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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 is present in Metarhizium anisopliae and plays an adsorptive role in infecting the insect epidermis. In order to obtain high-purity MAD1 protein, a polyclonal antibody was prepared to understand its function. In this study, the Mad1 gene was cloned from Metarhizium anisopliae and ligated into the prokaryotic expression vector for expression analysis. After induced by IPTG and metal chelation, The antibody was purified by chromatography and purified. The specificity of the antibody was detected by Western blotting. Western blot analysis showed that the antibody was highly specific, induced and purified, and the highly purified MAD1 protein product was obtained. The polyclonal antibody with a titer of 1:12 800 was obtained, which provided a good experimental material for functional verification and molecular detection of Mad1 gene .