猪IL-18成熟蛋白基因重组真核表达载体的构建及其抗病毒作用研究  

Construction of eukaryotic expression vector and antiviruses analysis of porcine mature interleukin-18

作　　者：曹素芳[1] 王岩[1] 

机构地区：[1]郑州牧业工程高等专科学校动物医学系,河南郑州450011

出　　处：《郑州牧业工程高等专科学校学报》2011年第3期1-5,共5页Journal of Zhengzhou College of Animal Husbandry Engineering

基　　金：河南省基础与前沿技术研究计划项目(072300430060);河南省重点科技攻关项目(072102130023);河南省高校科技创新人才支持计划

摘　　要：应用RT-PCR方法从三元猪脾脏淋巴细胞中扩增出猪白细胞介素-18成熟蛋白(pmIL-18)基因,经克隆测序表明,pmIL-18基因长474bp,编码157个氨基酸。序列分析发现,与已发表的pmIL-18基因序列同源性很高,均为99%以上。将pmIL-18基因插入表达载体pcDNA3.1,成功构建了pmIL-18基因的重组真核表达载体pcD-NA3/pmIL-18。将重组质粒转染COS7细胞,经SDS-PAGE及Western-blotting分析表明,pmIL-18基因在COS7细胞中获得了瞬时表达。收集转染细胞培养上清,检测pmIL-18抗病毒活性,结果表明,转染上清对猪繁殖与呼吸综合征病毒没有明显的抑制作用。After the total RNA extracted from lymphocyte of pig spleen, porcine interleukin-18 mature protein （pmIL-18） gene was amplified by RT-PCR. It was cloned into pGEM-T easy vector and then sequenced. The result showed that the pmIL-18 gene composed 474 bp and the predicted primary protein is composed of 157 amino acide. After comparing with pmIL-18 gene from Genbank,it showed that nueleotide sequence homology was beyond 99%. The f ragment pmIL-18 gene was obtained from the recombinant plasmid pGEM/pmIL- 18 by restrictive enzyme BamHI and XhoI digestion,and then was inserted into eukaryotie expression vector peDNA3. After restriction enzyme analysis, PCR identification and sequencing,the recombinant plasmid pcDNA3/pmIL-18 was constructed suceessf ully. The recombinant plasmid was transfected into COS7 cells by liposome. The pmIL-18 gene was expressed instantaneously in COS7 cells, which was confirmed by RT-PCR, SDS-PAGE and Western-blotting. The supernatant of transfeeted cells containing recombinant pmIL-18was collected, concentrated and filtration sterilization, and then detected antiviruses activity. The result showed that the recombinant pmlL-18 has no inhibition to PRRSV.

关 键 词：猪 白细胞介素-18 真核表达载体 抗病毒活性 

分 类 号：S852.61[农业科学—基础兽医学]