永生化绵羊瘤胃成纤维细胞系的建立     

永生化绵羊瘤胃成纤维细胞系的建立 《畜牧与饲料科学》2021,42(3):1-6

[目的]建立永生化绵羊瘤胃成纤维细胞系,为基础研究和动物病毒的研究提供稳定的体外细胞模型。[方法]采用组织块贴壁法培养绵羊瘤胃成纤维细胞（ovine ruminal fibroblasts cells,ORFCs）,利用PEI试剂将带有hTERT基因的pCI-neo-hTERT质粒转染ORFCs,经G418筛选得到阳性克隆细胞。采用RT-PCR、Western Blot检测F₁₅和F₃₅代阳性克隆细胞的hTERT基因的转录和表达情况;利用血球计数法绘制原代ORFCs和F₃₅代阳性克隆细胞的生长曲线比较其增殖能力。[结果]将hTERT基因成功转入ORFCs并稳定表达;阳性克隆细胞的增殖能力显著高于原代ORFCs。[结论]该试验成功建立了永生化绵羊瘤胃成纤维细胞系。  相似文献   

羊接触传染性脓疱性皮炎病毒核酸及蛋白类型分析     

王廷璞  薛双虎 《中国兽医学报》1998,18(4):328-333

对从中国不同地区采集的１１株羊接触传染性脓疱性皮炎病毒（ｃｏｎｔａｇｉｏｕｓｅｃｔｈｙｍａｖｉｒｕｓ,ＣＥＶ;ｏｒｆｖｉｒｕｓ）的痂块毒ＤＮＡ进行提取,用ＥｃｏＲＩ、ＢａｍＨＩ、ＨｐａⅠ、ＨｉｎｄⅢ、ＫｐｎⅠ酶切,琼脂糖凝胶电泳,确定了各毒株的核酸及分子量。结果表明,各毒株的核酸酶切片段在８～１５个之间,病毒基因组大小有１０～２５ｋｂ的差异。用ＳＤＳ－ＰＡＧＥ分析了经ＮＰ－４０和２－巯基乙醇裂解蔗糖密度梯度离心纯化的各病毒株的囊膜蛋白,结果显示,各毒株可分离出１８～３３个不同的蛋白区带,毒株间的差异主要集中在３６４００～８００００区带之间。结合２次交互免疫试验结果,将以上１１株羊接触传染性脓疱性皮炎病毒分为４大类。  相似文献   

牛传染性鼻气管炎间接ELISA诊断方法的建立   总被引：7，自引：0，他引：7  

朱远茂王海燕  薛飞辛九庆  赵立平童光志 郭巍李兆利  相文华 《中国兽医科技》2005,35(12):959-963

以牛肾细胞系（MDBK）培养牛传染性鼻气管炎病毒（IBRV）Bartha Nu/67株,经超速离心纯化病毒,再经超声破碎处理后作为诊断抗原,建立了检测牛血清IBRV抗体的间接酶联免疫吸附试验.该ELISA的判定标准为：血清D490 nm值大于0.369的判为阳性,小于0.295的判为阴性,在0.295与0.369之间的为可疑.特异性和重复性试验结果表明,该方法特异性高、重复性好.与法国进口ELISA抗体诊断试剂盒比较,其符合率为96.3%;与中和试验比较,符合率为95.8%,且敏感性更高.应用该诊断方法调查了我国部分地区IBRV的感染情况,结果显示,这些地区的IBRV感染率为67.1%.  相似文献   

新疆绵羊进行性肺炎的初步血清学调查   总被引：1，自引：0，他引：1  

邓普辉  焦天嗣  简子健  钟苡芳 《新疆农业大学学报》1987,(2)

对来自7个地区的784头绵羊血清样品进行对绵羊进行性肺炎(OPP)病毒的琼脂凝胶免疫扩散试验(AGID test,琼扩),共检出阳性1头、弱阳性10头,阳性率为1.4％。在763头新疆羊中,只检出1头阳性,其中来自伊犁、塔城、博尔塔拉、昌吉、乌鲁木齐的733头新疆羊的血清样品中,没有1头阳性反应羊。来自于田羊场的18头边区莱斯特羊和3头林肯羊的血清样品中,分别检出8头和2头阳性羊,阳性率分别为44.4％和33.6％。本文还对疾病的来源和检疫问题进行了讨论。  相似文献   

