001,63(9):1035-1038
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. 相似文献
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广西巴马小型猪卵泡抑素的克隆及原核表达 总被引：1，自引：1，他引：1
从广西大学巴马小型猪卵巢中提取总RNA，并以其为模板，应用逆转录-聚合酶链式反应（RT-PCR），在合成的特异性引物引导下，扩增获得广西巴马小型猪卵泡抑素（follistatin）cDNA的全序列，长度为1035 bp。将PCR产物克隆到pMD18-T载体后进行序列测定及分析，结果表明：广西巴马小型猪卵泡抑素cDNA序列与GenBank中已报道的家猪卵泡抑素同源性高达99.4%。同时，将目的基因插入到原核表达载体pET 32a+多克隆位点中，构建了follistatin的原核表达载体，并转化于大肠杆菌BL21。转化菌经异丙基硫代半乳糖苷（IPTG）诱导，变性聚丙烯酰胺凝胶电泳（SDS-PAGE）和蛋白质免疫印迹（Western blotting）分析，结果表明：试验已成功表达了follistatin的融合蛋白(约58.4 ku)。 相似文献
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Webb TL Burnett RC Avery AC Olver CS 《Veterinary clinical pathology / American Society for Veterinary Clinical Pathology》2010,39(4):424-432
Background: Characterization of anemia in horses presents a challenge, as they do not release reticulocytes into peripheral blood. Transferrin receptor (TfR) expression is highest on erythroid cells in people and rats, and measurement of a soluble serum form (sTfR) is used to quantify erythropoiesis in these species. We hypothesized that equine TfR (eTfR) expression is similar in quantity and distribution to that in these other species and thus has potential for characterization of the regenerative response in anemic horses. Objective: This study was conducted to clone and sequence the eTfR gene and measure expression levels using quantitative real‐time PCR and immunohistochemical (IHC) staining. Methods: Total RNA from equine bone marrow was used to produce cDNA. The eTfR gene was amplified using pooled gene‐specific primers, and PCR products were sequenced. Rapid amplification of cDNA ends was used to obtain the first 22 nucleotides of the coding sequence. Quantitative PCR was performed using eTfR gene‐specific primers, and IHC staining was used to localize TfR protein expression. Results: The deduced amino acid (aa) sequence (767 aa) of the eTfR was 75–83% identical with sequences of the receptor in several other mammals. As in people and rats, eTfR mRNA expression was highest in the bone marrow, and distribution in other tissues was also similar. Conclusion: The eTfR gene is similar to that of other mammals in structure and expression levels. We hypothesize that it is also similar in function and that, following development of an immunoassay, determining sTfR concentrations will be useful for identifying the regenerative response in anemic horses. 相似文献
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C R Engwerda R A Sandeman S J Stuart R M Sandeman 《Veterinary immunology and immunopathology》1992,34(1-2):115-126
A complementary DNA (cDNA) corresponding to sheep immunoglobulin E heavy (IgE H) chain messenger RNA (mRNA) was isolated and sequenced. A fragment of sheep IgE constant H (epsilon)-chain was initially synthesized using the polymerase chain reaction (PCR) and single-stranded cDNA prepared from the mRNA of parasite-stimulated sheep lymph nodes. This fragment was then used to probe a cDNA library, again prepared from a parasite-stimulated lymph node. A recombinant clone containing cDNA encoding a sheep IgE H-chain of 1802 base pairs was isolated and the H-chain cDNA-sequenced. The nucleotide sequence was found to code for a complete sheep IgE H-chain, consisting of both variable (V) and constant (C) regions. When the amino acid sequence derived from the nucleotide sequence was compared to other species epsilon-chains, interesting homologies and differences between corresponding domains were found, including conservation in cysteine and tryptophan residues and variable glycosylation sites. 相似文献
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Toribio RE Kohn CW Chew DJ Capen CC Rosol TJ 《American journal of veterinary research》2002,63(2):194-197
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. 相似文献
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Weber ER Helps CR Foster AP Perry AC Gruffydd-Jones TJ Hall L Harbour DA Duffus WP 《Veterinary immunology and immunopathology》2000,76(3-4):299-308
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. 相似文献
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Koga L Kobayashi Y Yazawa M Maeda S Masuda K Ohno K Tsujimoto H 《The Journal of veterinary medical science / the Japanese Society of Veterinary Science》2002,64(5):453-456
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. 相似文献
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Molecular cloning of the canine c-Met/HGF receptor and its expression in normal and regenerated liver 总被引：4，自引：0，他引：4
Neo S Kansaku N Furuichi M Watanabe M Hisamatsu S Ohno K Hisasue M Tsuchiya R Yamada T 《The Journal of veterinary medical science / the Japanese Society of Veterinary Science》2005,67(5):525-529
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. 相似文献
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试验旨在克隆山羊碱性氨基酸转运载体基因SLC3A1 cDNA序列,探究其表达的组织特异性及其在小肠中的表达发育模式。参考GenBank已发表的绵羊、牛SLC3A1基因mRNA序列设计引物,克隆山羊SLC3A1基因的cDNA序列,采用实时荧光定量PCR的方法分析其在1日龄山羊11种组织及其在1日龄、6月龄、8月龄、10月龄、12月龄山羊十二指肠、空肠、回肠中的mRNA表达情况。结果表明,成功克隆得到了山羊SLC3A1基因cDNA序列,其与绵羊、牛、野猪、人、小鼠、大鼠的同源性分别为99%、97%、88%、86%、80%和79%。SLC3A1基因转录表达有明显的组织特异性,其在1日龄山羊肾脏、回肠、空肠、结肠中表达量依次降低(P<0.05),其他组织中表达量很低。同一月龄山羊,SLC3A1基因在不同肠段的表达量数值上回肠>空肠>十二指肠。SLC3A1基因在不同月龄山羊十二指肠表达量以1日龄山羊为最高(P<0.05),6~12月龄山羊相同肠段之间表达量无显著差异(P>0.05);空肠表达量随山羊年龄的增加均呈现逐步降低的趋势;回肠表达量在各个月龄山羊之间差异不显著(P>0.05)。结果说明,山羊碱性氨基酸转运载体基因SLC3A1的主要表达部位在肾脏和肠道,推测b0,+碱性氨基酸转运系统转运氨基酸的主要部位为肾脏和肠道。SLC3A1基因在山羊小肠受肠段和发育阶段的影响,具有不同的表达发育模式。 相似文献