摘要
目的应用AOX1和GAP双启动子共表达体系提高人胰岛素原在毕赤酵母中的表达量。方法用限制性内切酶EcoRⅠ和NotⅠ双酶切pMD18-T-HMPIDesB30质粒,回收含短C肽AAK并去掉B链第30位苏氨酸(T)的人胰岛素原类似物HMPIDesB3(0Human mini-proinsulin des B30)片段,插入pGAPZαA载体中,构建重组质粒pGAPZαA-HMPIDesB30,电转化至经质粒pPIC9K-HMPIDesB30异位整合的胰岛素原分泌菌株FJ-H(1pPIC9K-HMPIDesB30/GS115)中,应用抗生素法筛选高表达菌株,并进行发酵试验。结果重组质粒pGAPZαA-HMPIDesB30经双酶切及测序证实构建正确;经Zeocin筛选获得高表达量菌株FJ-H3。经30 L发酵罐发酵,FJ-H3菌株人胰岛素原的表达水平可达1.6 g/L,分别为FJ-H1菌株的1.6倍,FJ-H2菌株(pGAPZαA-HMP-IDesB30/GS115)的5.3倍;经质谱分析,胰蛋白酶酶切后的目的蛋白相对分子质量与理论值相符。结论利用AOX1和GAP双启动子在毕赤酵母中高效表达了人胰岛素原类似物HMPIDesB30,为实现胰岛素的规模化制备奠定了基础。
Objective To increase the expression level of human proinsulin in Pichia pastoris by double promoter （AOX1 and GAP） co-expression system. Methods Plasmid pMD18-T-HuPIDesB30 was digested with EcoR I and Not Ⅰ , and the cDNA fragment of human mini-proinsulin des B（HMPIDesB30） containing short C peptide AAK, in which the Thr30（T） gene at site 30 of B chain was deleted, was recovered and inserted into vector pGAPZc^A. The constructed recombinant plasmid pGAPZαA-HMPIDesB30 was transformed to proinsulin-secreting FJ-H1 strain integrated by another recombinant plasmid pPIC9K-HMPIDesB30, i.e. pPIC9K- HMPIDesB30 /GSll5, based on which the strain for high expression of proinsulin was screened with antibiotic and suhjected to fermentation test. Results Restriction analysis and sequencing proved that recombinant plasmid pGAPZctA-HMPIDesB3~ was constructed correctly. FJ-H3 strain for high expression of proinsulin was obtained by screening with Zeocin. The expression level of proinsulin in FJ-H3 strain fermented in 30 L fermenter reached 1. 6 g/L, which was 1. 6 times higher than that in FJ-Ht strain and 5. 3 times higher than that in FJ-H2 strain （pGAPZαA-HMPIDesB30/GS115）. Mass spectrometry showed that the relative molecular mass of target protein digested with trypsin was consistent with the theoretical value. Conclusion HMPIDesB30 was highly expressed in P. pastoris by using AOX1 and GAP double promoters.
出处
《中国生物制品学杂志》
CAS
CSCD
2012年第4期422-425,共4页
Chinese Journal of Biologicals
基金
国家"十一五"重大专项候选药物(2009ZX09103-683)
