摘要
根据人胰岛素的氨基酸序列和酵母偏爱的密码子,采用基因半合成技术合成人胰岛素基因.将合成的胰岛素基因克隆到pP IK 9质粒,构建了毕赤(P ich ia)酵母重组表达载体pP IN S319,用B g lⅡ线性化后用电转化法导入毕赤酵母G S ll5菌株,经过营养筛选和PCR复筛,得到含人胰岛素基因的毕赤酵母工程菌株.经SDS-PAGR和密度扫描分析表明,合成的人胰岛素基因能够在毕赤酵母中高效分泌性表达,表达量可达0.563 m g/mL,表达产物无需转肽过程,只经过胰蛋白酶和羧肽酶B简单处理即得到优质重组人胰岛素,其体内活力约为30 IU/m g.
Insulin gene was synthesized according to the amino acid sequence of human insulin and yeast preferential codons. The insulin gene was cloned into pPIC9 vector and expression vector pPINS319 was constructed. The expression vector was digested with BgllⅠ and then used to transform Pichia Pastoris GSll5 by electroporation. The expression analysis showed that the insulin gene was able to expressed efficiently in Pichia Pastoris. SDS-PAGE and density scanning analysis indicated that the highest expression amout was about 0. 563 mg/mg. After treatment by trypsin and carboxypeptidase B, the endo-bioactivity of the recombinant in- sulin obtained was approximately 30 IU/mg.
出处
《南开大学学报（自然科学版）》
CAS
CSCD
北大核心
2006年第5期69-73,共5页
Acta Scientiarum Naturalium Universitatis Nankaiensis
基金
863项目(2002AA213061)
关键词
人胰岛素基因
毕赤酵母
分泌性表达
human insulingene
Pichia Pastoris
secretory expression
