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中国生物工程杂志

China Biotechnology
China Biotechnology  2011, Vol. 31 Issue (04): 1-6    DOI:
    
Improving The Secretory Expression of Vasonatrin Peptide in Pichia pastoris with Two Antibiotic Markers
FANG Cong, LI Peng, DING Yue-di, LEI Jian-yong, CHEN Yun, JIN Jian
School of Medicine and Pharmaceutics, Jiangnan University, Wuxi 214122, China
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Abstract  

Synthesised the Vasonatrin peptide coding gene according to the Vasonatrin peptide amino sequence and Pichia pastoris codon preference, and then the 5' end of Vasonatrin coding gene containing His tag marker was obtained by polymerase chain reaction (PCR) technology. The spliced gene was inserted into Pichia pastoris secretory expression vector pPIC9K and pPICZαB, linearized separately. First of all, transformed the linear pPIC9K-VNP into Pichia pastoris GS115 by electroporation, screened the excellent transformants on G418 plates. Similarly, transformed the linear pPICZαB-VNP into GS115/ pPIC9K-VNP, and then selected the transformants on Zeocin plates. The expression efficiency of the GS115/pPIC9K-VNP/pPICZαB-VNP strains was analyzed by Tricine-SDS-PAGE and Western blot. The results showed that the yield is about 70mg/L in shake flask, which is increased by 75% compared with the single antibiotic marker screening method. And the research of biological activity indicated that the purified peptides can inhibit lipopolysaccharide (LPS) to induce iNOS and TNF-α in vitro macrophages.



Key wordsVasonatrin peptide      Pichia pastoris secretory expression      Two antibiotic markers     
Received: 18 November 2010      Published: 26 April 2011
ZTFLH:  Q819  
Cite this article:

FANG Cong, LI Peng, DING Yue-di, LEI Jian-yong, CHEN Yun, JIN Jian. Improving The Secretory Expression of Vasonatrin Peptide in Pichia pastoris with Two Antibiotic Markers. China Biotechnology, 2011, 31(04): 1-6.

URL:

https://manu60.magtech.com.cn/biotech/     OR     https://manu60.magtech.com.cn/biotech/Y2011/V31/I04/1


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