Effects of Vibrio harveyi on plasma clotting protein (coagulogen) of white shrimp Litopenaeus vannamei

Blood clotting exhibits various important functions, including the prevention of body fluid loss and invasion of pathogens in shrimp. The effects of pathogenic Vibrio harveyi on plasma of white shrimp (Litopenaeus vannamei) in vitro and in vivo were investigated in this study. The clotting protein (coagulogen) in plasma of white shrimp pre‐incubated with extracellular products (ECP) of V. harveyi was found apparently decreased and fast‐migrated in crossed immunoelectrophoresis (CIE) gels. In addition, the coagulogen had been degraded to many low molecular‐weight protein bands in plasma pre‐incubated with ECP on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) gels. When pre‐challenged with bacterial cells and ECP of V. harveyi, the white shrimp began to die at about 30 and 16 h respectively. Moreover, plasma coagulogen was decreased more obvious in shrimp challenged with ECP than that with bacterial cells as visualized in CIE gels, and total plasma protein in both group of shrimp were all decreased. Haemolymph withdrawn from moribund shrimp pre‐challenged with V. harveyi or its ECP was observed unclottable. However, the addition of clotting factors (transglutaminase and/or Ca²⁺) to these unclottable plasma could apparently promote their re‐clotting ability as jelly‐like solid observed in microtubes. The recovery of clotting ability of plasma from moribund shrimp was due to the reformation of coagulogen (200 kDa) after adding the two clotting factors as shown on CIE and SDS‐PAGE gels. The present results suggest that the infection of V. harveyi in white shrimp may not only degrade coagulogen but also influence the presence of transglutaminase and Ca²⁺ ion.     

硫化物醌氧化还原酶定向表达载体的构建及细胞转染表达检测

为建立硫化物醌氧化还原酶(SQR)的肠道定向表达载体,同时通过转染小鼠肠道上皮细胞确定载体的有效性,本试验采用重叠PCR、中间载体法、酶切连接法获得重组载体,通过脂质体转染法将重组载体转入小鼠肠道上皮细胞,并鉴定重组载体的特异性表达。结果显示,成功构建了以肠脂肪酸结合蛋白质(IFABP)为启动子的SQR肠道定向表达载体pc DNA3.1(-)-IFABP-SQR-GFP,并在小鼠肠道上皮细胞中检测到表达的融合绿色荧光蛋白质(GFP)。表明IFABP启动子能够在肠道细胞中定向启动SQR的表达。     

对草地贪夜蛾高毒力的苏云金芽胞杆菌菌株筛选与杀虫活性研究

草地贪夜蛾是一种世界性重大农业害虫,给玉米等多种主要粮食和经济作物造成严重危害.为实现对草地贪夜蛾的高效、绿色、持续防控,亟需筛选高毒力苏云金芽胞杆菌菌株资源.本研究以前期实验室储备的对斜纹夜蛾、小地老虎具有杀虫活性的363株野生菌株库为候选菌株,基于菌株杀虫基因差异及进化关系,去除冗余菌株后获得172株候选菌株.通过...     

猪Jiv基因的克隆表达与多克隆抗体制备

【目的】克隆猪Jiv蛋白核心区的2 112bp的基因片段,构建其原核表达载体后进行蛋白的表达和纯化,制备抗猪Jiv蛋白的多克隆抗体。【方法】利用RT-PCR方法,从猪脾脏组织RNA中扩增得到猪Jiv基因,将其克隆到pMD19-T载体并测序,以此为基础构建原核表达载体pET32a-Jiv,转化E.coli Rosetta(DE3)感受态细胞中,利用IPTG诱导表达,获得大量包涵体形式的猪Jiv融合蛋白。通过镍离子螯合层析柱对表达的目的蛋白进行纯化,对纯化后的包涵体蛋白进行透析复性,以每只新西兰白兔400μg蛋白的初次免疫剂量和500μg蛋白的加强免疫剂量进行免疫,共免疫5次,采集血清,采用间接ELISA法检测抗体效价达到一定水平后,收集抗体,利用Western blot检测抗体特异性。【结果】克隆得到长度为2 112bp的猪Jiv基因片段;构建的pET32a-Jiv在E.coli Rosetta(DE3)感受态细胞中经诱导后高效表达,SDS-PAGE结果显示,纯化得到分子质量约为95ku的猪Jiv融合蛋白;测得的猪Jiv蛋白多克隆抗体效价达1∶6 400;Western blot检测结果证实,所获得抗体能够有效地应用于猪Jiv蛋白抗原的检测。【结论】成功地克隆了猪Jiv基因,制备了抗猪Jiv蛋白的多克隆抗体。     

