Journal of Jianghan University(Natural Science Edition) ›› 2017, Vol. 45 ›› Issue (1): 77-82.doi: 10.16389/j.cnki.cn42-1737/n.2017.01.013

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Construction of Recombinant Human Pramlintide Expression Vector and Its Expression in E. coli

YANG Wei1,2,WU Hongmei2,LI Jianan*1   

  1. 1. School of Life Sciences,Jianghan University,Wuhan 430056,Hubei,China;2. Technique Center of Wuhan Hiteck Biological Pharma Co.,Ltd.,Wuhan 430056,Hubei,China
  • Online:2017-02-28 Published:2017-02-28
  • Contact: LI Jianan

Abstract: Objective To construct the recombination human pramlintide prokaryotic expression vector and induce the expression of pramlintide in E. coli . Methods The human nature pramlintide gene whose genetic codon mutated to prokaryotic preference codon were chemically synthesized. The gene was inserted into pET-39b+ expression vector with double enzyme( KpnI、HindIII )digest to construct the recombination plasmid pET-39b+[Pramlintide],and then,the recombination plasmid was transferred into E. coli BL21 to construct high expression genetic engineering bacteria of pramlintide which was inducted with isopropyl β-D-thiogalactoside(IPTG). Results Pramlintide was inserted in plasmid at correct site,and gene sequencing indicated that the sequence of pramlintide was the same as exspected. Upon induction with 0. 1 mmol/L IPTG for 3 hours,the recombination protein was expressed in E. coli BL21 cells at 37 ℃ ,The expression of pramlintide reached the highest level and large amounts of recombination protein accumulated in cytoplasm. Conclusion The expression vector pET- 39b + [Pramlintide] was successfully constructed. Upon induction with IPTG,large amounts of recombination protein accumulated in E. coli BL21 cells. It will establish a foundation for producing large amounts of pramlintide in the future.

Key words: pramlintide, codon mutation, prokaryotic expression system, induced expression

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