西农萨能羊两个泌乳阶段差异表达基因SAA3的克隆和序列分析

为了研究不同泌乳期乳腺组织中差异表达基因，利用抑制性削减杂交技术构建西农萨能羊泌乳60d乳腺组织及泌乳28d乳腺组织中差异表达基因文库，用Real-Time PCR技术验证阳性克隆血清淀粉样蛋白A3（SAA3）基因，通过RT-PCR方法克隆西农萨能羊乳腺组织SAA3，并进行序列比对和功能预测。结果成功构建了西农萨能羊不同泌乳期乳腺组织中差异表达基因文库，筛选克隆了乳腺组织SAA3基因，GenBank登录号为：DQ839400，编码区长度为396bp，含有131个氨基酸。西农萨能羊乳腺组织中SAA3与牛（GenBank：NM_181016）、兔（GenBank：M64696．1）、人（GenBank：BC020795）、鼠（GenBank：NM_011315）核苷酸同源性分别为95％、84．3％、81.3％和81.9％，氨基酸同源性为93％、76％、72％、72％；在编码区261-287位较人、兔、鼠SAA编码区多27个碱基，连续大于5个氨基酸的保守区域有6个，较人SAA多3个潜在功能基序。     

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.     

Nucleotide and deduced amino acid sequence of equine retinal and pineal gland phosducin

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.     

Elevated extrahepatic expression and secretion of mammary-associated serum amyloid A 3 (M-SAA3) into colostrum.

Mammary-associated serum amyloid A 3 (M-SAA3) was secreted at highly elevated levels in bovine, equine and ovine colostrum and found at lower levels in milk 4 days postparturition. N-terminal sequencing of the mature M-SAA3 protein from all the three species revealed a conserved four amino acid motif (TFLK) within the first eight residues. This motif has not been reported to be present in any of the hepatically-produced acute phase SAA (A-SAA) isoforms. Cloning of the bovine M-Saa3 cDNA from mammary gland epithelial cells revealed an open reading frame that encoded a precursor protein of 131 amino acids which included an 18 amino acid signal peptide. The predicted 113 residue mature M-SAA3 protein had a theoretical molecular mass of 12,826Da that corresponded with the observed 12.8kDa molecular mass obtained for M-SAA3 in immunoblot analysis. The high abundance of this extrahepatically produced SAA3 isoform in the colostrum of healthy animals suggests that M-SAA3 may play an important functional role associated with newborn adaptation to extrauterine life and possibly mammary tissue remodeling.     

Cloning and nucleotide sequence of the equine and elk pituitary pre-prolactin cDNA

We report the equine (Equs equs) and elk (Cervus elaphus) pituitary pre-prolactin (PRL) cDNA cloning, and their nucleotide and deduced amino acid sequences. Pre-PRL cDNA was obtained by RNA ligation mediated-rapid amplification of cDNA ends (RLM-RACE) and polymerase chain reaction (PCR). The elk pre-PRL cDNA exhibits two polymorphisms at positions 96 and 672, which are silent since they encode for the same amino acids, proline and isoleucine, respectively. We found no polymorphisms in the equine pre-PRL cDNA. The deduced amino acid sequence of the equine pre-PRL is 99% identical to the previously reported protein sequence. Pre-PRL mRNA is <1 kb in length and is highly expressed in the anterior pituitary gland, as demonstrated by Northern hybridization analysis. In summary, we cloned and sequenced the equine and elk pre-PRL cDNAs. The deduced amino acid sequence of elk and equine pre-PRL appears to be moderately conserved among other mammalian species. The polymorphic sites found in the elk cDNA could potentially be used in parentage testing and gene mapping.     

Expression of recombinant feline serum amyloid A (SAA) protein.

Feline serum amyloid A (SAA) cDNA clone was isolated from a hepatic mRNA of a mixed-breed cat. The feline SAA cDNA clone contains 333 nucleotides and deduced amino acid sequence shows 87.4%, 73.9%, and 65.8% homology with dog, human and mouse SAA respectively. Relative to the human and mouse SAA proteins, an additional peptide of eight amino acids is specified in the feline cDNA clone. Recombinant feline SAA (rfSAA) was expressed at high levels using pGEX bacterial expression system. Cleavage from the fusion moiety, and purification using glutathione-sepharose yielded pure soluble form of rfSAA. Antibodies generated against rfSAA were specific for feline SAA and showed no cross-reactivity with human SAA. Furthermore, antibodies against human SAA did not react with feline SAA. These results indicate that antigenicity of feline SAA is totally different from human SAA.     

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.     

猪繁殖与呼吸综合征病毒辽宁分离株ORF5基因的克隆与序列分析

本试验以辽宁地区某猪场猪繁殖与呼吸综合征(porcine reproductive and respiratory syndrome,PRRS)发病猪病料为材料,采用RT-PCR方法特异性扩增编码猪繁殖与呼吸综合征病毒(porcine reproductive and respiratory syndrome virus,PRRSV)GP5蛋白的ORF5基因全长cDNA,结果该分离株ORF5基因编码区长603bp,可编码200个氨基酸。与美洲型代表株VR2332、欧洲型代表株LV进行同源性比较,氨基酸同源性分别为89.5%和55.7%。推测该辽宁分离株属于美洲型。     

猪新基因NPM1的克隆、序列分析与染色体定位

克隆了猪NPM1基因的cDNA和第4内含子序列，并利用生物信息学方法进行验证。所得cDNA序列全长1195bp且包含一个完整的开放阅读框，编码294个氨基酸。内含子序列长300bp，遵循GT／AG剪接法则。序列分析表明，该基因与已报道的人、大鼠和小鼠等物种的NPM1基因高度同源，氨基酸序列同源性分别为95％、91％和91％。该基因编码的蛋白具有Nucleoplasmin保守功能域。用体细胞杂种板将该基因定位在猪16号染色体q21区段，辐射杂种板定位结果表明该基因与猪16号染色体上SW977标记紧密连锁。     

Cloning and sequencing of a bottle-nosed dolphin (Tursiops truncatus) interleukin-4-encoding cDNA.

