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期 刊 ：植物保护学报 2010年

关键词：海芋凝集素；原核表达；多克隆抗体；

Keywords:Alocasia macrorrhiza lectin, prokaryotic expression, polyclonal antibody,

摘 要 ：为了制备海芋凝集素(Alocasia macrorrhiza lectin,简写AML)的多克隆抗体,分别构建了AML全长及部分片段的原核表达载体,并利用获得的原核表达的AML片段免疫新西兰雄兔获得抗血清。结果表明,含AML全长的原核表达载体pET30a (+) -AML cDNA的原核表达宿主菌株Rosetta (DE3),在加入不同浓度的诱导剂IPTG后,菌液浓度出现短暂增加后逐渐下降,且下降幅度与加入的IPTG浓度呈正相关,同时用SDS-PAGE电泳法未检测到预期融合蛋白的形成;而含AML片段的原核表达载体pET30a (+) -AML cDNA (270-613)的原核表达宿主菌株Rosetta (DE3),在IPTG诱导下能正常生长,且能诱导出预期大小的融合蛋白。利用制备型SDS-PAGE法纯化此融合蛋白作为抗原免疫新西兰雄兔获得抗血清,用该抗血清作为一抗进行Western blot,在海芋块茎、叶、叶柄和诱导菌体中均能检测到相应分子量的AML条带,表明制备的AML多克隆抗体可以用于检测AML。

Abstract:In order to prepare the polyclonal antibody of Alocasia macrorrhiza lectin (AML), the prokaryotic expression vectors containing partial and full length AML were constructed. The prokaryotic expressed partial AML fusion fragment was used as antigen to immune male New Zealand rabbit to produce antiserum. The results showed that the OD₆₀₀ of Rosetta (DE3) host cell with prokaryotic expression construct pET30a (+) -AML cDNA expressing the full length of AML increased shortly then decreased gradually, after the inducer IPTG was added. The higher the IPTG concentration was added, the lower the OD₆₀₀ of the broth was detected. Meanwhile no induced protein was detected by SDS-PAGE method. The OD₆₀₀ of Rosetta (DE3) host cell with prokaryotic expression construct pET30a (+) -AML cDNA (270-613) expressing partial of AML increased gradually, and the expected size fusion protein was detected by SDS-PAGE, under the induction of IPTG. The induced AML fragment was purified by preparation SDS-PAGE, and used as antigen to immune male New Zealand rabbit to produce polyclonal antibody of AML. By using this antiserum, the expected size protein was detected from the tuber, leaf, petiole of Alocasia macrorrhiza and the induced host cell containing pET30a (+) -AML cDNA (270-613) construct, through the Western blot, meaning that the prepared AML polyclonal antibody could be able to detect the AML specifically.

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