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

doi:10.1007/s11703-011-1144-9

Front Agric Chin

2011, Vol. 5

Issue (4) : 473-478 https://doi.org/10.1007/s11703-011-1144-9

RESEARCH ARTICLE

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

Lifeng ZHANG, Aihua YAN, Dong TIAN, Shengfang HAN, Dongmei WANG(

)

College of Life Science, Agricultural University of Hebei, Baoding 071001, China

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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

Corresponding Author(s): WANG Dongmei,Email:dongmeiwang63@hotmail.com

Issue Date: 05 December 2011

Cite this article:

Lifeng ZHANG,Aihua YAN,Dong TIAN, et al. Cloning and prokaryotic expression of translationally controlled tumor protein (TaTCTP) gene from wheat and preparation of antiserum[J]. Front Agric Chin, 2011, 5(4): 473-478.

URL:

https://academic.hep.com.cn/fag/EN/10.1007/s11703-011-1144-9
https://academic.hep.com.cn/fag/EN/Y2011/V5/I4/473

Fig.1 Integrity detection of total RNA of wheat leaves.

Fig.2 Amplification of gene. M is DNA marker; 1 means fragment.

Fig.3 Restriction enzyme analysis of pET30a-GST--His recombinant plasmid. M is DNA marker; 1 means digestion of pET30a-GST--His recombinant plasmid with RI and lI; 2 means the integrated pET30a-GST--His.

Fig.4 Exploration of prokaryotic expression conditions of TaTCTP. A: 1-6 mean supernatant, induced at 0 h, 1 h, 2 h, 4 h, 6 h and 20 h by 0.1 mmol/L IPTG treatment, respectively; 7–12 mean precipitate induced at 0 h, 1 h, 2 h, 4 h, 6 h and 20 h by 0.1 mmol/L IPTG, respectively. B: 1–5 mean supernatant induced at 0 h, 2 h, 4 h, 6 h and 20 h by 0.4 mmol/L IPTG treatment, respectively; 6-10 mean precipitate induced at 0 h, 2 h, 4 h, 6 h and 20 h by 0.4 mmol/L IPTG, respectively. C: 1–5 mean supernatant induced at 0 h, 2 h, 4 h, 6 h and 20 h by 0.8 mmol/L IPTG treatment, respectively; 6–10 mean precipitate induced at 0 h, 2 h, 4 h, 6 h and 20 h by 0.8 mmol/L IPTG, respectively. D: 1–4 mean supernatant induced at 0 h, 2 h, 4 h and 20 h by 1.0 mmol/L IPTG treatment, respectively; 5–8 mean precipitate induced at 0 h, 2 h, 4 h and 20 h by 1.0 mmol/L IPTG, respectively.

Fig.5 Purify of TaTCTP fusion protein. Note: 1-2 are bacterial lysis supernatant; 3-5 are wash buffer; 6-8 are elution buffer,respectively.

Fig.6 ELISA detection of antiserum titer

Fig.7 Western blotting verification of TaTCTP protein. Note: M is marker proteins with molecular masses in kilodaltons. 1-3 represent BSA, the purified recombinant protein TaTCTP, and wheat whole protein. M is from SDS-PAGE, Lanes 1-3 are from western bloting.

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