气肿疽梭菌CctA基因的原核表达及其间接ELISA检测方法的建立

研究旨在原核表达气肿疽梭菌细胞毒素A （CctA）基因,用纯化的重组蛋白建立其间接ELISA检测方法,a。利用大肠杆菌密码子的偏爱性优化CctA基因序列,克隆至原核表达载体pET-28a （+）,双酶切鉴定原核表达质粒pET28a-CctA并测序。将重组质粒转入大肠杆菌,经IPTG诱导得到高表达的重组CctA包涵体蛋白。包涵体蛋白变性后经镍（Ni）柱纯化,SDS-PAGE检测其纯化效果。以豚鼠抗气肿疽梭菌抗血清为一抗,用Western blotting方法检测重组CctA蛋白的反应原性。用棋盘滴定法建立间接ELISA检测方法,以复性后的重组CctA蛋白作为检测抗原,摸索抗原包被浓度、封闭液的种类及浓度、抗体的最适稀释度和反应条件等。选用3批气肿疽灭活疫苗免疫豚鼠后采集血清,同时用攻毒和间接ELISA两种方法验证疫苗的免疫效力。质粒双酶切结果显示,得到大小约853 bp的条带,与预期相符,且测序结果正确。SDS-PAGE结果表明,成功表达并纯化大小为35 ku的重组CctA蛋白。Western blotting结果显示,豚鼠抗气肿疽梭菌抗血清与重组CctA蛋白具有良好的反应原性。建立的间接ELISA法的最适条件为：抗原的包被浓度为0.5 μg/mL,于4 ℃包被过夜;封闭液选择10%胎牛血清,37 ℃孵育2 h;二抗的稀释度为1：8 000;室温避光显色10 min后终止反应,测定D_{450 nm}值。当P/N>4.6时,间接ELISA法检测结果与豚鼠攻毒试验结果拟合度较好。本研究成功表达CctA基因并纯化了重组CctA蛋白,建立的以重组CctA蛋白为检测抗原的间接ELISA检测方法,有望成为气肿疽灭活疫苗免疫效果验证的替代方法。     

羊源D型多杀性巴氏杆菌感染小鼠脾脏转录组分析

试验旨在探索羊源D型多杀性巴氏杆菌入侵宿主脾脏组织中引发的免疫应答途径。首先利用羊源D型多杀性巴氏杆菌(HN01菌株)感染小鼠,建立羊源D型多杀性巴氏杆菌感染的动物模型;之后利用转录组测序技术获得感染小鼠与正常小鼠的脾脏转录组数据,并使用COG、KOG、eggNOG、GO、KEGG数据库对测序结果中的差异表达基因(differentially expressed genes,DEGs)进行功能注释与分析,同时对于显著富集到关键免疫通路的差异表达基因使用STRING软件和KEGG mapper进行蛋白互作分析,筛选出核心通路中起关键作用的基因;最后选取关键的10个基因进行实时荧光定量RT-PCR验证。转录组分析结果显示,与正常组相比,感染组中筛选出3 380个差异表达基因(P<0.01,log₂|FoldChange|≥0.5),其中1 691个基因上调,1 689个下调。基因功能富集分析结果表明,感染组脾脏中的差异表达基因主要发挥信号转导的功能,其主要参与的生物途径包括细胞因子与细胞因子受体互作通路、趋化因子信号通路、HIF-1信号通路、TNF信号通路。蛋白互作分析筛选出约28个核心差异表达基因,结合实时荧光定量RT-PCR验证后,其中9个基因的表达结果与测序一致,分别是C3、Cd4、Cxcl13、Lck、Gnai1、Grap2、IL-6、Cxcr6及Serping1基因。本研究初步证明了脾脏在抵抗多杀性巴氏杆菌入侵中参与了一系列免疫应答反应,为进一步研究羊源D型多杀性巴氏杆菌与宿主之间相互作用的分子机制奠定理论基础。     

