定点突变的猪源性胰高血糖素样肽-2融合蛋白的原核表达与鉴定

doi:10.16431/j.cnki.1671-7236.2019.11.001

Abstract

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 D_{600 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

CLC Number:

HAN Fei, ZHAO Ruixiao, WANG Gang, JIANG Mingfeng. Prokaryotic Expression and Identification of Porcine Derived Glucagon Like Peptide-2 Fusion Protein with Site Directed Mutagenesis[J]. China Animal Husbandry and Veterinary Medicine, 2019, 46(11): 3137-3143.

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

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