Construction and Identification of Polycistronic Expression Plasmids of Antifungal Peptide CGA-N46 Gene

China Biotechnology

2011, Vol. 31

Issue (12): 93-98 DOI:

Construction and Identification of Polycistronic Expression Plasmids of Antifungal Peptide CGA-N46 Gene

LI Rui-fang, XIONG Qian-cheng, ZHANG Zong-wu, HUANG Liang, WANG Bin

College of Bioengineering, Henan University of Technology, Zhengzhou 450001, China

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Abstract

Objective: To improve the expression of antifungal peptide CGA-N46, the polycistronic expression of CGA-N46 were studied. Methods: The recombinant cloning plasmids pT-CAN46, pT-3CAN46, pT-5CAN46 and pT-8CAN46 were constructed firstly, in which one copy, three copies, five copies and eight copies of heterogeneous gene fragment which was pET-30a rbs sequence-initiation codon-CGA-N46 encoding sequence-termination codon was recombined with cloning plasmid pEASY-Blunt respectively. And then the recombinant expression plasmids pET-CAN46, pET-3CAN46, pET-5CAN46 and pET-8CAN46 were constructed, in which one cistron, three cistrons, five cistrons and eight cistrons of CGA-N46 gene were recombined with expression plasmid pET-30a at the down stream of rbs sequence of it respectively. The recombinant expression plasmids were transformed into the competent strains of E. coli Rosetta. The expression efficiency of CGA-N46 was researched. Results: Under the induction of IPTG, CGA-N46 gene cistrons expressed the monomers of CGA-N46 and transferred into the periplasm space of host strains, The percentage of each expression cassete of CGA-N46 in total protein was 3.4%, 5.0%, 12.4%, 17.1%. The results showed that the production of CGA-N46 gene increased with the icreasing of its cistrons in expression vector, the production of 8 cistrons was the most. Conclusions: Polycistronic expression can overcome the shortage of fusion expression and tandem expression. The technology of construction of polycistronic expression plasmid simplified the traditional gene engineering construction method by using the same end of endonucleases Nhe I, Spe I and Xba I, and sets up a sound foundation for efficient gene engineering expression of peptides.

Key words： Antifungal peptide CGA-N46 Gene engineering expression of peptide Polycistronic expression Plasmid construction

Received: 06 August 2011 Published: 25 December 2011

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LI Rui-fang, XIONG Qian-cheng, ZHANG Zong-wu, HUANG Liang, WANG Bin. Construction and Identification of Polycistronic Expression Plasmids of Antifungal Peptide CGA-N46 Gene. China Biotechnology, 2011, 31(12): 93-98.

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https://manu60.magtech.com.cn/biotech/ OR https://manu60.magtech.com.cn/biotech/Y2011/V31/I12/93

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