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.     

偏置双曲柄滑块机构设计

偏置双曲柄滑块机构不仅具有k>3的超急回运动特性,而且具有可变急回运动特性,机构中各构件尺寸一定时,仅仅改变其相对安装位置,便可获得不同的行程速比系数。本文分析偏置双曲柄滑块机构的急回运动特性,给出了按最小传动角设计偏置双曲柄滑块机构的设计方法和设计曲线。     

浙南地区杉木杂交组合再选择的研究

对4片定植于浙江和江西两地的全同胞试验林进行调查,评选出的若干优良杂交组合,进行重复制种和区域 化造林试验。于2001年11月份调查了遂昌试点3年生的杉木全同胞试验林,测定树高,地径,轮盘数,冠幅和成活 率5个性状,经过方差分析,方差一协方差分析和综合指数分析,初步评出9702(龙15×1339);9706(1419×1339); 9703(龙15×闽33)3个全同胞家系,其表聊突出,3年生树高超过223 cm,同时适应性也强,造林成活率在97．9％ 以上,值得在浙江省南部类似地区推广应用。     

探索低成本连续生产中、高密度纤维薄板和异型板的方式

连续生产中、高密度薄板的方式是采用连续双钢带冷热压机,而国内人造板设备制造行业尚无制造连续双钢带冷热压机的能力。本文依据木纤维薄板矫直原理,提出采用简易连续热辊压压机加矫直机的方案,探索低成本连续生产中、高密度纤维薄板和异型板的方式,以解决实际生产问题。     

Effects of different forms and origins of oilseeds on dynamics of ruminal biohydrogenation of long‐chain fatty acids in vitro

Dietary unsaturated fatty acids (FA) are intensively hydrogenated in the rumen, resulting in reduced amount of poly‐unsaturated fatty acids (PUFA) and accumulation of several biohydrogenation (BH) products. In this study, BH of PUFA originating from different oilseeds (linseed, soya beans, sunflower seed and rapeseed) present in crushed oilseeds or their free oils were assessed in vitro. The assay substrates were incubated in buffered rumen fluid for 0, 6, 12 and 24 h. After incubation, the FA pattern of the incubated samples was analysed using gas chromatography. Biohydrogenation is defined as disappearance of double bonds (DB) calculated from the contents of unsaturated FA. After 24‐h incubation, the DB contents of all oilseeds were reduced (p < 0.001) by 40–60%. The reduction was higher (p < 0.001) for the crushed form compared with the oil form. In addition, linseed and sunflower seed known as oilseeds with high contents of linolenic acid C18:3 c9,12,15 (LNA) and linoleic acid C18:2 c9,12 (LA), respectively, showed a higher (p < 0.001) accumulation of the BH intermediates conjugated linoleic acid (CLA, isomer C18:2 c9t11) and vaccenic acid (C18:1 t11) for the crushed form, when compared with the oil. These results suggest an inherent effect of the physical form of the assay oilseeds on in vitro BH. Changes in FA pattern during BH in vitro can be attributed to both source and physical form of the assay oilseeds. However, further investigations are warranted to ensure whether the observed in vitro effects on ruminal BH can be confirmed in vivo.     

PCV2 Rep蛋白N-糖基化位点突变对病毒复制的影响

N-糖基化对病毒的感染与增殖均具有重要作用,与PCV2复制相关的Rep蛋白含有三个N-糖基化位点。为了分析PCV2Rep蛋白N-糖基化位点突变对病毒复制的影响,本试验构建了三个双拷贝突变体感染性克隆2M23、2M256、2M286,并成功拯救病毒。通过间接免疫荧光检测病毒的拯救效果,TCID50测定病毒的感染力,荧光定量PCR检测细胞病毒的载量。结果显示,PCV2Rep蛋白的23~25aa、256~258aa N-糖基化位点突变后降低病毒的复制能力,而286~288aa突变后增强病毒的复制能力,为进一步阐明PCV2的复制及致病机制提供参考。     

