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篇目详细内容 |
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【篇名】 |
Cloning and prokaryotic expression in TaCaM2-3 of wheat and preparation of antiserum |
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【刊名】 |
Frontiers of Agriculture in China |
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【刊名缩写】 |
Front. Agric. China |
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【ISSN】 |
1673-7334 |
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【EISSN】 |
1673-744X |
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【DOI】 |
10.1007/s11703-010-0015-0 |
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【出版社】 |
Higher Education Press and Springer-Verlag Berlin
Heidelberg |
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【出版年】 |
2010 |
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【卷期】 |
4
卷3期 |
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【页码】 |
317-322
页,共
6
页 |
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【作者】 |
Wei LIU;
Aihua YAN;
Chunyan HOU;
Dongmei WANG;
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【关键词】 |
TaCaM2-3 cloning and expression; western blotting analysis; preparation of antiserum |
【摘要】 |
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Multiple calmodulin (CaM) isoforms exist in plant organisms and vary by their primary structures of 148 amino acids. They have different expression patterns and/or target enzyme activation abilities. To further understand the biological significance of TaCaM isoforms, total RNA was isolated from mature leaves of wheat and then TaCaM2-3 gene was amplified by PCR after reverse transcription. The PCR product was generated into T-easy vector to subsequently sequence. Then the recombinant expression vector (pET28a-TaCaM2-3) was constructed and transformed into E. coli strain BL21 to obtain a high level expression vector of CaM. SDS-PAGE analysis showed that the recombinant E. coli could express an approximate 20？kD protein. A western blotting analysis showed an anti-CaM monoclonal antibody specifically bound to the 20？kD band of expressed product. TaCaM-II was purified by Ni-NTA affinity chromatography from recombinant bacterial lysate. TaCaM-II protein was used to immunize New Zealand white rabbits to produce a polyclonal antiserum. The specificity of the anti-TaCaM-II antiserum was successfully verified by western blotting analysis. |
