茶树咖啡碱合成酶基因原核表达、及其抗体制备与鉴定

Prokaryotic Expression of Caffeine Synthase Gene( TCS1),Its Polyclonal Antibody Preparation and Identification

克隆出茶树咖啡碱合成酶基因,对其进行原核表达,并制备TCS1抗体,旨在从蛋白水平研究茶树体内TCS1的表达情况。根据Gen Bank登陆的TCS1基因的全长c DNA序列,找出其完整的ORF(开放阅读框),从茶树叶片c DNA中克隆了TCS1基因的开放阅读框,连接到p GEX-4T-2表达载体,经IPTG诱导表达重组蛋白p GEX-4T-2-TCS1。进行体外酶活检测后,亲和层析纯化重组蛋白,作为抗原免疫家兔,制备TCS1多克隆抗体。用ELISA方法检测抗体效价,Western blot检测抗体的特异性。通过优化诱导条件,得出重组蛋白的最佳表达条件为:30℃、4 h。诱导后的总蛋白、可溶性蛋白与包涵体蛋白均出现一条明显的外源蛋白条带。抗体经ELISA检测,效价为1∶2 000,Western blot检测表明抗体具有相对较好的特异性。构建了TCS1原核表达质粒,同时成功制备了抗TCS1的多克隆抗体。

Cloing and prokaryotic expressing of TCS1 （ Caffeine synthase 1 ） gene, preparing the antibody are the preliminary works for further research of the expression of TCS1 in tea plant. According to the cDNA full- length sequence of TCS1 from GenBank, we found out the complete ORF （ Open Reading Frame）. The open reading frame（ORF） of TCS1 gene from the cDNA of leaves of tea plant was amplified. The ORF was then ligated into the expression vector pGEX-4T-2. The recombinant protein was expressed by induction with IPTG. Then, we tested in vitro enzyme activity, and purified the recombinant protein subsequently by affinity chromatography. The TCS1 antibody was further refined by immunizing white rabbits with the purified protein. The antibody titer and specificity of the polyclonal antibody were confirmed by ELISA and Western blot. Induced by optimizing the conditions, the best expression condition of the recombinant protein was at 30℃ for 4 h. After induction of total protein, soluble protein and inclusion body protein showed an obvious foreign proteins stripes. ELISA detection of the antibody recommended dilution ratio of 1：2 000. We verified the favourable specific of the antibodies by western blot. The prokaryotic expression plasmid pGEX-4T-2-TCS1 was constructed, and the prepared polyclonal antibody had a high titer and good specificity.