作　　者： ; ; ; ; ; ; ; ;

机构地区： 遵义医学院

出　　处： 《生物技术通报》 2010年第3期168-172,共5页

摘　　要： 构建重组hIL-10真核表达载体,探索在毕赤酵母中的表达,为进一步研究hIL-10生物学功能及临床应用奠定基础。PCR扩增目的基因hIL-10cDNA,经EcoRI/XbaI双酶切后,将其亚克隆至同样双酶切的载体pPICZaA中,构建表达质粒pPICZaA-hIL-10;电转化法,将其转入毕赤酵母SMD1168,甲醇诱导Mut+型转化子基因表达;对目的蛋白进行检测及纯化。扩增出了目的基因hIL-10cDNA,重组质粒pPICZaA-hIL-10经酶切鉴定和序列测定正确;表达产物经SDS-PAGE分析在42.0kD处可见较浓染条带,Westernblotting分析可见特异条带;ELISA检测72h上清液中目的蛋白浓度可达1.973mg/L;纯化后目的蛋白纯度达67.0%。实现了重组hIL-10在毕赤酵母SMD1168中的成功表达和初步纯化。 It was to construct recombinant hlL-10 eukaryotic expression vector and explore its expression in Pichia pastoris SMDl168,which could lay the foundation for further studying hlL-10 biological function and clinical application. Interest gene hlL- 10cDNA was amplified by PCR and double-digested by the EcoR I / Xba I,and then was subcloned to pPICZaA which was digested by the same enzyme to construct expression plasmid pPICZaA-hIL-10. The recombinant plasmid was transformed into Pichia SMD1168 by electroporation. Interest protein in Mut^＋ transformants was induced expression with using methanol and purified. Results indicated that the gene hlL-10 cDNA was obtained, and recombinant pPICZaA-hIL-10 was corrected by digested identification and sequencing. The ex- pression supernatant showed a strong bands and specific bands at 42. 0 kD through SDS-PAGE and Western blotting analysis. Interest protein concentration after 72 h was up to 1. 973 mg/L by ELISA. Interest protein purity reached 67.0% after purification. Therefore,it proved that the recombinant hlL-lO in Pichia SMD1168 was expressed successfully and purified preliminarily.

关 键 词： 质粒构建 毕赤酵母 基因表达

领　　域： [生物学]