Preparation of Cathepsin L1 Monoclonal Antibody Against Fasclala gigantica and Establishment of Double Antibody Sandwich ELISA

【Objective】 This study was intend to obtain cathepsin L1(rFgCat L1) specific monoclonal antibody and construct the double antibody sandwich ELISA.【Method】 Five BALB/c mice were immunized with 1 mg/mL rFgCat L1 protein for four times.Mouse splenocytes were isolated and fused with SP2/0 cells to construct hybridoma cells.Strong positive hybridoma cell lines were screened, 1×10⁶ cells were injected intraperitoneally per mouse to prepare monoclonal antibodies.Antibody titer and antigenic epitope were detected using ELISA method, antibody subtype and specificity were identified using Western blotting method.The double antibody sandwich ELISA was constructed by combining the anti-rFgCat L1 polyclonal antibody, and its sensitivity and specificity were tested.The positive and negative critical value was screened by 20 negative sera with positive control, and the constructed double antibody sandwich ELISA was verified by 47 goat positive sera and 47 dairy cow positive sera.【Result】 After immunization, the antibody titers in serum of 4 mice were all more than 10⁴.After isolated mouse with the highest immune response spleen cells were fused with SP2/0 cells total of 8 of them were positive cell lines were obtained after selective culture.5D5 and 7G6 were identified as strong positive strains with stable antibody secretion.After multiple subcloning screens and subcultures, the antibodies secreted in the cell supernatant were stable, with titers of 2⁹ and 2¹⁰ respectively, with ascites titers of 10⁷ and 10⁸.Western blotting and antibody subtype identification kits identified that the two antibodies were IgG1 type and the light chain was kappa type, both of which could specifically bind FgESP.According to the same antigen site was recognized by the two kinds of antibodies, the antigen titer of the two monoclonal antibodies were comparied, 7G6 was used as the coating antibody, and anti-rFgCat L1 was used as the enzyme-labeled secondary antibody.The optimized condition of method was that 7G6 was coated at a concentration of 2 μg/mL, the dilution concentration of anti-rFgCat L1 polyclonal antibody was 25 μg/mL, the dilution of Don-HRP-conjugated was 1∶4 000, 5% skimmed milk powder was selected as the blocking solution and the color development time was 25 min.The method was proved that could recognize the lowest antigen concentration of 0.625 μg/mL, also could specifically recognize antigen of Fasciola fasciatus.The constructed sandwich ELISA method was used for antigen detection of 47 dairy cow positive serum and 47 goat positive serum infective samples kept in the laboratory and the positive antigen rate were 72.3% and 78.7%, respectively.【Conclusion】 Anti-rFgCat L1 monoclonal antibody was successfully prepared and the double-sheet sandwich ELISA method for fascioliasis was constructed, which provided a good theoretical basis and material basis for the development of low-cost and rapid diagnostic kits.     

IMAGING ASSESSMENT OF THE MODIFIED DOUBLE CONTRAST BARIUM ENEMA USING CARBOXYMETHYLCELLULOSE ON RADIOGRAPHY AND ULTRASONOGRAPHY IN DOGS

A modified double contrast barium enema using carboxymethylcellulose was evaluated in beagle dogs and compared with dogs receiving a conventional barium enema. The experimental group was divided into three groups (1, 2, and 3) and given 30 ml/kg of different volume ratios of a barium vs. carboxymethylcellulose mixture. Each group underwent sonography following radiography. The volume ratio of one part barium to three parts carboxymethylcellulose was judged to be the optimal mixture, resulting in a general distribution of contrast and bowel radiolucency on radiographs and adequate postradiography sonography. The modified barium enema using carboxymethylcellulose is useful for assessing the general morphology and mucosal layers of the colon simultaneously on radiographs and ultrasonographs.     

RNA干扰技术在动物繁殖中的应用

RNA干扰(RNA interference,RNAi)是一种由双链RNA诱发的转录后水平的基因沉默现象,是近几年发展起来的基因表达调节新机制,广泛地存在于真菌、植物和动物中。到目前为止,已有很多试验证明,RNAi作为一种基因功能研究的新手段,在动物繁殖研究中发挥了极其高效的作用。作者主要介绍了RNAi的研究历史、分子机制、作用特点、生物学意义及在动物繁殖中的应用。     

双电磁机构锤击系统的高频调控及试验

针对现有随焊锤击装置电磁锤锤击频率低(1～3Hz),难以满足生产要求的现状,该文开发了双电磁机构锤击系统。以电磁理论为基础,设计了双电磁锤击装置;以AT89C52单片机为控制核心,设计了高频锤击控制电路、键盘输入及显示电路,并对程序进行调试、试验。试验表明:锤击装置可实现6～8Hz的高频锤击;与对照组相比,当锤击频率为3Hz时,魏氏组织晶粒视场面积为原来的23.9%,晶粒仍较大,组织性能改善不太明显;当频率为7Hz时,魏氏组织晶粒视场面积为原来的3.8%,组织明显得到细化,晶粒细小,组织的塑性韧性提高,改善了焊缝的力学性能。     