羊脑多头蚴病ELISA试验用抗原制备的初步研究   总被引：1，自引：0，他引：1  

张西臣  胡力生 《中国兽医学报》1986,(4)

用羊脑多头蚴包囊头节、犬多头多头线虫节片经超速离心制备的抗原以及多头蚴包囊液抗原,分别对32头份羊脑多头蚴病阳性血清和63头份阴性血清进行EISA检测。头节抗原、节片抗原和囊液抗原的阳性率分别为81.25％、78.12％和53.12％,前两者阳性率均高于后者;其阴性率分别为82.54％、79.36％和80.95％。进而以头节抗原对32头份羊脑多头蚴病血清、25头份羊细颈囊尾蚴病血清、12头份羊棘球蚴病血清和7头份羊肝片吸虫病血清以及31头份羊肝片吸虫病阴性血清进行ELISA试验。结果,分别有26头份、9头份、4头份、1头份和1头份为阳性反应。经Dancan’s检验,多头蚴病血清S/N平均值明显地高于多头蚴病阴性血清。从而初步证明头节抗原用于诊断羊脑多头蚴病具有较高的敏感性和特异性。  相似文献   

The involvement of calpains in opacification induced by Ca2+-overload in ovine lens culture     

Hannah Y. Y. Lee  James D. Morton  Julie Sanderson  Roy Bickerstaffe  Lucinda J. G. Robertson 《Veterinary ophthalmology》2008,11(6):347-355

Objective To investigate biochemical changes accompanying Ca²⁺-induced lens opacification and the possible role of calpain activation in opacification within an ovine lens culture system. Methods Sheep lenses were cultured in minimal media. Lens opacification was induced by exposure to the Ca²⁺ ionophore, ionomycin, and graded by digital image analysis. Cell viability was estimated by the release of lactate dehydrogenase into the culture medium. Opaque lenses were fixed and stained for a microscopic view of the lens structural changes. Ionic changes in the lens were measured by atomic absorption spectroscopy. Calpain activation was determined by zymography on casein gels and proteolysis was investigated by SDS–polyacrylamide gel electrophoresis (SDS–PAGE), two-dimensional gel electrophoresis (2DE) and Western blotting. The calpain inhibitor, SJA6017, was used to investigate the involvement of calpains in lens opacification. Results Treatment of cultured ovine lenses with ionomycin increased total lens Ca²⁺ concentration and caused the cortical region of the lens to become opaque. Addition of the Ca²⁺ chelator, EGTA, inhibited the ionomycin-induced changes. Progress of opacification correlated with the death of lens cells and lens swelling in differentiating fiber cells. Autolysis of calpain 2, following ionomycin treatment, suggested activation of this protease. 2DE revealed that the ionomycin did not result in substantial proteolysis of the crystallins. However, Western blotting revealed significant breakdown of the cytoskeletal proteins, spectrin and vimentin. The pattern of the breakdown products was consistent with calpain proteolytic activity. SJA6017 retarded the cortical opacity induced by Ca²⁺-overload in the ovine lens. Conclusion The ovine lens with Ca²⁺-induced opacification by ionomycin is associated with calpain activation and the subsequent proteolysis of cytoskeletal proteins. These events could be initial factors contributing to cell death and the loss of lens transparency which occurs in this ovine model of cataractogenesis. The ovine model supports the hypothesis that cytoskeletal proteins and Ca²⁺ homeostasis play an important role in maintaining lens transparency.  相似文献   

Financial modelling of the potential cost of ovine Johne's disease and the benefit of vaccinating sheep flocks in southern New South Wales     

Bush RD  Windsor PA  Toribio JA  Webster SR 《Australian veterinary journal》2008,86(10):398-403

OBJECTIVE: To develop an enterprise gross margin (GM) model that predicts the on-farm financial impact of ovine Johne's disease (OJD) for various sheep enterprises in Australia. In addition, to estimate the benefits and costs of control through the use of the Gudair vaccination, including a breakeven point. DESIGN AND POPULATION: Data for the model was gained from an observational study conducted over a 3-year period from 2002 to 2004 using sheep from 12 OJD-infected flocks from southern New South Wales. Flocks ranged between 3500 and 20,000 sheep, with owner estimates of 5% or greater OJD mortality at the start of the study. PROCEDURE: A GM model was developed to predict the on-farm financial impact of OJD for various sheep enterprises in Australia, comparing non-infected, infected (status quo) and infected (vaccination) disease scenarios. RESULTS: Vaccination breakeven points are achieved within 2 to 3 years for breeding enterprises if OJD mortalities are high, rising towards 7 years for a Merino ewe enterprise if OJD mortalities are low. CONCLUSION: The GM model demonstrates the returns to investment of vaccination for Australian sheep producers with OJD-infected flocks.  相似文献   

