动物病原大肠杆菌K88的绿色荧光蛋白基因标记及其稳定性研究

Transformation and Expression of gfp Gene into Escherich coli K88

成功地将gfp/luxAB双标记基因整合到K88染色体上,得到绿色荧光蛋白基因标记的大肠杆菌K88∶gfp/lux,其菌体和菌落形态与原始菌株K88完全一致,引入的新质粒不影响菌株的基本形态。从含gfp基因的质粒DNA和K88∶gfp/lux基因组DNA上均可扩增出大小约700 bp的gfp基因片段。大肠杆菌特异性基因检测结果表明,从大肠杆菌K88和K88∶gfp/lux基因组DNA上均扩增出大小约260 bp的大肠杆菌特异性基因片段,说明gfp基因标记后的菌株均为大肠杆菌。在相同的培养条件下,K88∶gfp/lux和K88的生长曲线的变化趋势基本相同。通过检测肠毒性基因(estA)发现,从大肠杆菌K88和K88∶gfp/lux基因组DNA上均扩增出大小约158 bp的肠毒性基因片段,说明gfp基因标记后的菌株在肠毒性方面未发生变化。在无选择压力条件下将K88∶gfp/lux菌株每隔12 h连续转接10次后,所有菌落均保持着均匀并且强烈的绿色荧光,说明标记基因在K88∶gfp/lux中的表达稳定性很高。K88∶gfp/lux和K88在中性偏酸性的环境中生长较好,当初始pH值偏碱性时,生长较差。

The Escherich coli K88 strain was chromosomally tagged with the marker genes gfp and luxAB by electroporation.The transformant was screened for the green fluorescence by fluorescence stereomicroscopy.Morphologically,the cells of engineered strain K88∶gfp/lux were similar to wild type as determined by laser scanning confocal microscopy.A 700 bp fragment of the gfp gene was amplified from the genome DNA of the GFP-tagged strains,and a 260 bp fragment was amplified from the genome DNA of K88 and K88∶gfp/lux which showed that the GFP-tagged strain was E.coli.The green fluorescence could be kept until more than 10 times of subcultures.The tested of disease-causing virulent gene（estA gene）of K88 and K88∶gfp/lux showed that a 158 bp fragment could be amplified in them.The insertion of gfp gene did not change the expression of the virulent gene.It was the same for the GFP-tagged strain and the wild type on the culture pH value and growth curve.