猪源唾液乳杆菌对生长猪氮表观消化率及血清指标的影响

本研究旨在探讨猪源唾液乳杆菌对生长猪氮表观消化率和血清指标的影响。选用平均体重为(35.22±0.86)kg长×大二元杂交猪81头,随机分成3组,每组3个重复,每个重复9头。对照组饲喂基础日粮,试验组分别在基础日粮中添加0.20%、0.40%猪源唾液乳杆菌冻干制剂,试验期为30 d。结果表明:添加0.20%、0.40%的猪源唾液乳杆菌冻干制剂可显著提高生长猪氮表观消化率(P<0.05);与对照组相比,2个试验组猪血清白球比显著降低(P<0.05),尿素氮、乳酸脱氢酶、肌酸激酶含量分别比对照组降低了14.77%和13.42%(P<0.05)、16.92%和20.0%(P<0.05)、28.96%和23.00%(P<0.01),但2个试验组间差异不显著(P>0.05)。结果提示,日粮中添加猪源唾液乳杆菌能够提高生长猪氮表观消化率,改善血清指标,减少应激,且以添加剂量为0.20%时效果最好。     

对大豆新应用途径的研究——含蛋白成分洗涤产品的研究

[目的]为开发利用大豆功能性产品开辟新途径。[方法]以大豆粉为原料制成含蛋白洗涤剂,并测试含蛋白洗涤剂的理化性能及其对皮脂污布、蛋白污布、碳黑污布的去污力。[结果]大豆粉具有洗涤功能,其对皮脂污布的去污力强于其他洗涤剂,在大豆粉中加入表面活性剂后,其对皮脂污布的去污力降低。蛋白污布的洗涤效果与皮脂污布的洗涤效果相反,能形成较稳定蛋白膜的全豆粉不利于洗涤蛋白污布,而含蛋白洗涤剂对蛋白污布的洗涤效果优于对照组。含蛋白洗涤剂对碳黑污布的去污力显著高于其他洗涤剂,全豆粉对碳黑污布的洗涤效果好于含蛋白洗涤剂。[结论]大豆粉具有较好的去污洗涤作用,含蛋白洗涤剂既具有较好的洗涤效果,又具有保护皮肤的功能。     

黄萎病菌侵染下陆地棉Dirigent-like蛋白基因表达差异分析

目的发掘陆地棉Dirigent-like蛋白基因家族,明确不同黄萎病抗性品种间该基因家族成员的多态性及表达差异。方法利用Illumina Hiseq 2000测序平台,通过RNA-Seq测序技术,获得黄萎病菌侵染陆地棉抗病品种和感病品种的Dirigent-like蛋白基因家族成员Unigene序列;采用生物信息学软件对其进行核苷酸序列及编码蛋白的多重比对和系统进化分析;针对该家族成员设计特异的quantitative real-time PCR引物,在Applied Biosystems 7500 Real Time PCR System上使用SYBR green PCR试剂盒标记反应产物,棉花Gh ACTIN为内标,采用2-ΔΔCt法分析抗病品种冀79、感病品种TM-1中该基因家族成员在0 h及受侵染1、8、24、48、72和96 h后的表达差异。结果获得12个陆地棉Dirigent-like蛋白基因家族成员;该家族成员分属3个进化群,基因间存在4个保守结构域。其同源基因在D5基因组染色体上,主要集中于第2、3、6、7、9、10、13染色体。陆地棉受黄萎病侵染后,Dirigent-like蛋白基因抗病、感病品种间差异表达,抗病品种冀79受黄萎病菌侵染1 h时上调2倍以上的有Gh DIR4、Gh DIR6、Gh DIR7、Gh DIR9、Gh DIR10和Gh DIR11,其中,Gh DIR9上调6.5倍;侵染8 h时Gh DIR1、Gh DIR4、Gh DIR7和Gh DIR11上调,有2个上调2倍以上;侵染24 h时仅Gh DIR4上调2倍以上;侵染48 h时Gh DIR1、Gh DIR4、Gh DIR7和Gh DIR11上调达到2倍以上;侵染72 h时仅Gh DIR9上调2倍以上,达到6.4倍;侵染96 h时仅Gh DIR4上调2倍以上。感病品种TM-1受侵染1 h时仅Gh DIR7上调2倍以上;受侵染8 h时Gh DIR7上调325倍,Gh DIR10上调2倍以上;侵染24和48 h时Gh DIR4、Gh DIR5、Gh DIR6、Gh DIR7、Gh DIR9、Gh DIR10急剧上调,Gh DIR7、Gh DIR9、Gh DIR10上调10倍以上;受侵染72 h时Gh DIR4和Gh DIR7还保持较高的表达量;侵染96 h时Gh DIR5、Gh DIR6、Gh DIR7、Gh DIR9、Gh DIR10和Gh DIR11保持较高的表达量。由此看出,受黄萎病侵染时抗病种质冀79 Dirigent-like蛋白基因家族成员上调表达要早于感病品种,但是,该基因家族成员在感病品种受侵染8 h后上调倍数远远高于抗病品种,而且到96 h时其表达量还较高;抗病品种上调的Dirigent-like蛋白家族成员Gh DIR4、Gh DIR7、Gh DIR9和Gh DIR11值得关注;Gh DIR6、Gh DIR7、Gh DIR9和Gh DIR10在感病品种TM-1中上调显著。此外,抗病、感病品种间Gh DIR1、Gh DIR3、Gh DIR5、Gh DIR8、Gh DIR10和Gh DIR12存在核苷酸多态性。结论 Dirigent-like蛋白与黄萎病菌侵入后棉花植株的抗性反应相关。     

