人白介素12亚基p40和p35基因串联在毕赤酵母中的表达及表达产物活性分析

摘 要 利用毕赤酵母系统表达有活性的人单链白细胞介素12（hscIL-12），PCR法从质粒pBI121-IL-12中扩增hscIL-12基因，经酶切、连接构建重组表达载体pPIC9K-hscIL-12，SacI线性化后，PEG1000法转化毕赤酵母GS115，经G418筛选和菌落PCR鉴定，经甲醇诱导，hscIL-12在酵母中获得分泌表达，表达产物经Western Blot检测，显示该蛋白相对分子质量为70KDa，可与鼠抗人IL-12单克隆抗体特异性结合；定量分析结果表明，重组酵母培养上清中hscIL-12约占总蛋白的26％，表达量约为60mg／L；生物学活性实验表明，重组蛋白能促进人外周血淋巴细胞增殖。为利用rhscIL-12进行基因治疗奠定了基础。

Expression of Single Chain Human Interleukin-12 in Pichia pastoris and its bioactivity

Abstract　 Human single chain interleukin-12（hscIL-12）of bioactivity was expressed in methylotrophic yeast Pichia Pastoris expression system , hscIL-12 gene was amplified from plasmid pBI121-IL-12 by PCR. After digested by restricted enzyme, gene of interest was cloned into the yeast vector pPIC9K and obtained recombinant expression vector pPIC9K-hscIL-12. The pPIC9K-hscIL-12 was linearized with SacⅠ and then transformed into the Pichia Pastoris GS115 by PEG1000. Recombinant strains were screened by G418 resistant, and further confirmed by colony PCR. The hscIL-12 was induced to express in yeast by methano1. Western Blot analysis showed that the relative molecular weight of the expressed product was about 70kDa, the expression protein could bind to IL-12 monoclonal antibody specifically. Quantitative analysis showed that the target protein was in a level of 26% of the total protein of the culture supernatant，with a yield of 40mg/L. Bioactivity assay showed that the recombinant hscIL-12 could stimulate the lymphocyte proliferation. The successful expression of the rhscIL-12 in Pichia Pastoris can be potentially used in cancer gene therapy.