【作者】 赵伟春；

【导师】 程家安；

【作者基本信息】 浙江大学 ， 农业昆虫与害虫防治， 2001， 博士

【摘要】 本文在国内外首次制备出了分别针对褐飞虱Nilaparvata lugens和稻田多种弹尾虫Collembola复合种群的单克隆抗体，并应用褐飞虱、弹尾虫和白背飞虱Sogatellafurcifera的单克隆抗体结合田间调查资料评价了弹尾虫在稻田生态系统中的作用及主要捕食性天敌对褐飞虱和白背飞虱的控制作用。结果如下： 1)应用杂交瘤技术，制备了4株褐飞虱的单克隆抗体细胞株，分别命名为3B2、3B11、3H3和4B8。4种抗体的效价均在4.096×10⁶倍以上。它们只与褐飞虱所有虫态发生反应，不与稻田其它主要昆虫和蜘蛛发生交叉反应，其中以3H3和4B8的特异性更高。SDS-聚丙烯酰胺凝胶电泳和Western blot印迹分析表明，3B2和3B11只与褐飞虱287kDa的多肽结合，3H3和4B8与215、199和167kDa的三条多肽具有亲和性。在25℃下，1头褐飞虱怀卵雌成虫与3B2、3B11、3H3和4B8结合的多肽在拟环纹豹蛛肠道中的可测定时间分别为1.460d、0.477d、0.367d和1.913d，分别相当于35.04h、11.45h、8.81h和45.91h。4种单克隆抗体均为IgG₃亚类。根据上述结果，初步确定4B8用于捕食作用研究。 2)应用杂交瘤技术，制备了5株多种弹尾虫复合体的单克隆抗体细胞株，分别命名为1B5、2F10、2G12、3D8和4F8。5种单克隆抗体的效价均达到1.024×10⁸倍以上。它们只与弹尾虫发生反应，不与稻田中被检测的其他昆虫种类和蜘蛛发生交叉反应。应用免疫双扩散法鉴定抗体类型及亚类的结果表明，1B5、2F10、3D8和4F8为IgG₃亚类，2G12为IgG₁亚类。 3)应用褐飞虱单克隆抗体3B2和4B8比较了直接、间接和多种抗体夹心ELISA方法。结果表明：捕食者浓度对直接和间接ELISA检测结果的影响大于抗体夹心法。当拟环纹豹蛛浓度为0.042mg/ml（鲜重）时，直接和间接ELISA检测的OD值比不加拟环纹豹蛛的阳性对照仍降低5％以上。而当捕食者浓度为0.214mg/ml时，对同种单抗、异种单抗和两种单抗混合夹心ELISA检测OD值的影响均在5％以下。当捕食者浓度为0.214mg/ml时，抗体夹心ELISA的检测灵敏度高于直接和间接ELISA。同种单抗、异种单抗和两种单抗混合夹心ELISA检测灵敏度均为16.91ng/ml（1/17509头褐飞虱怀卵雌成虫/ml），直接和间接ELISA分别为67.59ng/ml（1/4378头/ml）和135.2ng/ml（1/2189头/ml）。建立了4B8稀释1000倍（28.130μg/ml），捕食者匀浆液定容至100ml/头，HRP-4B8稀释4000倍（1.045μg/ml）的夹心ELISA用于检测捕食者对褐飞虱的捕食作用。在此检测系统下，检测的灵敏度为每头拟环纹豹蛛含1石glpg褐飞虱猎物蛋白讨当于1／175头褐飞虱怀卵雌成虫人非目标抗原对检测结果的影响小于5％。 4)应用弹尾虫单克隆抗体 IBS、ZF10、2G12、3D8和 4F8比较了间接 ELISA和抗体夹心ELlsA方法。结果表明：2o12抗体夹心ELISA检测抗原的OD值和阴性对照的相当，无法使用。非目标抗原对间接ELISA的影响显著大于抗体夹心ELISA。当拟环纹豹蛛浓度为 0．428mg／mlD，对＊、ZF10、3D8和 4F8抗体夹心ELISA检测OD值的影响均在20％以下，对间接ELISA检测OD值的影响则在75％以上。在此非目标抗原浓度下，4种单克隆抗体的夹心ELISA检测的灵敏度均为89．727ng／ml （／！04头弹尾虫／ml），IBS、3D8和 4F8间接 ELISA的检坝灵敏度均为179．45ng／ml（l／52头／ml），ZF10间接ELISA的检狈灵敏度为89．727ng／ml（l／104头／ml卜建立了ZF10稀释4000倍o4．193pg／ml），捕食者匀浆液定容至50ml／头，H班上 稀释1500倍Q人63P咖l)的夹心ELISA检测系统用于检测捕食者对弹尾虫的捕食作用。在此检测系统下，检测的灵敏度为每头拟环纹豹蛛含4．487pg弹尾虫猎物蛋白K当于1a头弹尾虫L 非目标抗原对检测结果的影口向在20％以下。 5)应用单克隆抗体4B8研究了褐飞虱在拟环纹豹蛛体内的消化速率与温度、捕食量和捕食者个体大小的关系。结果表明，在温度为16oC、22t、25C、28C、34OC和 37’C及捕食量为 1头、2头。3头和 4头褐飞虱怀卵雌成虫的处理下，在 0～sd的消化时间内，猎物在拟环纹豹蛛肠道中均呈指数降解，猎物的残留率O)与消化时间（）可用y飞xp…V)进行拟合，所有24组处理的相关系数均达显著水平。温度是影响猎物降解速率的主要因子。当温度为16C时，4组捕食量处理的可测定时间分别为6，279d、6．686d、9．593d和 15．sl4d。当温度高于30C时，猎物的可狈定时间均短于id。在16℃～37oC的范围内，在捕食量为l、2、3、4头褐飞虱怀卵雌成虫的处理下，猎物的可测定时间（tDP）与温度厂)间的关系用方程＊广atCXP（化叮）拟合的相关系数均达显著水平。捕食量对猎物可测定时间也有显著的影响，除了 16oC和 28oC的处理外，其它温度处理的猎物可测定时间与捕食的猎物量呈线性相关。在 25 oC下，当捕食量从1头增至4头时，猎物的可测定时间从1．890d延长至5．980d。在体重为0．of－0J 头的范围内，捕食者个体大小对猎物消化速率的影，响不显著。根据试验结果，修正了 Sopp（1992）估讨捕食量的方程为 R＝Q0d／f(．1更多还原

