Cloning and prokaryotic expression of TaE3 from wheat and preparation of antiserum

Autor: Shengfang Han, Dongmei Wang, Yunwei Zhang, Xiang Gao
Rok vydání: 2011
Předmět:
Zdroj: Frontiers of Agriculture in China. 5:437-442
ISSN: 1673-744X
1673-7334
Popis: The E3 ubiquitin ligase is a multi-functional protein that performs vital roles, particularly in various stress environment. To further understand the biological significance of E3 ubiquitin ligase gene from wheat (TaE3), total RNA was isolated from wheat leaves and then TaE3 gene was amplified by PCR after reverse transcription. The PCR product was cloned into PMD19-T vector to sequence subsequently. And then the recombinant expression vector (pET30a-GST-TaE3-His) was constructed and transformed into E. coli strain BL21 (DE3). SDS-PAGE analysis showed that the recombinant E. coli could express a proximate 43 kDa protein. TaE3 fusion protein was purified by Ni-NTA affinity chromatography from recombinant bacterial lysate and was used to immunize rabbit to produce polyclonal antibody. The titer and specificity of the anti-TaE3 antibody were successfully detected by indirect ELISA and western blot analysis.
Databáze: OpenAIRE
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