拟南芥纤维素合酶的抗体制备与检测
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国家“973”前期项目(2010CB134401)

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    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-AtCesAs,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-CESA7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。

    Abstract:

    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 E.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.

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陶章生,徐 雯,张苗苗,彭良才,丰胜求.拟南芥纤维素合酶的抗体制备与检测[J].华中农业大学学报,2012,31(2):171-177

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  • 收稿日期:2011-03-22
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