拟南芥纤维素合酶的抗体制备与检测  

Preparation and detection of antibodies of cellulose synthase (CESA) in Arabidopsis

作　　者：陶章生[1,2,3] 徐雯[1,2,3] 张苗苗[1,2,3] 彭良才[1,2,3,4] 丰胜求[2,4] 

机构地区：[1]华中农业大学作物遗传改良国家重点实验室,武汉430070 [2]华中农业大学生物质与生物能源研究中心,武汉430070 [3]华中农业大学生命科学技术学院,武汉430070 [4]华中农业大学植物科学技术学院,武汉430070

出　　处：《华中农业大学学报》2012年第2期171-177,共7页Journal of Huazhong Agricultural University

基　　金：国家"973"前期项目(2010CB134401)

摘　　要：将拟南芥CesA家族基因的高变区克隆到融合表达载体pGEX-4T-3中,构建重组质粒pGEX-AtCe-sAs,在大肠杆菌JM109中经IPTG诱导表达谷胱甘肽巯基转移酶融合蛋白(GST-AtCESAs)。采用GST亲和层析法纯化GST-AtCESAs并制备了多克隆抗体。Western-blotting检测表明,抗体Anti-CESA4和Anti-CE-SA7存在明显的交叉反应,Anti-CESA1、Anti-CESA3、Anti-CESA6、Anti-CESA2、Anti-CESA5、Anti-CESA8均能在拟南芥原生质膜上检测到特异免疫条带,这为进一步深入研究拟南芥纤维素合成机制提供了有利条件。TPCR was used to amplify the high-frequent variable regions of AtCesA family genes,and then the PCR products were cloned into vector pGEX-4T-3, respectively. The fusion proteins composed of AtCESAs proteins and glutathione S transferase （GST-AtCESAs） in F.. coli JM109 were induced by IPTG. The GST-AtCESAs were purified using GST affinity chromatography, and polyclonal antibodies were prepared. Western-blotting analysis showed that specific bands of six antibodies Anti-CESA1, Anti- CESA3,Anti-CESA6, Anti-CESA2, Anti-CESA5 and Anti-CESA8 could be obviously detected in the plasma membrane of Arabidopsis thaliana, whereas cross-reaction bands of antibodies Anti-CESA7 and Anti-CESA4 were occurred, suggesting that the two antibodies were failed for further research. Preparation of Anti-CESAs antibodies is good for future research on mechanism of cellulose synthesis.

关 键 词：纤维素合酶 多克隆抗体 交叉反应 拟南芥 Western-blotting 

分 类 号：Q754[生物学—分子生物学]