Cloning and prokaryotic expression of translationally controlled tumor protein (TaTCTP) gene from wheat and preparation of antiserum

Abstract

The translationally controlled tumor protein (TCTP) is a multi-functioning protein that performs vital roles, particularly in various complicated life processes. To further understand the biological function of translationally controlled tumor protein (TaTCTP) gene from wheat, total RNA was isolated from wheat leaves and then TaTCTP gene was amplified by PCR after reverse transcription by using the anchored primers oligo(dT)₁₈. And then TaTCTP gene was connected into PMD19-T vector for sequence. The recombinant expression vector (pET30a-GST-TaTCTP-His) was constructed and transformed into E. coli strain Rosetta (DE3) subsequently, then a proximate 20 kDa protein in Rosetta (DE3) was expressed and characterized by SDS-PAGE. Moreover, TaTCTP 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-TaTCTP antibody were successfully detected by indirect ELISA and Western blot analysis.

Keywords

translationally controlled tumor protein (TCTP) / wheat / prokaryotic expression / Western blot analysis

References

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Acknowledgements

This research was supported by the National Natural Science Foundation of China (Nos. 30671244, 31171472), the Key Basic Research Project of Applied Basic Research Program of Hebei Province, China (No. 08965505D), and the Natural Science Foundation of Hebei Province, China (Nos. C2007000515, C2010000787).

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2014 Higher Education Press and Springer-Verlag Berlin Heidelberg