西农萨能羊凝乳酶原前体cDNA的克隆与序列分析

参照GenBank登录的绵羊凝乳酶原前体cDNA序列设计引物,以新生5d的西农萨能羊皱胃组织总RNA为模版,通过RT-PCR方法获得凝乳酶原前体cDNA,克隆测序后进行序列比对。结果表明,克隆基因为B型凝乳酶,该基因cDNA具有1292个碱基,编码381个氨基酸,包含16个氨基酸的信号肽序列和42个氨基酸的酶原序列。将其与已报道的山羊、绵羊和牛的凝乳酶原前体序列进行比对,发现核苷酸同源性分别为99.41%、98.74%和95.29%,氨基酸同源性为99.21%、98.42%和93.70%。     

Molecular cloning and sequencing of the cDNA encoding the bat CD4

Chiroptera is thought to be a vector or a natural reservoir of various pathogenic microbes. However, there are few basic studies on the subject of chiroptera immune systems. This is the first report to determine the sequence of bat CD4 cDNA. Comparison with other animals' CD4 and phylogenetic analysis have shown that bat CD4 had a higher homology to cat and dog CD4 than to human and mouse CD4. Moreover, from the analysis of the structure of the CD4 Ig-like C-type 1 region, in bat CD4 there was an insertion of 18 extra amino acids. In addition, bat CD4 lacked cystein, which suggested that the disulfide bond could not be formed. Human, monkey and mouse CD4 have the cystein and the disulfide bond, but pig, cat, whale and dog CD4, like that of the bat, lacked the cystein. We conducted the present study in order to help elucidate the infectious diseases derived from the bat as well as bat immune systems.     

绵羊孕酮受体基因的PCR-SSCP分析

作者设计3对引物,采用PCR-SSCP方法检测孕酮受体（progesterone receptor,PGR）基因DNA结合区和配体结合区序列在高繁殖力绵羊品种（小尾寒羊和湖羊）和低繁殖力绵羊品种（多赛特和特克赛尔）中的单核苷酸多态性,同时研究该基因对绵羊繁殖力的影响;对小尾寒羊PGR基因外显子5、6和9克隆测序,结合GenBank提供的绵羊PGR基因部分编码序列,拼接出绵羊PGR基因DNA结合区和配体结合区的完整序列,推导出编码的氨基酸序列;并将人、兔、狗、绵羊、小鼠和大鼠的PGR基因这两个区域的核苷酸与氨基酸序列进行比较。结果表明,PGR基因DNA结合区和配体结合区序列在所检测的4个绵羊品种中都不存在单核苷酸多态性;人、兔、狗、绵羊、小鼠、大鼠PGR基因DNA结合区和配体结合区的核苷酸序列同源性分别为88.6%~97.2%和84.4%~96.3%,氨基酸序列同源性分别为97.6%~100%和96.3%~99.6%。可见,哺乳动物PGR基因DNA结合区和配体结合区序列保守性很强,这两个区域可能不是影响绵羊高繁殖力的功能结构域。     

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.     

Molecular cloning and sequencing of canine T-cell costimulatory molecule (CD28)

T-cells express CD28 and CTLA-4, and through binding to their shared ligands (CD80/CD86) on antigen presenting cells, provide a potent co-stimulatory signal for T-cell activation and proliferation. To investigate the role of CD28 in canine immune system, we hereby report the molecular cloning and sequencing of the full-length complementary DNA (cDNA) coding for canine CD28, from pokeweed mitogen stimulated canine peripheral blood lymphocytes. The cloned cDNA contains an open reading frame of 663 nucleotides, encoding for a polypeptide of 221 amino acids. The amino acid sequence of the canine CD28 showed 91.9, 80, and 79.6% similarities with those of the cat, cattle, and human counterparts, respectively. Five sequence motifs of TATT or ATTTA involved in the regulation of gene expression by influencing mRNA stability are found in the 3' untranslated region. The hexapeptide motif (MYPPPY), five cysteine residues, a potential N-glycosylation site and a cytoplasmic phosphatidylinositol 3-kinase binding site in canine CD28 molecule are completely conserved in canine CTLA-4. The availability of full length canine CD28 will provide a useful molecule for studying its role in dog immune system.     

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.     

