摘要
目的:以鸭γ-干扰素(DuIFN-γ)基因为目的基因,研究同一启动子调控下,不同拷贝数的目的基因的表达状况。方法:采用基因重组技术构建含DuIFN-γcDNA单拷贝和双拷贝重组质粒并转入真核细胞COS-7中进行表达。DuIFN-γ活性采用Griess试剂检测。结果:双拷贝重组质粒和单拷贝重组质粒50%最大活性稀释度分别为1:320和1:160。双拷贝重组质粒转染上清稀释10240倍后,仍保留DuIFN-γ活性,而单拷贝重组质粒转染上清经1280倍稀释后,即丧失活性。结论:在同一启动子作用下,顺式插入双拷贝目的基因可明显增强目的基因在真核细胞中的表达量。
Objective:In this study ,duck interferon-gamma(DuIFN-γ) gene has been used as target gene to investigate the effect of copy number of cloned foreign gene on the expression of target protein under control of a single promoter .Methods:The 1.4kb size of DuIFN-γ cDNA was cleaved from plasmid pGEM-DuIFN-γ with EcoRI and subcloned into the EcoRI site of pcDNA1.1/Amp vector.The copy number and orientation of insert in recombinant plasmids were characterised by restriction enzyme analysis and transfected into COS-7 cells for expression with FuGENE6 transfection reagent.The biological activity of DuIFN-γ in cultured supernatant was assayed with Griess reagent for its capacity of activating chicken macrophage cell line HD11 to secrete nitric oxide. The recombinant plasmids with one or two copies of forward inserts were identified with restriction enzyme analysis.Results: The results of DuIFN-γ biological assay showed that 50% of maximal DuIFN-γ activity was reserved after 1∶160 dilution of cultured supernatant transfected by recombinant plasmid with single copy of DuIFN-γ cDNA,the cultured supernatant of transfected COS-7 cells remained half of the maximal activity of DuIFN-γ following 1∶320 dilution.In addition ,the DuIFN-γ was still active after 10240 fold dilution for plasmid wth double copies of DuIFN-γ genes ,but inactive after 1280 fold dilution for plasmid with single copy of DuIFN-γ gene.Conclusion:By sharing the same promoter ,the level of target protein (DuIFN-γ) expressed by recombinant plasmid with two copies of forward insert was obviously enhanced in eukaryotic cells.
出处
《重庆医科大学学报》
CAS
CSCD
2001年第4期368-371,共4页
Journal of Chongqing Medical University
基金
澳大利亚国家医学卫生研究基金(NH&MRC)资助