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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。
In order to explore the synthesis and transport mechanism of phenoloxidase (PPO), PPO was purified from hemolymph of Asian corn borer, larvae by ammonium sulfate precipitation, Blue Sepharose CL-6B affinity chromatography and Phenyl Sepharose CL-4B hydrophobic chromatography , The purification fold was 369.85, the recovery rate was 35.34%. The immunogenicity of purified PPO and the titer of its polyclonal antibody were determined by Western blotting and enzyme-linked immunosorbent assay. The distribution of PPO in the body wall and midgut of the Asian corn borer, larvae, was examined by transmission electron microscopy. The results showed that the relative molecular weight of purified PPO protein holoenzyme was about 158 kD. The indirect ELISA titer of pAb was 10-5 after 2 immunized mice, in which the highest titer of pAb in mouse No. 2 was 1: 4.08 × 104; The polyclonal antibody has a relatively high specificity. The gold particles in the midgut and body wall tissues of the 5th instar larvae of Asian corn borer are agglomerated and dotted distribution.