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.docVIP

动物病原大肠杆菌K88的绿色荧光蛋白基因标记(车建美，刘 波，蓝江林（福建省农业科学院农业生物资源研究所，福州 350003） 摘 要：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。 关键词：大肠杆菌K88；绿色荧光蛋白；gfp/luxAB基因 Transformation and Expression of Green Fluorescent Protein Gene in Escherich coli K88 CHE Jian-mei, LIU Bo, LAN Jiang-lin (1.Agricultural Bioresource Research Institute, Fujian Academy of Agricultural Sciences, Fuzhou 350003) Abstract：The Escherich coli strain was chromosomally tagged with the marker genes gfp and luxAB by electroporationransformant was screened for the green fluorescence by fluorescence stereomicroscopy. Morphologically, the cells of the engineered strain K88:gfp/lux were similar to wild type one 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 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 didn’t chang the expression of the virulent gene. It was the same for the GFP-tagged strian and the wild type one on the culture pH value and growth curve. Key words：Escherich coli，Green fluorescent protein，gfp/luxAB gene 肠毒素性大肠杆菌（Enterotoxigenic Escherichia coli．，ETEC）是一类引起人和幼畜腹泻的重要病原菌，发病率和死亡率均很高，是目前的研究热点之一，该菌的致病性与其具有黏附素和产肠毒素密切相关[1]，猪源产肠毒素性大肠杆菌的黏附素抗原主要有K88，K99