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期 刊 ：植物保护学报 2009年

关键词：亚洲玉米螟；酚氧化酶原；多克隆抗体；酶联免疫吸附；免疫电镜；

Keywords:Ostrinia furnacalis Guene, prophenoloxidase, polyclonal antibody, ELISA, immunoelectron microscopy,

摘 要 ：为探讨酚氧化酶原(PPO)的合成转运机制,采用硫酸铵沉淀、Blue Sepharose CL-6B亲和层析和Phenyl Sepharose CL-4B疏水层析等方法从亚洲玉米螟幼虫血淋巴中纯化得到PPO,纯化倍数为369.85,回收率为35.34%。采用免疫印迹和酶联免疫吸附进行纯化PPO的免疫原性及其多克隆抗体的效价测定,并以透射电镜检测PPO在亚洲玉米螟幼虫体壁和中肠中的分布。结果表明,纯化PPO蛋白全酶相对分子量约为158kD;免疫2只鼠后所得pAb间接ELISA效价均高达10^-5,其中第2号小鼠pAb效价最高为1∶ 4.08×10⁴;PPO多克隆抗体具有相对较高的特异性;在亚洲玉米螟5龄幼虫中肠和体壁组织中金颗粒呈团状和点状分布。

Abstract:To elucidate the mechanism of synthesis and transport of prophenoloxidase (PPO) in insect, PPO was isolated from the hemolymph of Ostinia furnacalis Guene larvae and purified to homogeneity by employing ammonium sulfate precipitation, Blue Sepharose CL-6B chromatography and Phenyl Sepharose CL-4B chromatography. The 369.85-fold purification and 35.34% recovery of activity were achieved, and the purified PPO exhibits a band on native PAGE with a molecular mass of 158kD. Balb/C mouse was injected with purified PPO to prepare polyclonal antibody. The antibody titer of two mouse is 10^-5, one of them is 1∶ 4.08×10⁴. Western blotting analysis also showed that we have got the effective polyclonal antibody of PPO. The antibody was used as a tool for the immunoelectron microscopy localization of PPO in the O.furnacalis larvae. The results showed that the colloidal gold particles were distributed in spotty group in the midgut and integument tissue of O.furnacalis, which indicated PPO existed in these tissue markedly.

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