猪IL-18在酵母中表达及活性检测  

Expression and bioactivity detection of porcine mature interkine-18 in Pichiapastoris

作　　者：曹素芳[1] 祁画丽[1] 

机构地区：[1]郑州牧业工程高等专科学校,河南郑州450011

出　　处：《中国兽医杂志》2013年第6期32-35,共4页Chinese Journal of Veterinary Medicine

基　　金：河南省教育厅自然科学研究计划项目(2011A230012)

摘　　要：以pGEMT/pIL-18为模板,应用PCR法扩增出猪IL-18基因,用EcoRⅠ和XbaⅠ双酶切后,插入酵母表达载体pPICZαA中,经酶切、PCR扩增及序列测定,成功构建了酵母重组表达载体pPICZ/pIL-18,电击转化毕赤酵母X-33,应用Zeocin筛选获得高拷贝重组转化菌株,甲醇诱导表达,经SDS-PAGE电泳分析重组pIL-18蛋白的表达,并用Sephadex G-200纯化表达的重组pIL-18蛋白。运用MTT法检测其生物学活性。试验结果表明,重组X-33酵母菌株能够表达分泌性pIL-18,诱导72h表达量最高,其表达量占总蛋白表达量的38%。而且纯化的重组pIL-18蛋白具有明显促进淋巴细胞增殖的活性。The porcine interleukin-18（pIL-18） mature protein gene was amplified from pGEMT/pIL-18 by PCR. The fragment pIL-18 was digested by EoRI and XbaI,and was then inserted into Pichiapastoris expression vector pPICZaA. After restriction enzyme analysis , PCR identification and sequencing , the recombinant expression plasmid pPICZ/pIL-18 was constructed successfully, and was transformed into Pichia pastoris X-33 by electroporation, multi-copy recombinant strains were screened by Zeocin. pPICZ/ pIL-18 could express fused protein via methanol induction. The expressed product was identified by SDS- PAGE, and the fusion protein was purified by Sephadex G-200 column. The bioactivity of pIL-18 was detected by MTT. The results showed that the fusion protein of pIL-18 could be secreted by Pichia pastoris X-33 and the expression reached the peak at 72h. It was about 38% of the total cell protein. After being purified,the recombinant pIL-18 could obvious increase lymphocyte proliferation.

关 键 词：猪 白细胞介素18 毕赤酵母 生物活性 

分 类 号：S852.61[农业科学—基础兽医学]