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Frontiers of Agriculture in China

ISSN 1673-7334

ISSN 1673-744X(Online)

CN 11-5729/S

Front Agric Chin    2011, Vol. 5 Issue (4) : 437-442    https://doi.org/10.1007/s11703-011-1146-7
RESEARCH ARTICLE
Cloning and prokaryotic expression of TaE3 from wheat and preparation of antiserum
Yunwei ZHANG, Xiang GAO, Shengfang HAN, Dongmei WANG()
College of Life Science, Agricultural University of Hebei, Baoding 071001, China
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Abstract

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.

Keywords E3 ubiquitin ligase (E3)      wheat      prokaryotic expression      western blot analysis     
Corresponding Author(s): WANG Dongmei,Email:dongmeiwang63@hotmail.com   
Issue Date: 05 December 2011
 Cite this article:   
Yunwei ZHANG,Xiang GAO,Shengfang HAN, et al. Cloning and prokaryotic expression of TaE3 from wheat and preparation of antiserum[J]. Front Agric Chin, 2011, 5(4): 437-442.
 URL:  
https://academic.hep.com.cn/fag/EN/10.1007/s11703-011-1146-7
https://academic.hep.com.cn/fag/EN/Y2011/V5/I4/437
Fig.1  Integrity detection of total RNA. Note: 1, 2 and 3 are the same RNA samples of wheat.
Fig.2  Amplification of gene. Note: M represents DNA marker; 1 represents the amplification products of RT-PCR from wheat gene.
Fig.3  Restriction enzyme analysis of pET30a-GST--His recombinant plasmid. M represents DNA marker. 1 represents the integrated pET30a-GST--His plasmid; 2 represents the enzyme digestion fractions of pET30a-GST--His by RI and dIII.
Fig.4  Induced expression of pET30a-GST--His in BL21. Note: M stands for marker, 1-5 stands for supernatant with 0.25 mmol/L IPTG induced at 0 h, 2 h, 4 h, 6 h and overnight respectively and 6-10 stands for supernatant,induced overnight by 0 mmol/L, 0.5 mmol/L, 0.25 mmol/L, 0.125 mmol/L, 0.1 mmol/L IPTG, respectively.
Fig.5  Western blotting verification of TaE3 protein. 1 and 2 stand for His-tag antibody against the supernatant of the bacterial lyses and negative control, respectively.
Fig.6  Purify of TaE3 fusion protein. 1 represents the bacterial lyses supernatant, 2 represents the supernatant after purified by Ni-NTA affinity chromatography, 3-4 represent the supernatant was washed twice by the wash buffer (15 mmol/L imidazole), 5-7 represent the supernatant was washed third times by the wash buffer (300 mmol/L imidazole); and M stands for marker.
Fig.7  ELISA detection of antiserum titer
Fig.8  Western blotting verification of anti-TaE3. Note: M stands for marker, 1 stands for TaE3 fusion protein and 2 stands for negative control.
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