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本研究旨在检验构建的stat5表达载体对奶牛乳腺上皮细胞泌乳能力的影响。构建真核表达载体pcD-NA3.1+-stat5-αS1,稳定转染到奶牛乳腺上皮细胞中。利用细胞活力分析仪、HPLC、Real-time PCR和WesternBlotting技术检测乳腺上皮细胞转染前后stat5基因和蛋白的表达量、细胞活力及乳糖和酪蛋白分泌情况。结果表明,与空白细胞和空白载体组相比,非磷酸化STAT5蛋白和stat5基因的表达量增加(P<0.01),乳糖含量提高(P<0.05),细胞活力和增殖能力增强(P<0.01),酪蛋白和磷酸化STAT5(pSTAT5)表达增多(P<0.05)。结果提示,构建的载体pcDNA3.1+-stat5-αS1在乳腺上皮细胞中高效表达,显著提高乳腺上皮细胞的泌乳能力。 相似文献
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《黑龙江畜牧兽医》2015,(9)
为了研究冷诱导RNA结合蛋白(CIRP)更深入的功能及意义,试验通过Lipo FiterTM将黑龙江八一农垦大学动物生理生化实验室之前已构建好的5组RNA干扰重组载体及空载体分别瞬时转染到大鼠肝细胞中,提取细胞RNA经反转录后进行荧光定量PCR试验,获得各组干扰载体对CIRP基因干扰后CIRP基因mRNA的相对表达量,确定效果最佳的干扰靶点。结果表明:在5组干扰试验组CIRP基因mRNA的表达量中,264组与空白对照组的表达量差异显著(P0.05),并且干扰效率达到70%以上;阴性对照组(空载体组)与空白对照组的表达量基本相同,差异不显著(P0.05)。说明试验成功筛选出有效干扰靶点。 相似文献
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《中国畜牧兽医》2016,(10)
为研究L型氨基酸转运载体1(L type amino acid transporter 1,LAT1)对奶牛乳腺中β-酪蛋白表达的作用,本试验采用PCR技术体外扩增奶牛LAT1基因并构建LAT1真核表达载体,采用脂质体转染技术将LAT1真核表达载体转染奶牛乳腺上皮细胞,并于转染48h后采用Western blotting技术检测LAT1过表达后乳腺上皮细胞中LAT1、4F2hc和β-酪蛋白的表达变化。荧光显微镜检测结果显示,LAT1真核表达载体成功转染奶牛乳腺上皮细胞;Western blotting检测结果显示,LAT1过表达的奶牛乳腺上皮细胞中LAT1极显著增加(P0.01),β-酪蛋白的表达显著升高(P0.05),4F2hc表达变化不显著(P0.05)。这些结果提示LAT1对奶牛乳腺上皮细胞乳蛋白合成具有促进作用。 相似文献
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通过设计和合成TNFRSF1A基因的3条靶向shRNA,并分别插入到pSGU6/GFP/Neo载体中,成功构建出了3个干扰载体,通过脂质体介导法将干扰载体成功转入到内蒙古绒山羊胎儿成纤维细胞中,并利用Real Time PCR检测TNFRSF1A基因的表达量。由结果可知,TNFRSF1A基因的干扰载体经测序鉴定证明构建成功,干扰载体转染内蒙古绒山羊胎儿成纤维细胞24 h后可见绿色荧光表达,当到达48 h时,转染效率达到最大, shRNA-1、 shRNA-2、 shRNA-3三组中TNFRSF1A的mRNA表达水平均低于转染空载体的NC组,且shRNA2对TNFRSF1A的mRNA表达具有最佳的干扰效果。综上所述,该研究成功构建了TNFRSF1A干扰载体,为进一步研究绒山羊TNFRSF1A基因在毛囊生长与周期调控的作用奠定基础。 相似文献
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研究旨在构建具有高效抗菌、活性持久的奶牛乳腺特异性表达质粒,探索研制新型抗菌制剂,为利用基因工程技术防治奶牛乳房炎提供重要材料。根据GenBank中奶牛溶菌酶基因(lysozyme,Lyz)mRNA序列(GenBank号:NM_180999.1)设计引物,从奶牛乳腺上皮细胞中RT-PCR扩增Lyz基因编码序列,将其克隆到携带增强型绿色荧光蛋白通用型表达载体pEGFP-N1中测序。重组质粒pEGFP-N1-Lyz经Ase I+Hind III双酶切除CMV启动子,将奶牛乳腺特异性表达β-乳球蛋白基因(BLG)启动子同源重组替换到Ase I和Hind III酶切位点,构建奶牛溶菌酶基因(Lyz)乳腺特异性表达质粒pBLG-EGFP-N1-Lyz,采用脂质体法转染奶牛乳腺上皮细胞(BMEC)、人肾脏上皮细胞(HEK293T),荧光显微镜检测转染细胞中绿色荧光蛋白的表达情况。结果表明,重组表达质粒pBLG-EGFP-N1-Lyz测序结果与目的片段长度相符,转染至BMEC细胞和HEK293T细胞,在奶牛乳腺上皮细胞观察到绿色荧光, HEK293T细胞未观察到绿色荧光,奶牛Lyz基因乳腺特异性表达质粒构建成功。 相似文献
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《黑龙江畜牧兽医》2016,(15)
为了抑制C2C12细胞中akirin2基因的表达,试验采用siRNA表达载体法构建了靶向akirin2基因的RNA干扰(RNAi)真核表达载体。首先针对akirin2基因设计并合成了相应的干扰序列,退火后将其与RNAi表达载体psiRNA-h7SKGFPzeo进行连接,构建成干扰载体akirin2-pshRNA0、akirin2-pshRNA1。将构建好的载体通过Turbofect转染试剂转染小鼠成肌细胞C2C12,通过荧光定量PCR检测其干扰效率,经酶切和测序鉴定所构建的akirin2基因RNA干扰载体与设计序列完全相符。结果表明:重组干扰载体akirin2-pshRNA0和akirin2-pshRNA1构建成功。将重组RNAi载体转染C2C12细胞,与正常对照组相比,转染akirin2-pshRNA1组的akirin2基因mRNA相对表达量显著降低,表达量下调了60.62%(P0.05)。说明试验构建的靶向akirin2基因的RNAi载体akirin2-pshRNA1可以有效地抑制C2C12细胞中akirin2基因的表达。 相似文献
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旨在研究重组克柔念珠菌(Candida krusei)14-3-3蛋白(rCK14-3-3)对奶牛乳腺上皮细胞(MAC-T)炎性反应的分子机制,为系统研究克柔念珠菌损伤奶牛乳腺上皮细胞的机制提供理论依据。采用原核表达的方法构建克柔念珠菌BMH1基因重组质粒并转化入大肠杆菌BL21(DE3)中,经诱导表达纯化后进行Western blot鉴定;CCK-8法筛选rCK14-3-3蛋白对MAC-T的最适作用浓度和时间;Western blot检测rCK14-3-3蛋白对MAC-T的Toll样受体、MAPK信号通路、NF-κB信号通路主要蛋白相对表达量的影响;ELISA检测rCK14-3-3蛋白对MAC-T中相关炎性因子IL-1β、IFN-γ、IL-18、TNF-α、IL-6、IL-12相对表达量的影响。结果显示:成功构建了pET-21a-BMH1重组表达载体,经诱导表达纯化后获得有生物学活性的rCK14-3-3蛋白,纯度达90%以上,浓度为0.2 mg·mL-1。与空白对照组相比,采用50μg·mL-1的rCK14-3-3作用MAC-T,TLR2、... 相似文献
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Beran GW 《Preventive veterinary medicine》2008,86(3-4):198-207
