马铃薯叶片特异性启动子克隆分析及其驱动的人白细胞介素-12表达载体构建

Cloning and Characterization of Leaf Specific Promoter from Potato(Solanum tuberosum L.) and its Application in Human IL12 Expression Vector Construction

利用植物生物反应器表达具有应用价值的药用蛋白是近年来生物技术研究热点。本研究利用PCR技术从马铃薯基因组中克隆其叶片组织特异性启动子Prbcs后,利用PLACE等数据库对序列进行启动子元件分析,接着通过重叠PCR、限制性内切酶酶切、体外连接、转化等技术,以双元载体pCambia-2301为骨架,构建Prbcs驱动的药用蛋白人白细胞介素12(hIL12)表达载体。结果显示,本研究成功从马铃薯基因组中扩增得到了623 bp大小的启动子片段,序列分析表明该片段含有典型的启动子元件如TATA-box,以及一些组织特异性的相关响应元件,符合组织特异性启动子的特征;成功获得了基于pCambia-2301的Prbcs驱动的hIL12植物表达载体。本研究获得的Ppatatin启动子及其驱动的hIL12表达载体,为提高hIL12在植物生物反应器中表达量以及优化马铃薯生物反应器打下了基础。

Expression of valuable pharmaceutical proteins using plant bioreactor was the research highlights of biotechnology. In this study, PCR technique was performed to amplify the Prbcs fragment using genomic DNA of potato as templates. The databases such as PLACE were used for analysis of promoter elements. Techniques of overlap PCR, restricted enzyme digestion, DNA ligation and transformation were performed to construct hIL12 expression vector driven by Prbcs using pCambia-2301 as basic vector. The results showed that the 623 bp of promoter fragment was obtained from potato genome. The sequence analysis showed that the typical promoter elements such as TATA-box and several tissue specific response elements were identified with the characteristics of tissue specific promoter. The hIL12 expression vector driven by Prbcs was then constructed successfully. The obtaining of Prbcs and hIL12 expression vector would play an important role in improving the expression level of hIL12 in plant bioreactor and optimizing the potato bioreactor.