GFP与水稻条纹病毒病害特异蛋白的融合基因在sf9昆虫细胞中的表达

 用重叠延伸PCR(overlap Polymerase Chain Reaction,overlap-PCR)方法获得了含有绿色荧光蛋白(Green fluorescence protein,GFP)和水稻条纹病毒(Rice stripe virus,RSV)病害特异性蛋白SP(Disease-specific protein)两者的融合基因(GFP-SP),并将其克隆至载体pMD18-T,得到的重组质粒pMD18-T-GFP-SP经Xba Ⅰ/Hind Ⅲ双酶切后与经相同方法处理过的杆状病毒转移载体pFastBacHTb相连接构建成重组转移质粒pFastBacHTb-GFP-SP。酶切和测序鉴定证明了其序列的正确性并且无移码现象发生。将此质粒转化入含穿梭载体Bacmid的感受态细胞DH10Bac,得到含有目的基因片段的重组杆状病毒穿梭质粒rb-GFP-SP。以之转染草地贪夜蛾(Spodoptera frugiperda,sf9)细胞,24~48h后在显微镜200倍可见光视野下观察到被感染细胞发生了细胞和细胞核变大、细胞内颗粒物增多、细胞脱落甚至裂解等一系列与正常的st9昆虫细胞形态有明显区别的变化。荧光显微镜下可见部分细胞发出清晰的绿色荧光,且大部分荧光集中在细胞质部分。这些结果表明,GFP-SP融合蛋白在st9昆虫细胞内成功表达。

Expression of fusionprotein of GFP and Rice stripe virus disease-specific protein in insect cells sf9

The GFP (green fluorescence protein) and disease-specific protein (SP) genes of Rice stripe virus (RSV) were combined as a fusion gene with overlap-PCR approach and the fusing gene was inserted into pMD18-T at Xba Ⅰ/Hind Ⅲ sites to produce pMD18-T-GFP-SP. A Xba Ⅰ/Hind Ⅲ fragment was released from pMD18T-GFP-SP and placed into a transposing vector pFastBacHTb predigested with Xba Ⅰand Hind Ⅲ restriction enzymes to produce pFastBacHTb-GFP-SP. Restriction endonuclease and sequencing analysis verified that the target genes were correctly inserted in right reading frame, pFastBacHtb-GFP-SP was introduced into the competent cells (E. coli DH10Bac) containing a shuttle vector bacmid, and recombinant bacmid rbGFP-SP was isolated and transfected into Spodoptera frugiperda (sf9) cells. An increased diameter, granular appearance and cells lysis, which were much different from the morphology of normal sf9 cells, were observed under fluorescence invert microscope 24 -48 h after infection. Meanwhile green fluorescence could be seen in cytoplasm of infected cells. The above rsults showed that GFP-SP fusion gene was sucessfully expressed in sf9 cells.