CAJ | 학술논문

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EcoR I/Xba I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-10 cDNA,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42.0 kD处可见较浓染条带,Western blotting分析可见特异条带;ELISA检测72 h上清液中目的蛋白浓度可达1.973 mg/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.
구건중조hIL-10진핵표체재체,탐색재필적효모중적표체,위진일보연구hIL-10생물학공능급림상응용전정기출.PCR확증목적기인hIL-10cDNA,경EcoR I/Xba I쌍매절후,장기아극륭지동양쌍매절적재체pPICZaA중,구건표체질립pPICZaA-hIL-10;전전화법,장기전입필적효모SMD1168,갑순유도Mut~+형전화자기인표체;대목적단백진행검측급순화.확증출료목적기인hIL-10 cDNA,중조질립pPICZaA-hIL-10경매절감정화서렬측정정학;표체산물경SDS-PAGE분석재42.0 kD처가견교농염조대,Western blotting분석가견특이조대;ELISA검측72 h상청액중목적단백농도가체1.973 mg/L;순화후목적단백순도체67.0%.실현료중조hIL-10재필적효모SMD1168중적성공표체화초보순화.
It was to construct recombinant hIL-10 eukaryotic expression vector and explore its expression in Pichia pastoris SMD1168,which could lay the foundation for further studying hIL-10 biological function and clinical application.Interest gene hIL-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 hIL-10 cDNA was obtained,and recombinant pPICZaA-hIL-10 was corrected by digested identification and sequencing.The expression 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 hIL-10 in Pichia SMD1168 was expressed successfully and purified preliminarily.