水稻LRR型类受体蛋白激酶胞外区的原核表达及多克隆抗体制备

Prokaryotic Expression and Polyclonal Antibody Preparation of The Extracellular Domain About Rice LRR Receptor-like Protein Kinase

前期研究表明,水稻根尖细胞质膜类受体蛋白激酶OsRLK的表达受盐胁迫诱导.为了进一步研究该激酶的生理功能,通过反转录PCR得到OsRLK胞外区cDNA片段,将其亚克隆至pET29a原核表达载体并在大肠杆菌中实现了高表达,表达量约为细胞总蛋白的30%.重组蛋白经SDS-PAGE分离,染色切胶收集后,作为抗原免疫新西兰家兔,分离抗血清,经纯化得到1∶20000效价的多克隆抗体.Western blot结果显示,该抗体能特异识别在原核表达系统内表达的抗原,以及水稻根尖细胞质膜组分中的LRR型类受体蛋白激酶,并且在蛋白质水平证实该激酶为盐胁迫响应蛋白.

Previous study indicated that the expression of a LRR receptor-like protein kinase OsRLK in root tips of rice could be induced by salt stress. In order to study the functions of OsRLK, the extracellular fragment of OsRLK gene was obtained through RT-PCR. The target fragment was subcloned into pET29a, and the recombinant plasmid pET29a-RLK was transformed into E. coli BL21 （DE3）. The target fragment over-expressed in host strain, and the expression level was about 30% of the total cellular protein. After separated by SDS-PAGE, the target band was excised from the gel, and was used as an antigen to raise the antibody in New Zealand rabbits. After separation and purification of antiserum, the anti-OsRLK polyclonal antibody with the titers of 1 ： 20 000 was successfully prepared. Western blot analysis showed that the antibody could specifically recognize the expressed fragment in E. coli and the OsRLK protein in root tips from rice. In addition, for the first time, the OsRLK was confirmed as a salt stress responsive protein.