Cloning of cDNA for canine interleukin-18 and canine interleukin-1beta converting enzyme and expression of canine interleukin-18.

Cloning of canine interleukin-18 (IL-18) and canine interleukin-1beta converting enzyme (ICE) cDNA was carried out by using murine IL-18 cDNA and human ICE cDNA, respectively, as probes. Sequence homology to known sequences of human, mouse, or rat genes was noted at nucleotide and amino acid levels. Canine IL-18 mRNA was expressed in various canine organs, whereas canine ICE mRNA was expressed in only a few, particularly in the brain and testis. Cloned canine IL-18 cDNA was expressed in Escherichia coli. The resulting protein promoted induction of canine interferon-y (IFN-y) from stimulated canine lymphocytes. Canine IL-18 and canine IL-12 produced canine IFN-gamma synergistically. Canine IL-18 suppressed the growth of tumor cells transplanted in SCID mice. Cloned canine IL-18 should prove useful as an anticancer agent.     

Cloning and expression analysis of an Atlantic salmon (Salmo salar L.) tapasin gene

Loading of the major histocompatibility complex (MHC) class I molecule with peptide is mediated by the multimeric peptide-loading complex in the ER where the glycoprotein tapasin (TAPBP) is required for stabilization of the complex and for control of peptide loading onto MHC class I. To expand our knowledge on antigen presentation genes in Atlantic salmon, we isolated a full-length salmon tapasin cDNA sequence (Sasa-TAPBP). It encoded a 443 bp amino acid sequence with two N-glycosylation sites, two conserved mammalian tapasin signature motifs, two Ig superfamily (IgSf) domains, a transmembrane (TM) domain and an ER-retention KK motif at the C-terminal end, indicative of a similar function as mammalian tapasins. We analysed the regulation of Sasa-TAPBP under immunostimulatory conditions and found an mRNA-upregulation during early infectious salmon anemia virus (ISAV) infection and poly I:C stimulation in vivo and in vitro, in line with our previous findings for other MHC class I pathway genes.     

人白细胞介素-21 cDNA克隆及其在大肠杆菌系统的表达

目的：克隆人细胞因子IL-2l全长cDNA及其在大肠杆菌中表达，并进一步检测其表达产物的功能。方法：抽取外周血，分离淋巴细胞；抗CD3抗体刺激后，提取总RNA，通过RT-PCR获得两个部分重叠的IL-21 cDNA片段(分别为5′端242bp，3′端425bp)，重组PCR扩增出IL-21全长cDNA：DNA测序正确后，将成熟IL-21 cDNA的编码框序列构建到原核表达载体pET28a( )中。卡那霉素平板筛选及PCR鉴定，挑出阳性克隆进行扩增、转化大肠杆菌进行IPTG诱导表达：SDS—PAGE、Western blotting鉴定，亲和层析分离纯化得到M，为18600的带有组氨酸标签重组人IL-21融合蛋白。透析法重折叠复性，MTT法测定其对T细胞增殖活性。结果：成功获得人IL-21全长cDNA，并以包涵体形式表达于大肠杆菌中。重折叠后的rhIL-21融合蛋白具有与抗CD3抗体共刺激T细胞增殖作用。结论：获得具有生物学活性的rhIL-21细胞因子，为进一步研究其功能奠定基础。     

藏猪白细胞介素4基因Cdna的克隆及序列分析

目的：克隆藏猪白细胞介素4基因cDNA。方法：从体外ConA刺激70小时的藏猪外周血淋巴细胞中提取总RNA，应用RT-PCR技术扩增，pMD-T载体连接，常规转化后，进行酶切及序列测定进行鉴定。结果：研究表明克隆得到的IL-4基因cDNA与成华猪的IL-4同源性达到99％，与长白杂交猪的同源率为98％。结论：从藏猪外周血淋巴细胞中成功地分离到IL-4基因。     

Neurotrophin and Trk neurotrophin receptors in the inner ear of Salmo salar and Salmo trutta

