蝶蛹金小蜂卵黄蛋白单克隆抗体的制备及其应用方法的建立  

Development of monoclonal antibodies to the vitellin in an endoparasitoid,Pteromalus puparum(Hymenoptera: Pteromalidae) and its application methods

作　　者：董胜张[1] 高秀云[1] 程正贤[2] 胡萃[1] 叶恭银[1] 

机构地区：[1]浙江大学昆虫科学研究所,杭州310029 [2]浙江大学生物技术研究所,杭州310029

出　　处：《昆虫学报》2007年第9期871-877,共7页Acta Entomologica Sinica

基　　金：国家自然科学基金项目(30270899);国家"973"项目(2006CB102005);教育部优秀人才支持计划(NCET-05-0513)

摘　　要：为深入研究寄生蜂卵黄发生及其内分泌调控，特采用杂交瘤细胞技术，制备4株能稳定分泌抗蝶蛹金小蜂Pteromalus puparum卵黄蛋白（vitellin，Vt）的单克隆抗体（mAb），即PpVtmAb1，PpVtmAb2，PpVtmAb3和PpVtmAb4。这4株单克隆抗体的重链和轻链的亚类均分别为IgG1和κ类型，不仅特异性识别Vt，而且识别雌蜂血淋巴中卵黄原蛋白（viteUogenin，Vg），但与雄蜂体液无反应。通过比较4种不同的ELISA方法，确定了微量检测蝶蛹金小蜂体内Vg／Vt的最适ELISA法，即双夹心ELISA法。该方法可用于单头雌蜂体内Vg／Vt的检测，其检测灵敏度为20ng／mL。用Western免疫印迹的方法证实了该蜂Vg的合成始于刚羽化的成虫，并在羽化后12～36h内含量达到高峰。In order to study vitellogenesis and its endocrine regulation, we used hybridoma techniques and developed four monoclonal antibodies to Pteromalus puparum soluble yolk proteins, named as PpVt mAb1, PpVt mAb2, PpVt mAb3, and PpVt mAb4, respectively. The four antibodies, each with a IgG1 heavy chain and a κ light chain, had high specificity and affinity not only to the ovarian vitellin （Vt）, but also to female hemolymph vitellogenin （Vg）. However, they showed no immunological reaction with the male substances. The indirect double antibody sandwich enzyme-linked immunosorbent assay （ELISA） was regarded as the most sensitive and accurate to measure Vg/Vt in the wasps, compared with other three methods of ELISA, namely, direct, indirect and double antibody sandwich ELISA. With this ELISA method it is possible to detect the Vg/ Vt titer in a single wasp with the sensitivity of 20 ng/mL. The Western blot analysis indicated that the Vg synthesis initiated just after eclosion, and peaked to high levels within 12 and 36 h after eclosion.

关 键 词：蝶蛹金小蜂 卵黄蛋白 单克隆抗体 双夹心ELISA 

分 类 号：Q965[生物学—昆虫学]