我国濒危家养动物细菌人工染色体基因组文库的构建及应用

细菌人工染色体(BAC)是新近发展起来的一种载体系统,具有克隆容量大,遗传特性稳定,转化效率高,易于操作等优点.在基因组文库构建和基因的快速克隆等方面已得到广泛应用.濒危家养动物细菌人工染色体基因组文库构建的目的在于有效、永久保存和利用其基因资源、快速克隆重要经济和抗逆性状基因以及为广泛、深入开展分子生物学研究提供宝贵的材料.     

犬基因组研究概况

美国科学家在2005年12月8日出版的《自然》杂志上公布了一头1岁拳师犬的完全基因组图谱,该图谱将有助于理解一些特定基因的演化,并将促进对人类和犬类共患疾病的研究。这次由政府资助、美国国家人类基因组研究所领导的“犬基因组计划”是于2003年6月正式启动的。研究小组用了一     

Construction of a canine bacterial artificial chromosome library for screening with PCR

We have constructed a canine bacterial artificial chromosome library amenable to PCR screening. The library consists of 96 768 clones and was initially screened with 112 microsatellites representing all canine chromosomes. For 87 primer sets (77%) one to seven positive superpools were identified. The library will be expanded by adding additional superpools in order to increase the genome coverage. Interested researchers can access the library following the rules published at http://www.dogmap.ch .     

Characterization of canine caspase-3

The canine caspase-3 gene was cloned and sequenced. The canine caspase-3 cDNA clone was 1473 base pairs in length and encoded 277 amino acids. The predicted amino acid sequence showed 88.4%, 88.0%, 85.9%, 65.9% and 60.6% homology with that of human, pig, mouse, chicken and zebra fish caspase-3, respectively. The caspase-3 mRNA was confirmed to express in skin, lymph node, bone marrow, small intestine and lung from a healthy dog by RT-PCR analysis.     

Characterization of an antibody panel for immunohistochemical analysis of canine muscle cells

Immunohistochemistry is an indispensable tool in the assessment and characterization of lineage-specific differentiation of grafted cells in cell-based-therapy. This strategy is under investigation for the treatment of many muscle disorders and different animals such as dogs are used as models to study the tissue regeneration. The aim of the present study was to characterize an antibody panel for the analysis of canine muscle cells, useful in routinely processed formalin-fixed paraffin-embedded tissues. Overall, 12 antibodies (8 mouse monoclonal and 4 goat polyclonal), validated for use on human tissues tested for cross-reactivity on canine smooth muscle (bladder, intestine, and uterus), skeletal muscle and heart. Specific staining was achieved with eight antibodies, of which six were cytoplasmic markers (desmin, HDAC8, MHC, SMA, Troponin I and Troponin T) and two were cardiac nuclear markers (GATA-4 and Nkx-2.5). This antibody panel may be useful not only for the evaluation of cell-based therapies in muscle disorders, but also for the evaluation of canine soft tissue neoplasms in veterinary pathology.     

Characterization of Escherichia coli strains associated with canine pyometra

The purpose of this study was to characterize E. coli isolates from canine pyometra which were isolated in pure culture. The E. coli strains were obtained in 128 cases, from 143 animals which were submitted to ovariohisterectomy. Biochemical analysis of all strains examined was possible on separation of 10 primary biotypes. The majority of the strains (87.5%) belonged to biotype 9, 1, 13 and 15. Dulcitol was fermented by 93% of all isolates. Haemolysin and colicin production was found in 53.9% and 26.6% of the strains, respectively. Approximately 37% of strains expressed resistance to two or more antibacterial agents. No plasmid was detected in 4.6% of the isolates. Plasmid profiles of all plasmid-containing isolates revealed plasmid bands corresponding to molecular weight ranging from 1 kb to 160 kb. Many of the strains examined had a single plasmid of 110 kb (46.1%), or two plasmids 110:65 kb (18.8%). Both plasmids appearing alone or in combination with other plasmids were detected in 90.1% of isolates with plasmid content. It was established that among haemolytic, colicinogenic and motile strains, the presence of both plasmids was 91.3, 94.1 and 91.4%, respectively. The appearance of both plasmids among dulcitol-positive and raffinose-negative strains was estimated at 88.2 and 88.3%, respectively. In a group of colicinogenic strains the presence of a single plasmid of molecular weight 110 kb was estimated at 5.9%. When both plasmids were present (profile 110:65), the percentage of these strains was 70.6%.     

Characterization of and radioimmunoassay for canine thyroglobulin

Canine thyroglobulin (cTg) has been isolated and purified. It has similar electrophoretic patterns as Tg from other mammalian species. The main fraction had a MW of 660,000, whereas also fractions of a MW of approximately 1,300,000 (dimer) and 330,000 (subunit) were present. The iodine content was 0.8 to 1.0 % (w/w). cTg did not cross-react with antibodies against human Tg to a degree that would allow the use of a radioimmunoassay for human Tg for the determination of cTg in serum or plasma. Therefore a polyclonal antiserum was raised against cTg and a homologous radioimmunoassay was developed, which was sensitive (0.4 μg/l) and specific (cross-reactivity in cTg assay of human Tg, goat Tg, T₄, T₃, and DIT < 0.01 %).

Plasma Tg levels in normal dogs of both sexes and aged 3–15 years amounted to 192 ± 73 μg/l (mean ± SD, n=30). There was no relation between plasma Tg and T₄ levels.     

