金龟子绿僵菌黏着蛋白MAD1原核表达及其抗体的制备

Prokaryocyte Expression and Antibody Preparation of Mad1 Gene Came form Metarhizium anisopliae

Mad1基因存在于金龟子绿僵菌(Metarhizium anisopliae),在侵染昆虫表皮时起到了吸附性作用。为获得高纯度MAD1蛋白,制备多克隆抗体,以便深入了解其功能,本研究从金龟子绿僵菌中克隆出Mad1基因并连接到原核表达载体对其进行表达形式分析,经IPTG诱导及金属螯合层析纯化免疫家兔制备抗体,最后利用Western印记检测抗体特异性。经Western印记检测表明抗体特异性极高,诱导表达及纯化制备出高纯度MAD1蛋白产物,并获得效价为1∶12 800的多克隆抗体,为Mad1基因功能验证及分子检测提供良好的实验材料。

Mad1 gene was ubiquitously exist in Metarhizium anisopliae, and responsible for adhering to insectsurface. To obtain high purity protein of MAD1 and its polyclonal antibody for studying the function of Mad1 gene, itwas cloned from M. anisopliae by RT-PCR, and then connected into the prokaryotic expression vector for the expres-sion. The protein was induced by IPTG and purified by metal chelate affinity, and then rabbits were immunized forantibody preparation. The specificity of polyclonal antibody were detected by Western-blot and indirect ELISA. Theresults indicated that high specificity antibody was obtained. The antibody titer was 1：12800, which will give goodexperimental materials for Mad1 gene function research and detection.