Neurotrophins (NTs) and their signal transducing Trk receptors play a critical role in the development and maintenance of specific neuronal populations in the nervous system of higher vertebrates. They are responsible for the innervation of the inner ear cochlear and vestibular sensory epithelia. Neurotrophins and Trks are also present in teleosts but their distribution in the inner ear is unknown. Thus, in the present study, we used Western-blot analysis and immunohistochemistry to investigate the expression and cell localization of both NTs and Trk receptors in the inner ear of alevins of Salmo salar and Salmo trutta. Western-blot analysis revealed the occurrence of brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3), but not nerve growth factor (NGF), as well as all three Trk receptors, i.e. TrkA, TrkB and TrkC, the estimated molecular weights of which were similar to those expected for mammals. Specific immunoreactivity for neurotrophins was detected mainly in the sensory epithelia. In particular, BDNF immunoreactivity was found in the maculae of the utricle and saccule, whereas NT-3 immunoreactivity was present in the sensory epithelium of the cristae ampullaris. As a rule the sensory epithelia of the inner ear lacked immunoreactivity for Trks, thus excluding possible mechanisms of autocrinia and/or paracrinia. By contrast, overlapping subpopulations of neurons in the statoacoustic ganglion expressed TrkA (about 15%), TrkB (about 65%) and TrkC (about 45%). The present results demonstrate that, as in mammals and birds, the inner ear of teleosts expresses the components of the neurotrophin-Trk system, but their roles remain to be elucidated.     

一个人类α-甘露糖苷酶全长cDNA的分子克隆△

用我们以往克隆到的1358bp 6A8 cDNA片段作探针,籍southern blot从一个人扁桃体细胞λgt11 cDNA文库筛选,再用RACE方法,克隆到了一个长3300bp的6A8全长cDNA。此cDNA内有一长3188bp（57～3245bp）的开放阅读框架,编码1064个氨基酸的蛋白质。此蛋白质的氨基酸序列与大鼠一个ER-α-甘露糖苷酶（1040个氨基酸）的相同性和相似性高达78%和82%,与一个酵母-α-甘露糖苷酶（1083个氨基酸）的相同性和相似性亦达33%和47%。另外,值得注意的是其260～432位氨基酸序列与人类、猪、大鼠和小鼠的多种α-甘露糖苷酶的相应氨基酸序列的相同性均高于20%,提示这段保守序列与α-甘露糖苷酶的功能可能有关。     

Coronary artery lesions in Atlantic salmon (Salmo salar)

One hundred and fifteen hearts and dorsal aortas were studied in anesthetized or moribund salmon for presence of histological alterations associated with spawning and changing environmental conditions. The control group consisted of 26 fish hatched and raised under experimental and controlled conditions. The study group included 61 prespawners and 23 postspawners. The lesions observed were divided into three grades in increasing order of severity. They consisted of focal or diffuse intimal proliferation of cells resembling smooth muscle cells and frequent alteration of the underlying elastica. The changes were more frequently seen in larger branches of coronary arteries, arterial conus, and atrioventricular sulcus regions. Both the relative incidence and severity of lesions appeared greater in prespawners as compared to postspawners. This study indicates a high incidence of coronary lesions in spawning Atlantic salmon. In addition, the severity of the lesions appeared reduced in postspawners exposed to prolonged starvation. It is suggested that the Atlantc salmon with its exposure to varying environmental conditions may be a useful model to study the epidemiology of coronary artery disease.     

Identification and characterization of a partial cDNA expressing interleukin-1-like activity in keratinocytes.

A partial cDNA has been isolated from the human keratinocyte cell line COLO-16 which is distinct from either IL-1 alpha or beta and encodes a protein which displays some of the biological properties associated with IL-1. The 720 bp partial cDNA hybridized on Northern blots of COLO-16 mRNA to a 1.6 kbp message of low abundance. Expression of the partial cDNA in COS-1 cells resulted in activity in three IL-1 assays: thymocyte co-stimulation, D10.G4.1 T-cell stimulation and fibroblast proliferation. Antisera generated against synthetic peptides based on inferred protein sequence from the cDNA reacted with a 20 kDa and 30 kDa species in both the COLO-16 cell line and PMA-stimulated normal human keratinocytes. These novel species were also present in PMA-stimulated and unstimulated human dermal fibroblasts and human T-cell lines.     