Sedative and cardiopulmonary effects of xylazine alone or in combination with methadone,morphine or tramadol in sheep          下载免费PDF全文

Leonardo L de Carvalho  Lilian T Nishimura  Luisa PB Borges  Sofia A Cerejo  Isadora OJ Villela  Adam Auckburally  Ewaldo de Mattos‐Junior 《Veterinary anaesthesia and analgesia》2016,43(2):179-188

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Growth differentiation factor‐9 improves development,mitochondrial activity and meiotic resumption of sheep oocytes after in vitro culture of secondary follicles     

Alane P. O. Monte  Jamile M. Santos  Vanúzia G. Menezes  Bruna B. Gouveia  Thae L. B. G. Lins  Ricssio S. Barberino  Joozito L. Oliveira  Nathalie J. Donfack  Maria Helena T. Matos 《Reproduction in domestic animals》2019,54(9):1169-1176

This study analysed the effect of growth differentiation factor‐9 (GDF‐9) on the in vitro culture of isolated ovine secondary follicles. The follicles were cultured in α‐MEM supplemented with BSA, insulin, glutamine, hypoxanthine, transferrin, selenium, ascorbic acid and FSH (α‐MEM⁺—control medium) or α‐MEM⁺ supplemented with 1, 10, 50 or 100 ng/ml GDF‐9. Next, the oocytes were destined to in vitro maturation (IVM). After 12 days of culture, there were no differences regarding the percentage of normal follicles, antrum formation and follicle diameter between the treatments (p > 0.05). The rates of fully grown oocytes (≥110 µm) were higher (p < 0.05) in 100 ng/ml GDF‐9 than other treatments, except for 10 ng/ml of GDF‐9 (p > 0.05). Treatment containing 100 ng/ml GDF‐9 showed higher (p < 0.05) mitochondrial activity than the control group. Moreover, 100 ng/ml GDF‐9 showed more oocytes in MI than α‐MEM⁺, 1 or 50 ng/ml GDF‐9 (p < 0.05). In conclusion, 100 ng/ml GDF‐9 increased the growth, mitochondrial function and meiotic resumption of oocytes from in vitro grown sheep secondary follicles.  相似文献   

酿酒酵母β-葡聚糖通过膜受体Dectin-1和TLR-2诱导绵羊瘤胃上皮细胞表达SBD-1的机制研究     

张曼  金鑫  王云鹤  魏方  温婧怡  杨银凤 《畜牧兽医学报》2019,(4):781-790

为了探索酿酒酵母β-葡聚糖诱导绵羊瘤胃上皮细胞(ovine ruminal epithelial cells,ORECs)β-防御素-1(sheepβ-defensin-1,SBD-1)表达过程中膜受体Dectin-1和TLR-2可能存在的作用。本研究首先利用免疫组化、RT-PCR、免疫荧光和Western blot等方法检测Dectin-1是否在ORECs内表达,并且采用qPCR和Western blot方法对β-葡聚糖刺激ORECs后细胞膜受体Dectin-1和TLR-2的表达变化进行检测。而后用不同浓度的Dectin-1阻断剂昆布多糖或TLR-2特异性封闭抗体分别预处理ORECs后,采用qPCR和ELISA方法检测SBD-1的表达变化,以确定β-葡聚糖诱导SBD-1表达过程中Dectin-1和TLR-2的参与情况。结果显示:1)绵羊瘤胃组织及ORECs内存在Dectin-1表达,且β-葡聚糖刺激ORECs后Dectin-1和TLR-2的表达水平显著增加(P<0.05);2)不同浓度的昆布多糖和TLR-2封闭抗体均可以极显著降低β-葡聚糖诱导SBD-1的表达(P<0.01),且随着阻断剂和封闭抗体浓度的增加,其抑制SBD-1表达的作用越明显。结果表明,Dectin-1在绵羊瘤胃组织及ORECs内均表达,并且酿酒酵母β-葡聚糖在ORECs中诱导SBD-1的表达是由Dectin-1和TLR-2介导产生的。  相似文献