长毛兔和獭兔白细胞介素-4基因的序列分析与真核表达

应用反转录—聚合酶链式反应（RT-PCR）技术,在国内首次从ConA诱导培养的长毛兔和獭兔外周血淋巴细胞总RNA中扩增出IL-4基因。将扩增基因克隆入pMD18-T载体,经序列测定分析表明,长毛兔和獭兔IL-4基因全长均为441 bp,两序列间碱基组成无差异（登录号：EF606761和EU639687）。长毛兔和獭兔IL-4基因编码147个氨基酸,其中前24个氨基酸组成信号肽,余下的123个氨基酸组成成熟肽。与已报道的犬、猫、人、猪、牛、羊、马、大熊猫和鼠的IL-4核苷酸同源性分别为61.8%、63.4%、69.8%、65.1%、64.1%、64.1%、63.1%、57.0%、60.5%,推导的氨基酸同源性分别为47.2%、50.0%、53.6%、52.1%、48.3%、46.9%、52.1%、43.7%、43.0%。将测序正确的阳性质粒采用脂质体法转染COS-7,用RT-PCR鉴定转染细胞中IL-4基因的表达,并通过MTT法检测到表达的IL-4蛋白具有一定的生物学活性。     

Identification of canine alpha-lactalbumin

The nucleotide sequence of canine alpha-lactalbumin cDNA from canine mammary tissue was determined by polymerase chain reaction with degenerate primers. A 742 base pairs nucleotide sequence cloned was similar to the size of mRNA in Northern blot analysis. The cDNA encodes 142 amino acid residues containing the conserved sequence motif of alpha-lactalbumin, demonstrating the highest homology with pig (73% identity-82% similarity) among the known amino acid sequences of alpha-lactalbumin. The canine cDNA also showed 71% identity-78% similarity with human, 58-73% with mouse, 60-74% with rat, 67-77% with goat, 66-77% with cattle, and 67-76% with sheep, respectively.     

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.     

Cloning of bovine CD69

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.     

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.     

Cloning of cervine interferon-gamma cDNA by polymerase chain reaction

As the first step in the development of a cervine IFN-gamma assay for the diagnosis of tuberculosis in deer, cervine IFN-gamma, cDNA was amplified by polymerase chain reaction using primers based on the bovine IFN-y sequence. A high level of amino acid homology was found between the cervine and the ovine and bovine sequences (94% and 91% respectively). There was less identity with the porcine, human, mouse and rat sequences (78%, 62%, 37% and 39%, respectively). The amino terminus of the mature IFN-gamma protein, which is critical for interaction with its receptor and for triggering biological activity, is highly conserved between the cervine, bovine and ovine proteins. A monoclonal antibody-based sandwich enzyme immunoassay (EIA) specific for bovine IFN-gamma also detects ovine but not cervine IFN-gamma. This suggests that the antibodies recognise epitopes common to the bovine and ovine protein but not cervine IFN-gamma. Seven amino acid residues that were common to the bovine and ovine sequence differed in the cervine sequence, suggesting that the specificity of the monoclonal antibodies may be dependent on one or more of these residues. The possibility of the development of an EIA for cervine IFN-gamma as a commercial in vitro diagnostic assay for tuberculosis in deer is discussed.     

Abnormalities in the axial skeleton of a Risso's dolphin,Grampus griseus

As the first step in the development of a cervine IFN-γ assay for the diagnosis of tuberculosis in deer, cervine IFN-γ cDNA was amplified by polymerase chain reaction using primers based on the bovine IFN-γ sequence. A high level of amino acid homology was found between the cervine and the ovine and bovine sequences (94% and 91% respectively). There was less identity with the porcine, human, mouse and rat sequences (78%, 62%, 37% and 39%, respectively). The amino terminus of the mature IFN-γ protein, which is critical for interaction with its receptor and for triggering biological activity, is highly conserved between the cervine, bovine and ovine proteins. A monoclonal antibody-based sandwich enzyme immunoassay (EIA) specific for bovine IFN-γ also detects ovine but not cervine IFN-γ. This suggests that the antibodies recognise epitopes common to the bovine and ovine protein but not cervine IFN-γ. Seven amino acid residues that were common to the bovine and ovine sequence differed in the cervine sequence, suggesting that the specificity of the monoclonal antibodies may be dependent on one or more of these residues. The possibility of the development of an EIA for cervine IFN-γ as a commercial in vitro diagnostic assay for tuberculosis in deer is discussed.