三孢布拉氏霉菌番茄红素环化酶多克隆抗体的制备及鉴定

Preparation and Identification of Polyclone Antibody Against Blakeslea trispora CarRA

对三孢布拉氏霉菌番茄红素环化酶(CarRA)进行抗原表位分析并制备兔多克隆抗体。运用GOLDENKEY分子生物学软件对三孢布拉氏霉菌CarRA结构蛋白的抗原表位及其二级结构进行了分析比较,结合现有的抗原表位选取原则从中筛选了一段显示表位特征的氨基酸残基序列,应用固相合成法合成三孢布拉氏霉菌CarRA多肽,然后将其与KLH偶联制成免疫原,应用于动物免疫及多克隆抗体的制备,对该多抗与三孢布拉氏霉菌CarRA的反应性进行了检测。获得了三孢布拉氏霉菌CarRA的多抗,该抗体经间接酶联免疫吸附测定法检测其效价为1:32000,Dot blot检测在三个稀释度下都出现了目的条带,证实该抗体具有较好的反应性和特异性。成功制备了三孢布拉氏霉菌CarRA多抗,为进一步明确发酵促进剂的作用机制及其对三孢布拉氏霉菌代谢网络的影响,对CarRA进行定量以及对其相关功能进行深入研究提供了有利条件。

To analyze the epitope of Blakeslea trispora CarRA protein and prepare its polyclonal antibody. Combined with existing antigen epitope selection principle, the Blakeslea trispora CarRA full-length sequence was analyzed by using the bio-informatics software GOLDENKEY to detect the structure of amino acids and select its epitopes. The Blakeslea trispora CarRA peptides fragment was synthesized by solid-phase peptide synthesis method and purified by HPLC. The synthetic antigen was conjugated with the keyhole limpet hemoyanin （KLH）, and then rabbits were immunized with this antigen and the antibody was examined. The results show that the antibody titer is as high as 1：32000. Through Dot blot analysis, it was proved that this peptide antibodies could react with Blakeslea trispora CarRA. All these prove that the antibody has wonderful reaction and speciality. A CarRA-specific antibody was succeddfully developed, which lays foundation for further studies of fermentation accelerator mechanisms, effects on Blakeslea trispora metabolic networks, CarRA quantitative research and the function of CarRA.