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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(+)-AMLcDNA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(+)-AMLcDNA(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。
In order to prepare the polyclonal antibody of Alocasia macrorrhiza lectin (abbreviated AML), the prokaryotic expression vector of AML full length and partial fragments was constructed, and antisera were obtained by immunizing New Zealand male rabbits with the prokaryotic expressed AML fragment. The results showed that Rosetta (DE3), a prokaryotic host strain of prokaryotic expression vector pET30a (+) - AMLcDNA with full length of AML, decreased in a short time after addition of different concentrations of IPTG, Which was positively correlated with the concentration of IPTG added, while the expected fusion protein was not detected by SDS-PAGE electrophoresis. The prokaryotic expression of the prokaryotic expression vector pET30a (+) - AMLcDNA (270-613) containing the AML fragment in the host strain Rosetta ( DE3), can grow normally under the induction of IPTG, and can induce the fusion protein of the expected size. The fusion protein was purified by preparative SDS-PAGE as an antigen to immunize New Zealand male rabbits to obtain antiserum. Western blot was performed using the antiserum as a primary antibody, and the corresponding proteins were detected in tubers, leaves, petioles and induced cells The molecular weight of the AML band indicates that the prepared AML polyclonal antibody can be used to detect AML.