人MBL相关丝氨酸蛋白酶-1 cDNA的克隆与鉴定

目的 获得人甘露聚糖结合凝集素（ＭＢＬ）相关丝氨酸蛋白酶－１（ＭＡＳＰ－１）基因。方法 以人胎肝组织总ＲＮＡ为模板,采用ＲＴ－ＰＣＲ获取目的ｃＤＮＡ片段,克隆入ｐＧＥＭ－Ｔ载体,进行酶切图谱分析和测序鉴定。结果 以ＲＴ－ＰＣＲ方法获得了含信号顺序的全长ＭＡＳＰ－１ｃＤＮＡ,将其与ｐＧＥＭ－Ｔ载体连接,转化大肠杆菌ＴＧ１,建立了ＭＡＳＰ－１的ｃＤＮＡ克隆,酶切图谱与微机分析结果一致。序列分析表明。与     

IN-1重组单链抗体基因的克隆和序列分析

目的　应用基因工程技术获得人工设计的IN - 1重组单链抗体 (IN - 1-scFv)的cDNA克隆。方法　参照 genebank中发表的IN - 1抗体的轻链重链序列 ,重新设计适于在大肠杆菌中表达的目的基因片段 ,将该基因双链分成 3 5个小片段合成 ,经退火、复性连接成目的片段后 ,克隆到经过BamHI和HindIII双酶切的克隆载体pUC18中 ,并转化大肠杆菌DH5a,抽提重组子 pUC18/744进行克隆PCR、酶切鉴定及测序分析。结果　测序结果证明获得的基因序列与实验设计仅差一个碱基。结论　正确设计并合成了IN - 1重组单链抗体 (IN - 1-scFv)的cDNA ,为深入研究其生物活性奠定了基础     

Cloning and characterization of a cDNA specific for bovine retina

A retina-specific cDNA clone (pCR18) was selected from a bovine retinal cDNA library and characterized. The clone pCR18 consisted of 905 base pairs and hybridized to the mRNA of about 12S from the bovine retina, but not that from the brain or liver. The nucleotide sequence revealed a long open reading frame which encodes a 147 amino acid polypeptide of about 15,700 Da. No significant sequence homology with the predicted protein was found in the protein sequence library of about 3500. Messenger RNA which hybridized to pCR18 translated a polypeptide of about 19,000 Da in a reticulocyte translation system. Southern blot analysis indicated that the bovine genome contains a single copy of this gene. Furthermore, RNA dot analysis showed that the poly(A)+ RNA from the human retinoblastoma cell lines (Y79 and WERI) hybridized to pCR18, whose intensity was comparable to that of the bovine retina. In situ hybridization revealed that pCR18 was expressed mostly in some ganglion cells of the rat retina. The results suggest that cDNA clone (pCR18) encodes a protein specific for the retina and mRNA for pCR18 is mostly localized in the retinal ganglion cells and also expressed in the human retinoblastoma cells, although its function remains to be elucidated.     

Cloning of a cDNA encoding phosphofructokinase from Haemonchus contortus.

Phosphofructokinase (PFK), the key regulatory enzyme in glycolysis, has been cloned from the pathogenic parasitic nematode Haemonchus contortus by functional complementation in Escherichia coli. An E. coli strain deleted for both PFK loci (strain DF1020) was transformed with plasmid DNA from a lambda ZAP II H. contortus cDNA library. Two out of 3 x 10(7) transformants were able to grow on minimal medium with mannitol as the sole carbon source. A plasmid, pPFK, containing a 2.7-kb insert, was isolated from one of these transformants and conferred on DF1020 the ability to grow on mannitol (the PFK phenotype). The complemented cells contain PFK enzyme activity, absent in the E. coli mutant, at levels considerably higher than in wild type E. coli. Sequence analysis of the 2.7-kb insert shows an open reading frame that predicts a 789-amino acid protein that has approximately 70% similarity to mammalian PFKs. The amino acid sequence around asp182, thought to be the catalytic site, is completely conserved from nematodes to mammals.     

Toxicity of Aeromonas salmonicida cells to Atlantic salmon Salmo salar peritoneal macrophages.

Several strains of Aeromonas salmonicida were toxic to cultured peritoneal macrophages of Atlantic salmon, Salmo salar, in minimum doses of 5 x 10(6) CFU, whereas other strains were not. There was no correlation between cytotoxicity and in vivo virulence of the bacteria, the presence or absence of the two major surface components of A. salmonicida (namely, the A-layer and the O-polysaccharide chain of the LPS) nor, finally, with the ability of the strains to produce the following enzymes in vitro: protease, hemolysin, elastase and lecithinase. Toxicity was only observed with metabolically active bacteria and not with formalin- or heat-killed bacteria. The exact nature of the toxic factor remains unknown but is most likely associated with the surface of the bacteria. It is not extracellular since 24 and 48 h culture supernatants of the cytotoxic strains had no apparent effect on macrophages. The cytotoxic effect was found to be severe and rapid, it is likely a major virulence factor of A. salmonicida, but the exact role of such a potent toxin in the pathology of furunculosis has yet to be clarified.