铜绿微囊藻生物钟蛋白的表达纯化与抗体制备  

Expression, purification and antibody preparation of biological clock proteins in Microcystic aeruginosa

作　　者：罗志勇[1] 徐虹[1] 章军[1] 杨秀娜[1] 王立红[1] 

机构地区：[1]厦门大学生命科学学院细胞生物学与肿瘤细胞工程教育部重点实验室,厦门361005

出　　处：《高技术通讯》2006年第7期725-729,共5页Chinese High Technology Letters

基　　金：国家自然科学基金（40306024）资助项目.

摘　　要：为了获得融合表达的铜绿微囊藻（Microcystic aeruginosa）生物钟蛋白KaiA、KaiB、KaiC并制备其相应的多克隆抗体,将kaiA、kaiB、kaiC基因分别克隆到原核表达质粒pET-His中.重组质粒pET-His-KaiA,pET-His-KaiB和pET-His-KaiC经酶切和测序鉴定后,分别转化E.coli BL21（DE3）进行融合表达.经SDS-PAGE分析可知,融合表达的KaiA、KaiB和KaiC蛋白表达量可分别达到菌体总蛋白的25%、40%和20%.经亲和层析后融合蛋白KaiA和KaiB的纯度分别达95%和92%,而KaiC经胶回收纯化后纯度也可达93%.将纯化后的三种Kai蛋白作为抗原分别免疫小鼠制备多克隆抗体,经ELISA检测抗体滴度表明,制备的抗KaiA、抗KaiB和抗KaiC的多克隆抗体效价高,分别可达到1：50000、1：60000和1：100000.Western blotting结果表明：获得的多克隆抗体具有较高的效价,抗体能识别相应的Kai蛋白,具有较高的特异性,能用于铜绿微囊藻生物钟蛋白KaiA、KaiB和KaiC的表达节律检测.To obtain the fusion expressed circadian clock proteins KaiA, KaiB, KaiC of Microcystic aeruginosa and prepare their polyclonal antibodies, the genes encoding KaiA, KaiB and KaiC were cloned into pET-His vector respectively. Three recombinant expression plasmids pET-His-KaiA, pET-His-KaiB and pET-His-KaiC were verified by restriction endonudease analysis and sequencing,and then transformed into E. coli BL21 （DE3） respectively. Expression analysis by SDS-PAGE, each of Kai proteins was highly expressed and the expression amounts of KaiA, KaiB and KaiC in transformed strains were 25%, 40% and 20% respectively. The purities of KaiA and KaiB were up to 95% and 92% respectively by affinity chromatography, and the purity of KaiC was up to 93 % by gel extraction purification. The high purity target proteins were used to immunize mice to prepare polyclonal antibodies. By means of ELISA determination, the titers of anti-KaiA-IgG, anti-KaiB-IgG and anti-KaiC-IgG were 1：50000, 1：60000, 1 ： 100000 respectively. The results of Western blotting showed that the three prepared polyclonal antibodies could specifically bind to the relevant Kai proteins. These antibodies exhibit satisfactory specificity and reactivity against KaiA, KaiB and KaiC respectively, and can be used to examine the expression rhythms of circadian clock proteins KaiA, KaiB and KaiC in Microcystic aeruginosa.

关 键 词：铜绿微囊藻 生物钟蛋白 多克隆抗体 酶联免疫吸附 免疫印迹 

分 类 号：Q78[生物学—分子生物学] X524[环境科学与工程—环境工程]