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| Prokaryotic Expression, Purification of NteIF2α and Preparation and Application of Polyclonal Antibody in Nicotiana tabacum |
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Abstract Eukaryotic translation initiation factor (eIF2α) plays an important role in the regulation of protein synthesis in eukaryotes. To investigate the function of eIF2α and explore its regulatory mechanism of protein synthesis in tobacco (Nicotiana tabacum), the recombinant vector pET15b-NteIF2α for prokaryotic expression of NteIF2α was constructed. The results showed that NteIF2α of tobacco K326 was successfully produced by Escherichia coli BL21-CodonPlus-(DE3)-RIPL strain under induction of 0.2 and 0.5 mmol/L isopropyl-β-d-thiogalactoside (IPTG) for 4 h at 28 ℃, and the obtained protein was identified by Western blot using anti-His antibody. Further, the expression condition was optimized and the soluble protein was obtained under induction of 0.2 mmol/L IPTG for 13 h at 16 ℃. Also, this protein was purified by Ni2+-NTA agarose colunm and gelfiltration column, and a single protein band was obtained. Finally, the polyclonal antibody of NteIF2α was prepared and its antibody titer was 1∶400 000 by enzyme-linked immuno sorbent assay (ELISA). The antibody was used for detection of NteIF2α in different organs of tobacco K326, and the results showed that the NteIF2α level was the highest in leaf and the lowest in root, stem and flower. Prokaryotic expression and purification of NteIF2α protein was successful, and highly specific NteIF2α antibody was prepared in this study. The results lay a foundation for further study the function of eIF2a and its roles in regulation of protein synthesis in plant.
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Received: 08 January 2016
Published: 24 December 2016
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