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

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.