拷贝数变异在畜禽中的研究进展

拷贝数变异（copy number variation,CNV）具有多种形式的变异结构,在品种多样性、生物进化和疾病相关性等研究中起着重要作用,并具有片段长度大、覆盖范围广等特点。随着分子生物学的发展及DNA测序技术的日渐成熟,人们对遗传变异的研究不断向DNA分子水平深入,多态性标记在畜禽育种中已逐渐成为动物育种研究的趋势和主流。由于CNV对基因的调控和表达所造成的影响更为显著,因此,CNV在重要畜禽中的研究越来越多。目前,已检测出大量有关畜禽重要经济性状的基因序列变异,并有许多研究均表明CNV与动物的重要经济特征及疾病的发生有关。笔者主要通过参考国内外相关的研究报道,简述了CNV的相关研究背景、概念、突变机制,归纳总结了CNV对牛、羊、猪、鸡的经济性状、繁殖性状和疾病调控的影响,以期通过对这些重要畜禽的基因组学研究揭示其适应性遗传机理和表型性状差异的遗传基础,开发相应的分子遗传标记,为畜禽的标记辅助选育提供理论基础。     

库水剧烈变化对砌石高坝水平位移

对建于峡谷中的砌古高坝，一般中型水库居多，这类水库调节库容小，库水位变化快，水位引起的位移对大坝中部各坝段的水平位影响是主要的，分析了石门水库大坝水平位移的变化状态，主要是由库水位的变化引起的弹性位移。通过计算发现，库水位剧烈变化时，大坝水平位移就大，但由于大坝弹性较好，有逐步稳定的趋势。     

基于LLWin3.0控制Fischertechnik模型实例

随着计算机的普及，特别是软件技术的发展，基于电脑控制且充分展示个性化创新实验，成为当今高等学校素质教育的重要方式和发展方向。为此，详细介绍了德国慧鱼公司的专利产品“创意组合模型(fischertechnik)的应用实例。结合LLWin3．0图形化编程技术。为教育行业应用提供了有效的途径。     

基于灰色关联法的小型农田水利工程建设项目绩效评价研究

运用灰色关联法原理,采用均方差法计算指标权重,根据近年来湖北省进行小型农田水利工程建设项目绩效评价的实际情况,建立了小型农田水利工程建设项目评价定性定量混合指标体系和模型,应用该模型和方法对湖北省2012年第二批20个小农水重点县工程建设项目进行了评价(作为研究,各县、市名以汉语拼音字头代替)。结果表明:LHK、SSS、ZJS、DJK、XTS等14个县为优秀,XCX、TMS、JSX等5个县为良好,CYX为一般,无及格等级的重点县。评价结果与实际基本相符。证明本文所提出的模型和方法可应用于实际,是一种快速便捷的评价工具。     

灰关联决策在水质面板数据处理中的扩展及应用

针对面板数据灰色关联决策分析评价问题,在经典灰色关联分析和灰色综合评价方法的基础上,构建面板数据灰色关联决策评价模型。将灰色关联决策方法扩展到面板数据分析中,解决了小样本面板数据的灰色评价分析问题。将这种方法应用于水环境面板数据质量评价中,经实例计算验证了面板数据灰色关联决策评价模型的稳定性、合理性和实用性,为水环境面板数据质量分析评价提供了可行的计算思路和方法。     

Blood Typing of Asiatic Black Bear (Ursusthibetanus)

In this study,we aimed to determine whether bear had different blood group systems.Whole blood samples were collected from 40 Asiatic Black bears in Fujian province,China.Tube method was used to detect antibodies in plasma,and antibody isotype was determined with 2-mercaptoethanol.Plasma was further analyzed by mass spectrometry.The plasma from four bears had antibodies,possibly IgM isoform,which could agglutinate RBCs from 30 bears.Blood samples from 10 bears were tested by human blood typing reagents.The results showed that 4 bears had blood type like human type O,while 6 bears had like human type B.Plasma protein had extensive homology to serum albumin-like isoform 1 found in giant panda (Ailuropodamelanoleuca).We suggested that Asiatic Black bear might have at least one blood group system with two blood types.If the sick bear needs blood transfusion,a cross-matching test was necessary.Moreover,giant panda might receive blood from Asiatic Black bear in case of emergency.     