【Abstract】 Using the monoclonal antibodies developed respectively against rice planthoppers, brown planthopper, Nilaparvcua lugens (St釯), white-backed planthopper, Sogatella furc~fera (Horv醫h) and collembola, field predation of the major natural enemies of rice planthoppers and collembola were evaluated. The results were as follows: 1) Four monoclonal antibodies (McAbs), namely 3B2, 3B 11, 3H3 and 4B8, were developed against N. lugens using hybridoma technique. All McAbs had high absorption values even they were diluted over 4.096x 106 times. They reacted with all the stages of N. lugens while not cross-reacted with other insect pests and predators in the rice paddy. The 3H3 and 4B8 were the most specific. SDS-PAGE and Western blot analysis indicated that 3B2, 3B 11 bound specifically to one polypeptide with molecular weight of 287 kDa and 3113, 4B8 reacted with the polypeptides with molecular weights of 215, 199 and 167 kDa. At the temperature of 25 ~慍, the 3B2, 3B 11, 3H3 and 4B8 could detect the epitopes after one female of N. lugens had been ingested by Pardosa pseudoannulate for about 35.04h, 1l.45h, 8.81h and 45.91h, respectively. Immunodiffusion showed that all McAbs belong to IgG3 subclass. Based on the results as above, 4B8 was chosen to study the interactions between N. lugens and their predators in the field. 2) Five McAbs, namely 1B5, 2F10, 2Gl2, 3D8 and 4F8, were developed against the collembola using hybridoma technique. They had high absorption values even they were diluted over 1.024 X 108 times. All of the McAbs only reacted with the collembola while not cross-reacted with other insect pests and predators. Immunodiffusion showed that the 1 B5, 2F 10, 3D8 and 4F8 belong to IgG3 subclass, while the 2G12 belong to IgG?subclass. 3) Three variations of ELISA, direct, indirect and double antibody sandwich methods of ELISA were examined for their abilities to detect N. lugens antigens in the predator guts. The results showed that the direct and indirect ELISA were affected more strongly by the not-target antigens than on the double antibody sandwich ELISA. When the negative effects of the non-target were about 5%, the concentration of the non-target antigens was 0.042mg/mi with the direct and indirect ELISA methods and 0.214mg/mi with the double antibody sandwich ELISA methods respectively. The double antibody sandwich ELISA methods were the most sensitive when the 140 concentration of the non-target antigens was 0.214mg/mi. They could detect about 16.91 nglml (1/17509 individuals of the female/mi) of N. lugens antigens compared with 67.59ng/ml (1/4378 female/mI) for the indirect ELISA method and 135.l8ng/ml (1/2189 female/mi) for the direct method. The double antibody sandwich method of ELISA was established with the 4138 diluted 1000 times (28. 1 30 jig/mI), enzyme-linked antibody HRP-4B8 diluted 4000 times (1.045jig/ml) and sample diluted 100 times (1 OOml/individual) for detection of N. lugens antigens. The negative effect of the non-target antigens was lower than 5% and about 1.691 jig prey antigens (1/175 female) in a predator gut could be detected under this detecting system. 4) Indirect methods and double antibody sandwich methods of ELISA with 1 B5, 2F10, 2012, 3D8 and 4F8 were examined for their abilities to detect the collemboia antigens in predator guts. The results showed that the double antibody sandwich ELISA with 2012 was too insensitive to be更多还原