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Preparation and Preliminary Application of Polyclonal Antibody of NdhO Protein in the Cyanobacterium Synechocystis sp. Strain PCC 6803 |
ZHAO Jiao-hong, GAO Fu-dan, WEI Lan-zhen, WANG Quan-xi, MA Wei-min |
College of Life and Environment Sciences, Shanghai Normal University, Shanghai 200234, China |
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Abstract Cyanobacterial NADPH dehydrogenase (NDH-1) is an important photosynthetic membrane protein complex, and is essential to CO2 uptake, cyclic electron transport around photosystem I and cellular respiration. This enzyme accepts electrons from NADPH and consists of at least 17 subunits, i.e., NdhA to NdhQ. Recently, an ndhO gene inactivation mutant, ΔndhO, has also successfully been obtained. However, little is known regarding the functional roles of NdhO subunit in cyanobacteria. Therefore, the encoding gene, ndhO, was PCR amplified from the unicellular cyanobacterium Synechocystis sp. strain PCC 6803, the expression plasmid pET32a(+)-ndhO was constructed and transformed into BL21(DE3)pLysS, and the expression of NdhO protein was induced by IPTG. After purification, the fusion protein pET-NdhO was used to immunize Japanese white rabbit to obtain the polyclonal antibody. The titer of the polyclonal antibody was detected by ELISA and its specificity was analyzed by immunoblotting. The titer of polyclonal antibody was found to be up to 1 ∶ 1 025 000, and thus possessed a high specificity. Further, immunoblotting results using the polyclonal antibody showed the presence of NdhO in active NDH-1 mediumcomplex, and not active NDH-1 supercomplex. Therefore, the antibody of NdhO obtained will further help us to reveal the functional roles of cyanobacterial NdhO subunit.
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Received: 03 February 2011
Published: 28 June 2011
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Corresponding Authors:
Weimin Ma
E-mail: wma@shnu.edu.cn
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