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中国生物工程杂志

China Biotechnology
China Biotechnology  2015, Vol. 35 Issue (4): 54-59    DOI: 10.13523/j.cb.20150408
    
Prokaryotic Expression and Function Analysis of SoHb from Spinach
GUO Zhao-lai, BAI Xue-gui, YAN Jin-ping, CHEN Xuan-qin, LI Kun-zhi, XU Hui-ni
College of Life Science and Technology, Kunming University of Science and Technology, Kunming 650500, China
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Abstract  

To further investigate the function of non symbiotic hemoglobin of spinach (SoHb), the full length cDNA encoding SoHb was amplified by RT-PCR from spinach root and cloned into inducible expression vector pET32a. The recombinant prokaryotic expression plasmid was transformed into E.coli BL21 star (DE3) strain for genetic engineering strain. The transformed strain was induced with isopropyl-beta- -D-thiogalactoside (IPTG) for expressing fusion protein. SDS-PAGE analysis showed that the recombinant protein with a molecular mass about 38 kDa was highly expressed in E.coli and presented both in the supernatant and the pellet part of E.coli lysates. The pET32a-SoHb strain was more resistant to nitrosative stress than the pET32a empty vector strain. The supernatant was further purified by Ni2+ NTA affinity chromatography and immunized white mouses as antigen. The polyclonal antibody was obtained and analyzed by Western blot. It laid foundation for investigating the function of SoHb.



Key wordsNon-symbiotic hemoglobin      Escherichia coli expression      Nitrosative stress     
Received: 14 November 2014      Published: 25 April 2015
ZTFLH:  Q78  
Cite this article:

GUO Zhao-lai, BAI Xue-gui, YAN Jin-ping, CHEN Xuan-qin, LI Kun-zhi, XU Hui-ni. Prokaryotic Expression and Function Analysis of SoHb from Spinach. China Biotechnology, 2015, 35(4): 54-59.

URL:

https://manu60.magtech.com.cn/biotech/10.13523/j.cb.20150408     OR     https://manu60.magtech.com.cn/biotech/Y2015/V35/I4/54


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