摘要
以杆状病毒pFastBacTM Dual为载体,将O型口蹄疫病毒(FMDV)VP1基因和山羊白介素18(gIL-18)基因,分别插入到双表达载体的P10启动子和多角体蛋白启动子PH的下游,构建重组质粒pFastBacTMDual-gIL18-VP1,然后转化DH10Bac感受态细胞,经三重抗性与蓝白斑筛选,获得杆状病毒重组质粒Bacmid-gIL18-VP1,并利用昆虫细胞进行转染。间接免疫荧光试验(IFA)检测表明gIL-18基因和VP1基因同时在同一细胞中独立表达。将共表达产物进行生物学活性分析表明,重组蛋白能够诱导山羊脾淋巴细胞生成IFN-γ抑制水疱性口炎病毒(VSV)在牛肾细胞(MDBK)上生长。本试验用Bac-to-Bac系统成功构建了同时含有gIL-18基因和VP1基因的重组杆状病毒,并在昆虫细胞中得到了高效表达,且具有良好的生物学活性,从而为下一步研究gIL-18在口蹄疫(FMD)中的免疫调节作用奠定了坚实的基础。
The VP1 gene of foot-and-mouth disease virus（FMDV） type O and goat interleukin-18（gIL-18） gene were inserted into the downstreams of P10 promoter and PH promoter of baculovirus expression plasmid pFastBacTM Dual,respectively.Then the constructing eukaryotic expression plasmid pFastBacTM Dual-gIL18-VP1 was transformed into DH10Bac,and screened by three antibiotics and blue-white patch.Finally,the recombinant Bacmid-gIL18-VP1 was transfected into Sf9 insect cells.The coexpression of gIL-18 and VP1 protein in sf9 cells was identified by IFA.Then identified bioactivity of the coexpressed production,the result showed that the recombinant protein could inhibit the growth of VSV in MDBK by inducing IFN-γ production in goat spleen lymphocyte.The recombinant baculovirus Bacmid-gIL18-VP1 can be successfully constructed by Bac-to-Bac expression system,with high expression and bioactivity in sf9 cells.The study laid a foundation for gIL-18＇s immunoloregulation in FMD.
出处
《中国兽医学报》
CAS
CSCD
北大核心
2011年第2期151-155,共5页
Chinese Journal of Veterinary Science
基金
山东省科技攻关重大专项(SDSP2005041005)
作者简介
作者简介：王婷婷（1983-），女，在读硕士。
通讯作者
