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中国生物工程杂志

China Biotechnology
China Biotechnology
China Biotechnology  2011, Vol. 31 Issue (8): 29-34    DOI:
    
Construction of Trypsin Inhibitor KSTI3 Gene New Eukaryotic Expression System and Expression in Dunaliella salina
XU Wen-qi, CHAI Xiao-jie, ZHANG Ting, DAI Jing-yu, ZHANG Xiao-lin
Key Laboratory of Marine Bio-resources Restoration and Habitat Reparation in Liaoning Province,Dalian Ocean University, Dalian 116023,China
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Abstract  

To construct cloning vector pMDNos,terminator Nos was amplified from the template pBI121 plasmid by PCR. pMDKN vector was formed by combination between pMDNos fragment and KSTI3 from plasmid pMDKSTI3 by restriction enzyme digestion. Finally,promoter CaMV35S and chloramphenicol resistance gene Cat cloned from pCAMBIA2201 were all introduced to the vector pMDKN. DNA sequencing result showed that KSTI3 gene,promoter CaMV35S ,terminator Nos and Cat gene were completely consistent with the original sequence,so new eukaryotic expression vector pMDCKN-Cat was successfully constructed. pMDCKN-Cat was transformed into Dunaliella salina ( D. salina )with LiAc/PEG method,and recombinant D. salina was selected under chloramphenicol pressure and by PCR detection. Western blotting ultimately showed that a clear strip with the molecular weight of 20.1kDa appeared on the nitrocellulose membrane,and proved that trypsin inhibitor KSTI3 was expressed in D. salina cells.

Key words： Trypsin inhibitor KSTI3 gene      Dunaliella salina      LiAc/PEG mediated method      Eukaryotic expression system     
Received: 03 February 2011      Published: 25 August 2011
ZTFLH:  Q786  
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Cite this article:

XU Wen-qi, CHAI Xiao-jie, ZHANG Ting, DAI Jing-yu, ZHANG Xiao-lin. Construction of Trypsin Inhibitor KSTI3 Gene New Eukaryotic Expression System and Expression in Dunaliella salina. China Biotechnology, 2011, 31(8): 29-34.

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https://manu60.magtech.com.cn/biotech/     OR     https://manu60.magtech.com.cn/biotech/Y2011/V31/I8/29


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