畜牧兽医学报 ›› 2007, Vol. 38 ›› Issue (4): 395-399.doi:

• 基础兽医 • 上一篇    下一篇

基于RNA复制子GHRH基因表达载体的构建及在293细胞中的表达

任晓慧;乔继光;刘松财;张明军;章倩倩;侯峰;吕铁钢;周立光;吴琼;欧阳松应;张永亮   

  1. 1.吉林大学畜牧兽医学院,长春 130062; 2.华南农业大学动物科学学院,五山510642; 3.河北农业大学海洋学院,秦皇岛 066003;4.中国协和医科大学,北京 100730;5.深圳市肉品卫生检验所,深圳 518004
  • 收稿日期:1900-01-01 修回日期:1900-01-01 出版日期:2007-04-25 发布日期:2007-04-25

The Construction of GHRH Gene Expression Vector Based on RNA Replicon and Its Expression in 293 Cell

  • Received:1900-01-01 Revised:1900-01-01 Online:2007-04-25 Published:2007-04-25

摘要: 本研究将GHRH基因克隆到塞姆利基森林病毒(SFV)复制子质粒型表达载体pCMV-Rep-lacZ,构建了真核表达载体pCMV-Rep-GHRH。采用脂质体介导将表达质粒pCMV-Rep-GHRH转染293细胞,经RT-PCR、IFA、Tricine-SDS-PAGE和Western Blot检测,证明表达质粒pCMV-Rep-GHRH在293细胞中正确地表达了GHRH多肽分子。本研究还利用放免法检测质粒载体转染细胞培养上清中GHRH浓度,初步比较了RNA复制子载体pCMV-Rep-GHRH和普通载体(pIRES-GHRH和pCDNA3-GHRH)的表达效率,结果表明,转染后48h,RNA复制子载体表达GHRH的含量显著高于普通载体,分别比pIRES-GHRH和pCDNA3-GHRH高出36.12%(P<0.05)和67.94%(P<0.05)。结论:构建的真核表达载体pCMV-Rep-GHRH能够在真核细胞293中表达GHRH多肽分子,且表达水平显著高于普通载体,为下一步更好地调控动物生长以提高动物生产性能打下基础。

Abstract: In the present study, GHRH gene was cloned into pCMV-Rep-lacZ which digested by BamHⅠin order to replace the LacZ gene with our gene of interest and dephosphorylated by shrimp alkaline phosphatase, creating pCMV-Rep-GHRH. pCMV-Rep-GHRH was transfected by Lipofectin to prepared 293 cells. Results of RT-PCR, Tricine-SDS-PAGE ,Western Blot and immunofluorescence antibody assay(IFA) showed the our interested gene was expressed efficiently in transfected 293 cell. In order to compare the expressed efficacy between RNA replicon vector(pCMV-Rep-GHRH) and ordinary vetor(pIRES-GHRH and pCDNA3-GHRH), we also detected the concentration of GHRH from transfected cell supernatant by RIA in this study. The results showed that the concentration of GHRH from pCMV-Rep-GHRH transfected cell supernatant was 36.12%(P<0.05)and 67.94%(P<0.05)higher signifiantly than that of GHRH from pIRES-GHRH and pCDNA3-GHRH transfected cell supernatant respectively. We conclude that that GHRH can be expressed efficiently in eukaryotic expression vetor pCMV-Rep-GHRH transfected 293 cell. We can also draw another conclusion that the express level of RNA replicon vector was superior to ordinary vetor. It may be a potential way to increase animal productivity.