摘要
目的构建肝片吸虫组织蛋白酶L(CatL)的真核表达载体,研究重组蛋白的生物学活性。方法以构建好的重组质粒pET30a-FhCatL为模板,利用PCR技术扩增肝片吸虫组织蛋白酶L基因(catL),连接真核表达载体pEGFP-N1,构建重组质粒pEGFP-N1-CatL,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-N1-CatL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CatL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,具有免疫反应性,可做为分子疫苗的候选和诊断抗原进行进一步的研究。
In this research we constructed eukaryotic expression plasmid expressing Fasciola hepatica cathepsin L-like proteases （CarL） and analyzed the immunogenicity of recombinant protein. CatL gene was amplified by PCR with the template of recombinant pET30a-FhCatL plasmid and cloned into pEGFP-N1 vector to construct recombinant plasmids pEGFP-N1-CatL. The recombinant plasmid pEGFP-N1-CatL was transfected into HeLa cells and fluorescent signal was detected by fluorescence microscope. The immunogenicity of recombinant protein identified by western blotting demonstrated that the recombinant pro- tein specifically reacted with serum from goat infected by Fasciola hepatica and showed robust immunoreactivity. The eukary- otic expression plasmid may be a potential gene vaccine or diagnose preparations in further study.
出处
《中国人兽共患病学报》
CAS
CSCD
北大核心
2013年第1期51-53,58,共4页
Chinese Journal of Zoonoses
基金
黑龙江省教育厅青年骨干项目(No.1251G042)
黑龙江省科技计划项目(GZ11B208)联合资助~~
关键词
肝片吸虫
catL基因
真核表达
活性分析
Fasciola hepatica
cathepsin L-like proteases
eukaryotic expression
activity analysis