When early people made their appearance, zoonotic infectious diseases were already waiting, but epidemic diseases did not appear in human history until people began to live in large numbers under conditions of close contact, mainly during the last 10,000 years. Disease has decimated urban populations, conquered armies, and disrupted society. The focus here is on (1) the plague of Athens and the Black Death; (2) smallpox, influenza, and rabies; (3) avian influenza prion diseases, and foot & mouth disease; and (4) emerging and re-emerging diseases. All have veterinary public health associations. In Athens, Greece, in 430 BC, when the Spartans ravaged the countryside, hordes crowded into Athens so that orderly movements, space in which to live, and adequate supplies of food became impossible. Crowding of any population fosters disease transmission; chaos and disorder enhance it all the more. Out of northern Egypt came a terrible plague from across the Mediterranean Sea. The identity of the plague of Athens remains unsure, but the well-considered conclusion is Rift Valley Fever, a mosquito borne, viral zoonosis. The Black Death, also called the Plague, raged in Asia for centuries. In 1347, the Black Death was brought by a ship out of Asia to Sicily. The scenes of devastation were repeated throughout Europe, with 90% or more of the people dying in city after city. Influenza, too, has been a cause of periodic human epidemics, but the great pandemic of influenza occurred in the last months of World War I. In the years of highest occurrence, more than half the world's population became clinically infected. If veterinary public health had been born earlier, it could have led to elucidating the epidemiology of influenza and the plagues of Athens, Europe, and Asia. In turn, smallpox had also caused continual tragedy. In 1796, Edward Jenner began to harvest pustules of cowpox from children or infected cows and inject them into susceptible children. In 1980, the World Health Organization declared that smallpox had been eliminated from the world. Rabies, though, still strikes terror. A number of animal diseases, broadly termed emerging and re-emerging diseases, need surveillance because they have the potential to impact human health. From late in 2003 to 2007, the highly pathogenic H5N1 influenza virus in poultry infected at least 121 people and caused 62 deaths in four countries. The prion diseases, too, all have very high numbers in concentrated contacts. To control these diseases, veterinary public health is essential, with diagnosis, epidemiological surveillance, clinical manifestations, and prevention as primary measures. 相似文献
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Dekker A Dercksen D Snoep J van Wuyjckhuise L 《Tijdschrift voor diergeneeskunde》2007,132(18):695-701
On 26th of july 2007 a new case of bluetongue was notified in the Netherlands and on 2nd of august 2007 foot-and-mouth disease was diagnosed in Surrey, England, which raised the threat of having both infections simultaniously in one area. Bluetongue and foot-and-mouth disease have a different pathogenesis, but symptoms may resemble each other at a later stage of infection. The pathogenesis and possible clinical symptoms of both infections are discussed and illustrated with pictures. 相似文献
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在动物的异常行为中以刻板行为最为常见,而咽气癖又是马最常见的口部刻板行为之一。作者就咽气癖在生理方面和心智方面对马体产生的影响、行为基础、诱因的研究进展及常见的防治措施进行综述。 相似文献
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