“双壁面射流”燃烧系统性能的试验研究

为了改善直喷柴油机的排放与经济性能,该文开发了一种直喷柴油机"双壁面射流"燃烧系统。通过试验的方法研究了燃烧室结构参数、喷油器结构参数、转速、负荷及喷油时刻等运行条件对"双壁面射流"燃烧系统燃烧过程的影响。试验结果表明:随转速的增加,着火始点向后推迟,缸压峰值呈现出先增大后减小的趋势,瞬时放热率峰值、缸内燃烧平均温度和累计放热量都降低;随负荷的增加,着火点提前,缸压峰值对应的曲轴转角向后推迟,缸压峰值、缸内燃烧平均温度和累计放热量都增加,总指示热效率随负荷的增加而降低。不同燃烧室结构参数和喷油器参数的"双壁面射流"燃烧系统燃烧持续期,在2100r/min的负荷特性中,小负荷主要受压缩比的影响,大负荷主要受预混合燃烧量的影响;总指示热效率主要受压缩比和壁面油膜生成量的影响;而NOx与碳烟排放受预混合燃烧量和贯穿度的影响。将"双壁面射流"燃烧系统静态供油提前角提前到18°CA BTDC,能实现扭矩点与功率点同为0.21BSU的低碳烟排放。     

风水两相侵蚀对坡面产流产沙特性的影响

为了研究黄土高原风蚀水蚀交错区两相侵蚀的交互作用,该试验采用风洞和模拟降雨相结合的方法,探讨了风水两相侵蚀条件下坡面降雨入渗、产流产沙特征。结果表明:相对于未风蚀处理,中度风蚀处理(风速14m/s)使坡面产流时间延长,轻度风蚀处理(风速11m/s)对产流时间影响不大(P>0.05);风蚀处理(风速11m/s、14m/s)使坡面入渗率降低2.9%～10.0%,径流强度增加4.5%～21.7%,侵蚀率增加7.3%～39.0%,不同风速间差异不显著(P>0.05),且影响程度均随雨强的增大而降低;风力、水力两种侵蚀营力相互促进导致坡面糙度增大,风速14m/s、雨强100mm/h时,坡面糙度是风速11m/s、雨强60mm/h时的3倍。研究结果为评价风水两相侵蚀效应提供理论依据。     

有机无机肥配施对双季稻田土壤养分利用与渗漏淋失的影响

采用田间小区试验,研究稻草、猪粪、猪粪堆肥或沼渣沼液与化肥配施对双季稻田土壤养分利用与渗漏淋失的影响。结果表明,有机肥与化肥配施有利于提高稻田土壤中N、P、K养分和有机质含量,降低稻田渗漏液中TN、TP、NH4+-N、NO3--N浓度,促进水稻养分吸收与利用。其中,以20%的猪粪堆肥氮与化肥配施效果较好,与施纯化肥相比,1年后土壤中全氮、全磷、全钾和有机质含量分别提高了2.59%,0.87%,0.15%和21.45%,碱解氮、速效磷、速效钾含量分别提高3.31%,3.22%和12.24%;氮、磷、钾肥料利用率分别提高5.22%,0.55%,3.50%;水稻地上部氮、磷、钾养分累积量分别提高7.83,0.33,3.14g/kg;水稻生长期稻田渗漏液中TN、TP、NH4+-N、NO3--N浓度明显降低;早稻增产19.65%,晚稻无明显减产。     

液压限位缓冲结构在汽车减振器上的应用

在分析普通双筒式减振器结构和工作原理基础上,提出一种加装双活塞液压限位缓冲器的汽车减振器,并对其工作机理进行分析;建立了具有液压限位缓冲结构的汽车减振器等效液压系统模型,对其阻尼特性进行分析.分析结果表明：在汽车减振器内部加装液压限位缓冲结构,可以产生附加阻尼力,使减振器具有更好的减振功能,有效地提高汽车恶劣行驶工况下的平顺性.     

玉米全膜双垄沟直插式精量穴播机设计与试验

为适应我国西北地区旱地玉米全膜双垄沟播作业需求,解决传统膜上播种机具作业存在挑膜、撕膜、种穴与幼苗错位等问题,设计了一种玉米全膜双垄沟直插式精量穴播机。该机采用凸轮-曲柄滑块机构、运动放大机构实现了成穴器的投种控制与定点强制开启。仿真分析与田间验证试验表明,直插式精量穴播机在覆膜垄沟播种作业时,成穴器位于播种段与出土段的水平分速为零,播种株距可在一定范围内调节,穴孔布置、成穴器运动轨迹均合理,满足玉米全膜双垄沟播作业的农艺技术要求。