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.     

Molecular characterisation of carbohydrate digestion and absorption in equine small intestine

Dietary carbohydrates, when digested and absorbed in the small intestine of the horse, provide a substantial fraction of metabolisable energy. However, if levels in diets exceed the capacity of the equine small intestine to digest and absorb them, they reach the hindgut, cause alterations in microbial populations and the metabolite products and predispose the horse to gastrointestinal diseases. We set out to determine, at the molecular level, the mechanisms, properties and the site of expression of carbohydrate digestive and absorptive functions of the equine small intestinal brush-border membrane. We have demonstrated that the disaccharidases sucrase, lactase and maltase are expressed diversely along the length of the intestine and D-glucose is transported across the equine intestinal brush-border membrane by a high affinity, low capacity, Na+/glucose cotransporter type 1 isoform (SGLT1). The highest rate of transport is in duodenum > jejunum > ileum. We have cloned and sequenced the cDNA encoding equine SGLT1 and alignment with SGLT1 of other species indicates 85-89% homology at the nucleotide and 84-87% identity at the amino acid levels. We have shown that there is a good correlation between levels of functional SGLT1 protein and SGLT1 mRNA abundance along the length of the small intestine. This indicates that the major site of glucose absorption in horses maintained on conventional grass-based diets is in the proximal intestine, and the expression of equine intestinal SGLT1 along the proximal to distal axis of the intestine is regulated at the level of mRNA abundance. The data presented in this paper are the first to provide information on the capacity of the equine intestine to digest and absorb soluble carbohydrates and has implications for a better feed management, pharmaceutical intervention and for dietary supplementation in horses following intestinal resection.     

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.     

水牛TLR4 cDNA全长的克隆及其生物信息学分析

本研究利用RT-PCR方法分段克隆水牛TLR4 cDNA后拼接成全长,并克隆于pMD20-T载体中,同时运用生物信息学软件对其核苷酸序列及编码蛋白质的结构进行分析和预测。结果表明,克隆的TLR4 cDNA ORF全长2526 bp,共编码841个氨基酸,N端具有由25个氨基酸组成的信号肽,该蛋白预测的分子质量为95.98 ku,等电点为6.37;水牛TLR4与GenBank中登录的水牛TLR4(DQ857349)核苷酸序列同源性达99.01%,与黄牛、绵羊、野猪、马、人和黑猩猩的同源性超过80%,与小鼠和狗的同源性次之,分别为72.17%和61.30%,与鸡的最低,仅为53.94%;该蛋白是由胞外区(1-634位氨基酸)、跨膜区(635-657位氨基酸)和胞内区(658-841位氨基酸)3部分构成的跨膜蛋白,胞外区含有12个LRR串联重复结构域、胞内区具有TIR结构域。本试验成功克隆了水牛TLR4 cDNA全长,并获得了核苷酸及其蛋白质的生物信息学分析数据,为进一步研究其功能奠定了基础。     

小尾寒羊CIB1基因全长cDNA克隆及原核表达

本研究旨在克隆小尾寒羊钙粘和蛋白1(Calcium and integrin binding protein 1,CIB1)基因cDNA全长,并在原核载体中表达.参照已发表的CIB1基因的核苷酸序列,设计了1对特异性引物,采用RT-PCR法,从小尾寒羊睾丸组织中扩增获得CIB1基因全长cDNA.将其克隆到pMD19-T载体,并进行测序分析.将该基因编码区重组于融合表达质粒pET32a中,构建了重组原核表达载体(pET32a-CIB1).将其转化到BL21(DE3)pLysS宿主菌中,用IPTG进行诱导表达.结果表明,克隆的CIB1基因cDNA与GenBank上登录的牛、猪、猕猴、人、小鼠、大鼠、黑猩猩等动物该基因序列的同源性达90%以上,编码氨基酸的同源性在93%以上,并且该序列包含有完整的开放阅读框,大小为576 bp.实现了高效特异性融合表达,表达产物的分子质量约为38 ku.本研究结果为进一步研究CIB1蛋白功能打下良好的基础.     

Genomic characterization of equine interleukin-4 receptor alpha-chain (IL4R)

Three overlapping fragments of the equine interleukin-4 receptor alpha chain gene (IL4R) were cloned and sequenced. The resulting 3553 bp cDNA sequence exhibited homology to human, murine and bovine IL4R. The equine IL4R exhibits many conserved features when compared to other species, including intron-exon boundary positions and amino acid sequence motifs characteristic of type I cytokine receptors. The IL4R gene was localized to horse chromosome ECA13 by synteny mapping on a somatic cell hybrid panel. Evidence for an alternative splice variant of IL4R was found in the genomic sequence and subsequently verified using RT-PCR on equine monocyte RNA. A polymorphism screen of the largest exon, homologous to exon 12 of the human IL4R gene, was performed using DNA from 60 horses of various breeds which yielded 11 coding-region single nucleotide polymorphisms (SNPs), 7 synonymous and 4 non-synonymous. Three of the four non-synonymous SNPs occur at high frequencies and are found very near a conserved tyrosine residue.