Effect of Aerosol OT and Paraformaldehyde Supplement on the Voluntary Intake,Digestion and Metabolism of Late-pregnant Ewe Fed with Ground Diet

The Small-tail Han ewes with 3 years old, (48.7±2.0)kg body weight, carring the multi-lamb gene, were fed with the same diet, mating after one month.18 pregnant ewes were selected and divided into 3 groups(groups 1(control group), 2 and 3).From 90 days of pregnancy, groups 2 and 3 were supplemented with aerosol OT(800 mg/kg) and paraformaldehyde(300 mg/kg), respectively, and the experiments of digestion and metabolism were carried on from 121 to 130 days of pregnancy to study the effect of aerosol OT and paraformaldehyde supplements on the voluntary intake, digestion and metabolism, lambing of late-pregnant ewes.The results showed that when fed with aerosol OT, the voluntary intake of dry matter was increased by 13.0%(P<0.01);The amount of digestion of DM, OM, CP, cellulose and GE was increased by 12.4%(P<0.05), 14.4%(P<0.01), 23.2%(P<0.01), 15.2%(P>0.05) and 27.4%(P<0.01), respectively;The retention of N, Ca and P was increased by 31.1%(P<0.01), 48.5%(P<0.01) and 31.9%(P<0.01), respectively;The serum triglyceride and free fatty acid content tended to decrease;but the apparent digestibility of the dietary nutrients were not significantly affected(P>0.05);The loss of ewe postpartum weight was reduced by 61.9%(P<0.01), a nest of lamb weight was increased by 4.2%(P>0.05).In the condition of feeding paraformaldehyde, the voluntary intake of dry matter was increased by 12.6%(P<0.01);The amount of digestion of DM, OM, CP, cellulose and GE was increased by 15.4%(P<0.01), 16.2%(P<0.01), 22.4%(P<0.01), 14.2%(P>0.05) and 29.0%(P<0.01), respectively;The retention of N, Ca and P was increased by 33.5%(P<0.01), 48.9%(P<0.01) and 36.3%(P<0.01), respectively;But the apparent digestibility of the dietary nutrients were no significantly affected(P>0.05);The serum β-hydroxybutrate and free fatty acid content tended to decrease;The loss of ewe postpartum weight was reduced by 57.1%(P<0.01), a nest of lamb weight was increased by 11.1%(P<0.05).This study showed that by feeding aerosol OT or paraformaldehyde, the intake, digestibility of Ca, P and N could be improved in late-pregnant ewe, the body weight loss, body fat mobilization could be reduced, and the birth weight of litter could be increased.     

Analysis on Differential Expression Genes in Porcine Aortic Vascular Endothelial Cells Induced by Apolipoprotein C Ⅲ

The aim of this study was to investigate the differential expression genes induced by ApoCⅢ,and study the function of ApoCⅢ.Porcine aortic vascular endothelial cells were successfully isolated using enzyme digestion,and then screened the differential expression genes induced by ApoCⅢ using the Solexa high-throughput sequencing technology.The results showed 647 differential expression genes,including 390 up-regulated genes and 257 down-regulated genes.The qRT-PCR results verified that the gene expression results from Solexa sequencing data were reliable.GO and Pathway analysis showed that the function of differential expression genes were related to immune response,cell apoptosis and death.These findings suggested that ApoCⅢ affected the physiological function of porcine aortic endothelial cells by the molecular pathways of inflammation,cell adhesion and apoptosis,which provided a theoretical basis for further understanding the molecular mechanisms of atherosclerosis caused by ApoCⅢ.