Characterization of lymphocytes in canine gastrointestinal lymphoma

Primary canine gastrointestinal lymphoma has been believed to be of B-cell origin based on the morphology and behavior of the neoplastic cells and the evidence from the human medical field. However, the neoplasms have not to date been characterized as to the origin of the cell population. Forty-four cases diagnosed as canine gastrointestinal lymphoma were retrieved from the records of the Veterinary Teaching Hospitals at the University of Minnesota and the University of Wisconsin-Madison. Four of the cases have been previously identified as epitheliotropic T-cell gastrointestinal lymphoma. Twenty-three of the dogs were female, with 11 intact and 12 neutered, and 21 of the dogs were male, with 12 intact and nine neutered. Sixteen breeds as well as individuals of mixed breeding were represented. The Boxer and the sharpei were the most commonly represented breeds with six individuals each. The age range of the dogs was 1.5-14.66 years, with two dogs identified as adult and two of unknown age. Archived tissue blocks of gastrointestinal samples were sectioned in duplicate and prepared for immunohistochemical staining with CD3 (T-cell marker) and CD20 (B-cell marker). In 75% of the cases examined under light microscopy, 50-95% of the neoplastic cells stained positively with CD3 and exhibited marked epitheliotropic behavior. In three of the cases, from 10% up to 50% of the neoplastic cells stained positively with CD20, with widely scattered CD3(+) cells. In the remainder of the cases, few to none of the neoplastic cells stained with either of the markers. This retrospective study shows that canine primary gastrointestinal lymphoma is more commonly of T-cell origin, rather than B-cell origin.     

Characterization of circulating lymphocyte subpopulations in canine leishmaniasis throughout treatment with antimonials and allopurinol

Canine leishmaniasis (CL) is a systemic parasitic disease with a wide variability of response to specific therapy: the majority of patients apparently improve with treatment, some of them respond but later relapse, and few of them do not respond at all. It has been demonstrated that the immune response plays a key role in the development and outcome of Leishmania infection in the dog and in the response to the treatment, although this response is not well understood. Some authors have suggested that ill dogs show a reduction in the percentage of circulating CD4+ lymphocytes and in the CD4+/CD8+ ratio, both of which normalize after treatment and clinical recovery. The present paper discusses the variation of the different lymphocyte subpopulations (CD3, CD4, CD8, CD21) of peripheral blood mononuclear cells (PBMC) in 28 dogs diagnosed with CL and submitted to conventional treatment with meglumine antimoniate (Glucantime) for 1 month and with allopurinol (Zyloric) for 1 year, in order to evaluate the usefulness of these parameters as indicators of the immunological condition of the ill animals and of the prognosis of their evolution during the treatment. It is concluded that circulating lymphocyte subpopulations are similar in dogs with leishmaniasis and in healthy dogs and that there is no correlation between the clinical status or response to therapy and the values of the counts of the different lymphocyte subpopulations. Therefore, the percentage of different lymphocyte subpopulations cannot be used as a parameter to predict the evolution of an individual patient in a clinical context.     

Hemagglutination by canine parvovirus: serologic studies and diagnostic applications

Conditions for canine parvoviral hemagglutination (HA) and hemagglutination-inhibition (HI) reactions were defined. The HA phenomena were used to differentiate canine parvovirus (CPV) from feline panleukopenia virus (FPV), mink enteritis virus (MEV), and minute virus of canines. Serologic comparisons of the CPV, FPV, and MEV by HA-HI and serum-neutralization tests indicated that CPV, FPV, and MEV were antigenically similar but were different from minute virus of canines. Diagnostic application of HA tests to fecal samples from acute cases of enteritis was discussed. Combinating HA tests with HI tests on fecal samples provided a rapid and specific diagnostic method for CPV infection. Secular seroprevalence studies indicated the emergence of CPV infeciton in the United States dog population-at-large in 1978.     

mRNA差异显示技术及其在猪基因组研究中的应用

差异显示是一种快速分离、鉴定不同细胞类型基因表达差异的有效方法。此技术自建立以来便广泛应用于比较研究中 ;本文简要介绍了有关差异显示的原理及在猪基因组研究中的应用     

貂,貉,犬多种免疫球蛋白的研究与应用

利用犬温热，细小病毒性肠炎，犬传染性肝炎，狂犬病等弱毒株，以香菇多糖和动物的核糖，多肽做为免疫增强剂，对异源动物羊，猪，犊牛，家兔进行了免疫应答，筛选出了猪做为异源免疫动物。在免疫增强剂的参与下，完成了多次免疫应答，达到了理想的免疫球蛋白（ＩｇＧ），通过试验摸清了该制剂的免疫程序，确定了生产工艺，经重复性试验，稳定性试验及含量测定，以及临床应用试验和现志应用试验均达以了预期效果，该制剂经冷冻干燥后     

犬瘟热病毒弱毒和野毒的全基因组扩增及序列测定

分析比对了GenBank中已公布的23株犬瘟热病毒全基因组序列,针对保守区设计了11对特异性通用引物,用于扩增犬瘟热病毒野毒株和疫苗株全基因组。通过优化11对引物的工作浓度及退火温度等条件,对保存的3株疫苗毒CDV3株、OR12株、CDV/R-20/8株和2株野毒HBF-1株和SD(14)07株进行了全基因组扩增。取5μL扩增产物进行1%琼脂糖凝胶电泳,结果显示11对通用引物对以上5株犬瘟热病毒扩增后,均获得11个特异性目的片段,条带大小与预期相符。选取疫苗毒OR12株和野毒HBF-1株的进行全基因组序列测定,与GenBank中已公布的序列进行比对,同源性均大于99.0%,证实这11对特异性通用引物能够实现对犬瘟热病毒野毒和弱毒全基因组的准确扩增,并获得准确的全基因组序列。