GLP－1毕赤酵母表达载体的构建及重组菌株的筛选

摘要

In this paper,a human glucagon like peptide(GLP)-1 was selected as the study object. The GLP-1 gene was synthesized by the whole gene sequence,and the pUC57 plasmid was obtained by restriction enzyme XbaI and XhoI double enzyme digestion.With XbaI and XhoI double enzymes cutting expression plasmid pPICZαA, the target gene connected to pPICZαA plasmid with T4 DNA ligase to construct the vector into Pichia pastoris GS115.Synthesis of the target gene sequence was 128bp.The positive recombinant was amplified by GLP-1 specific primers,and the fragments of 120bp were obtained. The specific fragments were recovered and sequenced.The sequence was found to be the GLP-1 gene, which was successfully transferred into Pichia pastoris.The result laid a solid foundation for the further study of the high efficient expression of GLP-1 in Pichia pastoris.%选择了一种人胰高血糖素样肽（ GLP ）－1作为研究对象，全基因合成GLP－1基因序列，用限制性内切酶XbaI和XhoI双酶切合成后的pUC57质粒获得目的基因。以XbaI 和XhoI 双酶切表达质粒pPICZαA，将目的基因与表达质粒pPICZαA用T4连接酶连接，构建pPICZαA－GLP－1质粒，转入毕赤酵母GS115。合成的目的基因序列为128bp，用GLP－1特异性引物扩增阳性重组子，得到120bp左右的片段。将此特异性片段扩增回收，测序发现确为GLP－1基因，表明成功转入毕赤酵母中。为进一步研究GLP－1在毕赤酵母中的高效表达奠定了坚实的基础。