水稻秸秆双轴深埋还田机设计与试验

针对北方寒地稻田在秸秆还田作业时,传统单轴机具难以适应覆有大量秸秆的湿黏土壤条件,作业质量难以满足实际作业需求的问题,设计了一种前轴正旋、后轴反旋的新型水稻秸秆双轴深埋还田机。结合实际农艺要求及土壤运动过程确定前后刀轴中心水平距离650 mm、竖直距离100 mm,并对整机进行配置。运用EDEM仿真软件模拟还田机工作过程,以前进速度、前轴转速、后轴转速为试验因素,以秸秆还田率和机具功耗为评价指标进行正交试验,建立秸秆还田率及机具功耗回归方程。利用Design-Expert分析软件得到最优参数组合,根据仿真优化结果及实际加工需求确定最优工作参数为：前进速度1.5 km/h、前轴转速274.2 r/min、后轴转速219.4 r/min,为后续田间试验提供理论支撑。田间试验结果表明,在留茬高度为15～20 cm、地表秸秆覆盖量为468～578 g/m²、拖拉机前进作业速度为低速1挡(1.5 km/h)时,水稻秸秆双轴深埋还田机还田率为88.7%～91.2%、地面平整度为1.8～2.4 cm、碎土率为97.7%～98.8%、耕深为16.6～19.5 cm,各项指标均满足...     

农业机械推广鉴定证后监督的实践与思考

为总结利用农机证后监督工作成果,进一步创新完善监督制度,深入推进农业机械化领域“放管服”改革,回顾农机证后监督发展历程,定义为4个阶段：起步摸索阶段、常态规范阶段、创新发展阶段、完善提升阶段。介绍证后监督总体实施情况,包括监督检查内容、工作程序及历年抽查数量、完成率、抽查产品的地域分布及种类分布分析。基于结果数据分析发现整改确认后合格占比呈下降趋势、不合格占比呈上升趋势、证后监督权威日益显现等趋势性变化,并结合政策文件制修订背景分析证后监督结果趋势性变化的原因。通过深入分析不合格及整改等原因,发现获证企业对政策了解不足、对产品一致性管理不足、对证书和标志的动态管理不足、生产经营异常现象日益增多等经营管理现状。针对证后监督总体实施情况及发现问题,提出需加大抽查证书数量及产品种类、加强政策宣传及人员培训、建立信用风险分类监管制度、进一步挖掘潜力强化证后监督作用的政策建议。     

麦/玉两熟区农作模式分析及轮耕模式探讨

从20世纪90年代初期起,以少/免耕为主体的耕作模式在麦/玉两熟区得到大面积推广应用。经过近15年的耕作栽培,少/免耕充分显示了它省时省工、高效环保的作用,但是与此同时一系列弊端也日益显露。对宁晋县百户农民耕作意愿调查的结果显示,在长期少/免耕后小麦产量和经济效益有所下降,而应在免耕3～5年以后对土壤进行深耕翻。对比麦/玉种植模式与马铃薯/棉花套作的经济效益,以轮作实现轮耕是麦/玉两熟区耕作模式发展方向。     

木芙蓉的繁育系统及传粉生物学

以木芙蓉为研究材料,运用杂交指数、花粉数/胚珠数、座果率、种子萌发率等指标对木芙蓉的繁育系统及传粉生物学等进行研究。结果表明:木芙蓉单花的花期1 d,整体花期7—11月份;木芙蓉的杂交指数为4,繁育系统为异交、部分自交亲和、需要传粉者;木芙蓉的花粉数/胚珠数为415±11.3,繁育系统属于兼性异交类型;木芙蓉自然座果率为74%;同株同花授粉的座果率为40%;人工异株异花授粉座果率为71%;各种授粉方式下,单个果实的种子个数为128~148,差别不大;各种授粉方式下,平均种子萌发率为47%~60%,没有显著差异;传粉昆虫有蜂类、甲虫,其中中华蜜蜂、黑小蜜蜂为主要传粉昆虫。