不同蛋白质含量大豆结瘤特性与根瘤氮代谢物含量的比较分析

为了明确高蛋白野生大豆氮素积累规律,本试验选取3类不同蛋白质含量的大豆材料,在各生育期对根瘤生长特性及根瘤氮代谢物的含量变化进行研究,结果表明：籽粒蛋白质含量与R2-R8的根瘤数呈显著正相关。V6-R2高蛋白野生大豆根瘤数增加较快,整个生育期根瘤重不断增加。三种类型大豆的血红蛋白含量、单株血红蛋白总量在R2-R8与籽粒蛋白质含量呈显著正相关。在V6到R8期高蛋白野生大豆血红蛋白含量、总量都明显高于其它两种类型大豆材料,差异性均达到显著水平。R2-R8期三种类型大豆游离氨基酸含量、酰脲含量均呈下降趋势,高蛋白野生大豆游离氨基酸含量低于、而酰脲含量却又高于其它两种类型材料,且差异性显著。这可能说明高蛋白野生大豆根瘤衰老较慢、根瘤内氮代谢物含量丰富,生育后期仍具有较强的酰脲合成与运输能力是形成该类型大豆籽粒高蛋白质含量的原因之一。     

奶牛乳房炎乳腺膜蛋白的比较蛋白质组学研究

【目的】通过分析临床健康和乳房炎奶牛乳腺组织膜蛋白的差异表达,以期在蛋白质水平探索奶牛乳房炎的发生机理。【方法】采用二维凝胶电泳技术分离了临床健康和乳房炎奶牛的乳腺组织膜蛋白,经考马斯亮蓝染色后PDQuest 7.4软件匹配、检测凝胶中差异表达的蛋白点,对12个差异蛋白点采用高效液相色谱串联离子阱质谱分析,SEQUEST软件搜索NCBInr数据库。半定量RT-PCR分析差异表达蛋白的mRNA水平。【结果】12个差异蛋白点鉴定为11种蛋白质,其中9个蛋白点在临床型乳房炎奶牛乳腺组织膜蛋白中表达量上调而3个蛋白点表达量下调,主要涉及细胞膜骨架系统、信号转导、物质结合和传递等生物学功能。半定量RT-PCR分析表明,乳房炎奶牛乳腺组织中钙粒蛋白B的mRNA表达水平是健康奶牛的1.6倍。【结论】推测这些差异蛋白的表达变化与奶牛乳房炎发生相关,为从蛋白质水平揭示奶牛乳房炎的发生机理以及发现潜在的治疗目标蛋白提供了理论依据。     

Outbreak of anaplasmosis associated with novel genetic variants of Anaplasma marginale in a dairy cattle

During early lactation, dairy cows may present a transient immunosuppressive state and develop anaplasmosis caused by Anaplasma marginale. In this study, clinical anaplasmosis in dairy cattle in the Thrace region of Turkey was investigated with respect to within-herd prevalence, vertical transmission, and genetic diversity. In March and September 2015, thirty lactating cows showed primary clinical signs of anaplasmosis, including fever, anaemia, decreased milk yield, anorexia, and laboured breathing. Symptoms disappeared in most cows after administration of long-acting oxytetracycline, but nine of them (30%) died. Following diagnosis based on clinical signs, microscopy and molecular findings, blood samples were collected from apparently healthy lactating cows (n = 184), pregnant heifers (n = 39) and newborn calves (n = 24). DNA was extracted from each sample and analyzed for the presence of major surface proteins (MSPs) of A. marginale, followed by sequencing to assess diversity of isolates. Microscopic examination of erythrocytes revealed A. marginale inclusion bodies in symptomatic cows. Examination of thin blood smears showed 3.8% of the lactating, clinically asymptomatic, cows to be infected with A. marginale, while nPCR detected 31.0% positive. A. marginale infection was not detected in pregnant heifers by either method. Congenital infection was found in one calf by nPCR. This is the first report of transplacental transmission of A. marginale in Turkey. The MSP4 sequence analyses showed high genetic diversity among the isolates, presenting 97.6-99.6% homology at the amino acid level. The sequences of MSP1a amplicons revealed genetic diversity providing three new tandem repeats.