肝片吸虫GST真核表达载体构建及重组蛋白活性分析  

Construction on eukaryotic expression plasmid of Fasciola hepatica GST gene and analysis on recombinant protein

作　　者：冉旭华[1,2] 闻晓波[1,2] 王春仁[2] 刘娣[1] 孙中武[1] 李晓娟[2] 王密[2] 

机构地区：[1]东北林业大学博士后科研流动站,黑龙江省农业科学院博士后科研工作站,哈尔滨150086 [2]黑龙江八一农垦大学动物科技学院,大庆163319

出　　处：《中国人兽共患病学报》2011年第11期1005-1007,共3页Chinese Journal of Zoonoses

基　　金：黑龙江省教育厅青年骨干项目(No.1251G042);黑龙江省农科院博士后进站项目;黑龙江八一农垦大学博士科研启动基金(校启D2007-2)联合资助

摘　　要：目的构建肝片吸虫谷胱甘肽S-转移酶(GST)的真核表达载体,研究重组蛋白的免疫原性。方法以构建好的重组质粒pET30a-FhGST为模板,利用PCR技术扩增肝片吸虫谷胱甘肽S-转移酶基因(GST),连接真核表达载体pEGFP-N1,构建重组质粒pEGFP-GST,转染Hela细胞,荧光显微镜下观察绿色荧光,Western blotting检测重组蛋白表达情况。结果重组质粒pEGFP-GST在Hela细胞中获得了表达,Western blotting结果表明真核表达质粒表达的重组蛋白能与自然感染肝片吸虫的山羊阳性血清发生特异性反应。结论肝片吸虫GST真核表达载体构建成功,真核表达产物可与自然感染的山羊阳性血清发生特异性反应,具有生物学活性,可做为分子疫苗的候选进行进一步的研究。In this research,we constructed eukaryotic expression plasmid of Fasciola hepatica GST gene and analyzed the immunogenicity of recombinant protein.The glutathione S-transferase（GST）gene of F.hepatica played the importanty protective role against the worms infection.In this research,GST was amplified by PCR from the pET30a-FhGST,and then inserted into pEGFP-N1 vector to construct recombinant plasmids pEGFP-GST.The recombinant plasmid pEGFP-GST was transfected into Hela cells and fluorescent signal was detected by fluorescence microscope.Western blotting analysis was done to analysze immunogenicity of recombinant protein.And the results demonstrated that eukaryotic expression plasmid of Fasciola hepatica GST gene was constructed successfully.Recombinant protein could be specifically recognized by goat serum infected by Fasciola hepatica,which proving its immunoreactivity.It＇s suggested that the eukaryotic expression plasmid might be used as gene vaccine in further research.

关 键 词：肝片吸虫 GST基因 真核表达 活性分析 

分 类 号：S383.2[农业科学—农艺学]