猪IL-18成熟蛋白在大肠杆菌中的表达及生物学活性鉴定  

Expression of porcine IL-18 mature protein in Escherichia coli and identification of its bioactivity

作　　者：陈红英[1] 郑兰兰[1] 金钺[1] 李新生[1] 段廷云 崔保安[1] 

机构地区：[1]河南农业大学牧医工程学院,河南郑州450002 [2]漯河出入境检验检疫局,河南漯河462000

出　　处：《分子科学学报》2009年第4期284-289,共6页Journal of Molecular Science

基　　金：国家"十一五"科技支撑计划专项资助(2006BAD06A08)

摘　　要：以含猪IL-18全基因的重组质粒pGEM-IL-18为模板,PCR扩增猪IL-18成熟蛋白基因.将IL-18成熟蛋白片段定向插入原核表达载体pET-28a(+)中,构建重组表达质粒pET-IL-18,转化大肠杆菌BL21(DE3),在IPTG诱导下表达融合蛋白(His-IL-18),并进行融合蛋白的纯化、生物学活性鉴定.结果表明,SDS-PAGE可检测到相对分子质量约为2.1×104的融合蛋白,Western blot证实His-IL-18能与猪IL-18单克隆抗体发生特异性反应.重组猪IL-18经纯化后,能明显刺激猪脾脏T淋巴细胞增殖反应,在Marc-145细胞上抗猪繁殖与呼吸综合征病毒的活性为2.50×103IU/mg,在PK-15细胞上抗猪伪狂犬病毒、猪细小病毒的活性分别为2.00×103和2.24×103IU/mg.表明建立的表达系统能够表达重组猪IL-18,表达的重组猪IL-18具有一定的生物学活性.Porcine IL-18 mature protein gene was amplified with recombination plasmid pGEM-IL-18 as template and inserted into prokaryotic expression vector pET-28a（ ＋ ） to generate a recombinant plasmid pET-IL-18, pET-IL-18 was transformed into E. coli BI21（DE3）,which can express fusion protein in E. coli BI21（DE3） by IFFG induc- ing. The expressed product was identified by SDS-PAGE and Western blot. After purification, the bioactivity of.recombinant porcine IL-18 was identified by recombinant porcine interleukin-18 on lymphocyte proliferation assay and the replication of viruses in host cells. The results showed that fusion protein （ His-IL- 18） was about 2.1 ×10^4 and it could react with monoclonal antibody against porcine IL-18.Recombinant porcine IL-18 induced in vitro proliferation of porcine splenocytes. The anitviral activity of recombinant porcine IL-18 against porcine reproductive and respiratory syndrome virus at Marc-145 cell was 2.50×10^3 IU/mg.The antiviral activities of recombinant porcine IL-18 against pseudorabies virus and porcine parvovirus in PK-15 cells were 2.00×10^3 and 2.24 ×10^3 IU/mg,respectively. Expression system pET-IL-18 can express recombinant porcine IL-18,and recombinant porcine IL-18 has bioactivity.

关 键 词：猪IL-18 原核表达 生物学活性 抗病毒活性 

分 类 号：O785[理学—晶体学]