摘要
【目的】构建能在真核细胞中共表达斯氏艾美耳球虫微线蛋白5(MIC-5)和兔IL-15重组载体,为进一步研究抗斯氏艾美耳球虫的免疫调节性DNA疫苗奠定基础。【方法】将斯氏艾美耳球虫MIC-5和兔IL-15基因,克隆到真核双表达载体pBudce4.1中,应用脂质体介导法将构建的重组质粒pB-MIC-5-IL-15转染BHK-21细胞。在转染后,用RT-PCR法和间接免疫荧光试验(IFA)分别检测MIC-5和IL-15基因在BHK-21细胞中的转录和表达情况。【结果】在重组质粒pB-MIC-5-IL-15转染BHK-21细胞24 h后即可检测到MIC-5、IL-15基因进行了转录;在转染36、48、72h后,MIC-5和IL-15基因可同时在BHK-21细胞中表达。【结论】成功构建了含有斯氏艾美耳球虫MIC-5和兔IL-15基因的重组载体,实现了两种基因在真核细胞中的共表达,为抗斯氏艾美耳球虫的免疫调节性DNA疫苗研制奠定了基础。
【Objective】 The aim of this study is to construct a recombinant co-expression vector expressing Eimeria stiedai microneme protein 5（MIC-5） and rabbit IL-15 in eukaryotic cells,which may lay a foundation for further study immune regulatory DNA vaccine against infection of Eimeria stiedai.【Method】 E.stiedai MIC-5 gene and rabbit＇s IL-15 gene were respectively cloned into plasmid pBudce4.1 to construct recombinant vector pB-MIC-5-IL-15 for co-expression,and then the recombinant plasmid pB-MIC-5-IL-15 was transfected into BHK-21 cell line for expression.The RT-PCR and indirect fluorescent immunoassay were used to detect the transcription and expression of interesting genes in BHK-21 cell line,respectively.【Result】 The results showed that E.stiedai MIC-5 gene and rabbit＇s IL-15 gene were successfully transcripted.And interesting genes of E.stiedai MIC-5 and rabbit＇s IL-15 were successfully expressed at 36,48 and 72 hour post-transfection,respectively.【Conclusion】 The recombinant co-expression vector was successfully constructed,which can express E.stiedai MIC-5 and rabbit IL-15 gene in eukaryotic cells.
出处
《中国农业科学》
CAS
CSCD
北大核心
2011年第19期4096-4101,共6页
Scientia Agricultura Sinica
基金
科技部家畜疫病病原生物学国家重点实验室项目(2008-001)
石河子大学高层次人才专项基金(2009-011)
