China Animal Husbandry and Veterinary Medicine ›› 2019, Vol. 46 ›› Issue (11): 3137-3143.doi: 10.16431/j.cnki.1671-7236.2019.11.001

• Biotechnology • Previous Articles     Next Articles

Prokaryotic Expression and Identification of Porcine Derived Glucagon Like Peptide-2 Fusion Protein with Site Directed Mutagenesis

HAN Fei1, ZHAO Ruixiao1, WANG Gang2, JIANG Mingfeng1   

  1. 1. Institute of Qinghai-Tibet Plateau, Southwest Minzu University, Chengdu 610041, China;
    2. National Engineering Research Center for Biomaterials, Sichuan University, Chengdu 610064, China
  • Received:2019-05-05 Online:2019-11-20 Published:2019-11-25

Abstract: In order to improve the production efficiency of GLP-2 to meet the application needs of animal husbandry,the porcine derived[Gly2]GLP-2(p[Gly2]GLP-2) was induced by site directed mutagenesis using genetic engineering technology.After replacing alanine at the second amino end of GLP-2 with glycine residue,the codon sequence of p[Gly2]GLP-2 was optimized according to the codon preference of Escherichia coli,and enterokinase recognition sites were added to the N end of the target peptide sequence to synthesize the gene sequence.Prokaryotic recombinant expression vector p[Gly2]GLP-2-pET-40b(+) was constructed by connecting Kpn Ⅰ and Xho Ⅰ digestion sites to pET-40b(+).The recombinant plasmid was transformed in E.coli BL21(DE3) competent cell to induced the expression of recombinant protein.The optimum expression parameters were that 0.2 mmol/L IPTG was used to induce the expression of p[Gly2]GLP-2-pET-40b(+) recombinant plasmid for 5 h when the bacterial solution D600 nm value was 0.6,and the recombinant strain with high expression of p[Gly2]GLP-2 was finally obtained.High purity p[Gly2]GLP-2 fusion protein was obtained by purification with nickel ion affinity chromatography column and gradient elution,which laid a foundation for further functional research and application in animal husbandry.

Key words: GLP-2; prokaryotic expression; induction conditions optimization; affinity chromatography; gradient elute

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